Journal of Ophthalmology
Volume 2017 (2017), Article ID 7598140, 23 pages
https://doi.org/10.1155/2017/7598140

Research Article

Inhibition of Rho Kinase Induces Antioxidative Molecules and Suppresses Reactive Oxidative Species in Trabecular Meshwork Cells

Tomokazu Fujimoto,¹ Toshihiro Inoue,¹ Saori Ohira,¹ Nanako Awai-Kasaoka,¹ Takanori Kameda,² Miyuki Inoue-Mochita,¹ and Hidenobu Tanihara¹

¹Department of Ophthalmology, Faculty of Life Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto 860-8556, Japan
²Department of Ophthalmology and Visual Sciences, Kyoto University Graduate School of Medicine, Yoshida-Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan

Correspondence should be addressed to Toshihiro Inoue; noel@da2.so-net.ne.jp

Received 21 February 2017; Revised 22 May 2017; Accepted 30 May 2017; Published 19 July 2017

Academic Editor: Ciro Costagliola

Copyright © 2017 Tomokazu Fujimoto et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

Purpose. To investigate the effect of rho kinase inhibitors on oxidative stress in trabecular meshwork (TM) cells. Methods. TM cells were isolated from the eyes of cynomolgus monkeys. Y-27632 and menadione were used to inhibit rho kinase and induce production of reactive oxygen species (ROS), respectively. The cynomolgus monkey array and 12,613 probes were used in DNA microarray analysis, and the affected genes were categorized using gene ontology analysis. The mRNA levels of the target genes were confirmed by real-time RT-PCR. Intracellular oxidative stress was detected using a fluorescent reagent sensitive to ROS. Cell viability was assessed by the WST-8 assay. Results. Gene ontology analysis revealed upregulation of genes involved in antioxidant activity, and upregulation of catalase was confirmed by real-time RT-PCR after 30 min treatment with Y-27632. Production of ROS was increased by menadione, and the effect was partly suppressed by pretreatment with Y-27632. At a lower dose of menadione, Y-27632 stimulated TM cells and significantly increased their viability following menadione treatment compared to control cells. Conclusion. Using microarray analysis, Y-27632 was shown to upregulate antioxidative genes including catalase and partially reduce ROS production and cell death by oxidative stress caused by menadione.

1. Introduction

Oxidative stress is a major physiological phenomenon, mediated through the production of reactive oxygen species (ROS), such as peroxides, superoxide, hydroxyl radical, and singlet oxygen. ROS play an important role in cell homeostasis and pathogen response and are therefore essential in biological processes. In contrast, increases in ROS are seen in various age-related diseases including glaucoma [1]. For instance, in the aqueous humor of glaucoma patients, the levels of oxidative stress markers are significantly increased [2–5]. Additionally, oxidative DNA damage is reportedly increased in the trabecular meshwork (TM) of glaucoma patients [6, 7]. These findings indicate that the TM of glaucomatous eyes is continuously exposed to oxidative stress, and therefore, damage to TM may increase outflow resistance and the risk of glaucoma progression. In line with this, lower systemic antioxidant capacity is related to higher intraocular pressure (IOP) levels in open-angle glaucoma patients [8]. Moreover, glaucoma-related genes, such as CYP1B1 and FOXC1, are reportedly linked to oxidative stress in the eyes [9–12]. Taken together, control of oxidative stress in the eye may be a therapeutic target to slow glaucoma progression.

Rho-rho kinase (ROCK) signaling controls polymerization of actin and thereby mediates various cell functions, such as contraction, migration, phagocytosis, and mitosis. Inhibition of ROCK increases aqueous outflow by depolymerizing F-actin in TM cells and Schlemm’s canal endothelial cells [13, 14]. A ROCK inhibitor, ripasudil, has been approved as an IOP-lowering drug in Japan [15]. Ripasudil significantly reduces the IOP of glaucoma patients upon either single or multiple administration [16, 17]. However, ROCK inhibitors have drawn attention as antioxidative drugs against cardiovascular diseases and chronic renal injury [18, 19]. Indeed, ripasudil (also known as K-115) has been reported to have a neuroprotective effect on the optic nerve by suppressing oxidative stress in an animal model [20]. Thus, the effect of ROCK inhibitors on oxidative stress in TM cells is of interest from a therapeutic point of view against glaucoma.

Here, we show the results of an exhaustive investigation using a microarray, revealing that treatment with Y-27632, a well-known ROCK inhibitor, upregulates antioxidative molecules in TM cells, inhibits ROS production, and promotes cell survival.

2. Materials and Methods

2.1. Cell Culture

Trabecular meshwork (TM) cells were isolated from the eyes of cynomolgus monkeys (Shin Nippon Biomedical Laboratories, Kagoshima, Japan) according to the method described previously [21]. Primary TM cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Wako, Osaka, Japan) supplemented with 10% FBS, 2 mM glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, and 0.5 μg/mL amphotericin B at 37°C in 5% CO₂. These cells were used after 2–5 passages. The character of the isolated cells in the present study was confirmed by expression of specific TM markers (caveolin 1, collagen 4α5, matrix gla protein, tissue inhibitor of metalloproteinase 3, and vascular cell adhesion protein 1), phagocytosis function, and myocilin induction by dexamethasone as described previously [22].

2.2. DNA Microarray Analysis

Custom cDNA microarray analysis was performed using a CombiMatrix microarray (CombiMatrix, Mukilteo, WA) as described previously [23]. Briefly, the cynomolgus monkey array was designed to detect directly labeled mRNA from 12,613 probes. Confluent TM cells in 100 mm dishes were treated with 25 μM Y-27632 (Merck Millipore, Darmstadt, Germany) or vehicle (deionized water) for 30 min. Total RNA was extracted from the cells, and the integrity and concentration of total RNA was measured using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). Fluorescence-labeled antisense RNA was synthesized by direct incorporation of Cy5-UTP or Cy3-UTP, using each RNA sample and an RNA Transcript SureLABEL Core kit (Takara Bio, Shiga, Japan). Labeled antisense RNAs were hybridized simultaneously with the microarray chips. DNA microarray preparation, hybridization, processing, scanning, and analyses were performed according to the manufacturer’s instructions (Filgen, Nagoya, Japan). Fluorescent images of hybridized microarrays were obtained with a GenePix 4000B Scanner (Molecular Devices, Sunnyvale, CA). Array-Pro Analyzer Ver4.5 (Media Cybernetics, Silver Spring, MD) was used to determine the signal intensity of each spot and its local background. Scanned images were analyzed using Microarray Data Analysis Tool Ver3.2 software (Filgen). Signals from Y-27632 treated cells were compared with those from vehicle-treated cells, and genes that showed greater than 3/2-fold change in expression in at least one of the pairwise probe comparisons were considered upregulated, whereas those that showed a change of expression smaller than 2/3-fold were considered downregulated. These analyses were performed three times using TM cells from three different monkeys independently, and genes with common differences in expression among the three experiments were identified as affected genes. The affected genes were further analyzed by gene ontology, in which putative functions of gene products were categorized as “biological process,” “cellular component,” or “molecular function” by a BLAST homology search of EST sequences available from the National Center for Biotechnology Information.

2.3. Real-Time RT-PCR

Total RNA was isolated from cultured TM cells treated with Y-27632 for 30 min using NucleoSpin RNA (Macherey-Nagel, Düren, Germany). Total RNA was reverse transcribed (PrimeScript RT Master Mix; Takara Bio Inc., Shiga, Japan) according to the manufacturer’s protocol. Quantitative real-time RT-PCR was performed using an ABI Prism 7000 (Life Technologies). Reactions were performed in 20 μL of reaction mixture containing 10 μL PCR master mix (SYBR Premix Ex Taq II; Takara Bio Inc.), 0.4 μM primer pairs, and 2 μL cDNA samples. The gene-specific primer pairs were as follows: monkey catalase, forward (F) 5′-GCA AAT CTG TGA GGC CGG GG-3′; reverse (R) 5′-GCG CAT CTA GCA CCG GAG AA-3′ and 18S ribosomal RNA, (F) 5′-GCC CGA AGC GTT TAC TTT GA-3′; (R) 5′-CCG CGG TCC TAT TCC ATT ATT-3′. The thermal cycling conditions were 95°C for 30 s and 40 cycles of 95°C for 5 s and 60°C for 31 s. All PCR reactions were performed in duplicate.

Relative expression of catalase in the Y-27632-treated samples was compared to that in control samples using the comparative CT method (ΔΔCT method); 18S ribosomal RNA was used as an endogenous control. The threshold cycle, CT, was determined after setting the threshold in the linear amplification phase of the PCR reaction and ΔCT was defined as ΔCT = CT (target gene) − CT (18S rRNA). Relative expression of the target gene was calculated as: 2−ΔΔCT, ΔΔCT = ΔCT (treated sample) − ΔCT (control).

2.4. Intracellular Oxidative Stress Detection

The effects of Y-27632 on the production of ROS were evaluated using CellROX® green reagent (Life Technologies) in the TM cells. These cells were cultured on 6 cm dishes in DMEM containing 10% FBS and antibiotics at 37°C in 5% CO₂. After cells had grown to confluence, they were pretreated with Y-27632 for 30 min and then stimulated with 100 μM menadione (Sigma, St. Louis, MO) for 1 h. CellROX reagent was then added to each dish to give a final concentration of 5 μM and incubated for 30 min at 37°C. After incubation, TM cells were washed in PBS and detached by trypsin/EDTA solution and centrifuged at 1200 rpm for 3 min. The supernatant was removed, and cells were fixed in 4% paraformaldehyde in PBS for 15 min and then centrifuged twice at 1200 rpm for 3 min, resuspending in PBS after each spin. FITC fluorescence of TM cells was analyzed using a Cell Sorter SH800 (Sony Biotechnology, Tokyo, Japan).

2.5. Cell Viability Assay

The effects of Y-27632 on TM cell viability were evaluated using the WST-8 assay (Cell Counting Kit-8, Dojindo Laboratories, Kumamoto, Japan). Cells were seeded on 96-well plates (1 × 10⁴ cells/well) and incubated at 37°C under 5% CO₂ overnight. After pretreatment with Y-27632 for 30 min, cells were stimulated with H₂O₂ or menadione for 24 h. CCK-8 reagents were added into each well and incubated for 2 h at 37°C. Absorbance at 450 nm was determined using a microplate reader (Multiskan FC, Thermo Fisher Scientific). Cell viability was expressed as a percentage of control (vehicle-treated) cells.

2.6. Direct Antioxidant Activity of Y-27632

Direct antioxidant activity was assessed by 2-methyl-6-p-methoxyphenylethynylimidazopyrazinone (AB-2950 MPEC; ATTO, Tokyo, Japan), a superoxide-sensitive luminescent reagent, and reagents for xanthine-oxidase-induced superoxide production (AB-2970 CLETA-S, ATTO) following the manufacturer’s protocol. Briefly, 10 μL of 300 μM MPEC/ethanol and 80 μL of 1.25 unit/mL xanthine oxidase/HEPES were mixed. Then, 10 μL of 25 μM Y-27632 or 20 mM n-acetyl cysteine (positive control) was added into each well of a 96-well plate. Subsequently, 90 μL of the mixture of MPEC and xanthine oxidase and 200 μL of xanthine were added to each well. The luminescent signal was measured for 10 s by a luminometer (AB-2270 Octa; ATTO).

2.7. Statistical Analysis

Data are presented as means ± standard error. Statistical comparisons of multiple groups were performed using the Tukey-Kramer HSD test and Dunnett’s test, and those of two groups were performed using Wilcoxon rank sum test and Wilcoxon signed rank test. Differences were considered statistically significant at .

3. Results

3.1. Microarray Expression Profile in Y-27632-Treated TM Cells

Among the 12,613 genes analyzed by microarray, the affected genes are listed in Tables 1 and 2; 444 genes were upregulated, and 56 were downregulated. Significantly upregulated and downregulated gene categories based on gene ontology analysis in Y-27632 treated TM cells are listed in Tables 3 and 4. Gene ontology analysis revealed that the upregulated genes were related to various cellular functions including antioxidant activity (), and downregulated genes were related to integrin complexes (), and calcium ion transport into the cytosol (). In the category of antioxidant activity, upregulated genes were homologous to human gene coding catalase (), thioredoxin domain-containing 2 (also known as spermatozoa; ), nucleoredoxin (), albumin (probe 1, ; probe 2, ), and glutathione transferase zeta 1 (). Upregulation of the mRNA of catalase, an extensively investigated antioxidant, was confirmed by real-time RT-PCR and found to be 1.5 times higher in TM cells treated with Y-27632 compared to the control TM cells (; Figure 1(a)). In contrast, four other genes involved in antioxidant activity were not significantly affected after treatment with Y-27632 (data not shown).

Table 1: Genes that are upregulated in TM cells.

Accession number
Human RefSeq description
Fold change

DW528016
gi|75750485|ref|NM_004773.2 Homo sapiens zinc finger, HIT type 3 (ZNHIT3), mRNA
6.79928

CJ434702
gi|20986504|ref|NM_002753.2 Homo sapiens mitogen-activated protein kinase 10 (MAPK10), transcript variant 1, mRNA
5.85538

AB168851
gi|224586874|ref|NM_033124.4 Homo sapiens coiled-coil domain-containing 65 (CCDC65), mRNA
5.77453

AB169150
gi|223555972|ref|NR_026827.1 Homo sapiens hypothetical LOC84856 (LOC84856), noncoding RNA
5.01086

DW523643
gi|225903398|ref|NM_001146152.1 Homo sapiens cytochrome P450, family 51, subfamily A, polypeptide 1 (CYP51A1), transcript variant 2, mRNA
4.6977

AK240630
gi|4503754|ref|NM_002021.1 Homo sapiens flavin-containing monooxygenase 1 (FMO1), mRNA
4.64946

BB894083
gi|154689768|ref|NM_020840.1 Homo sapiens folliculin-interacting protein 2 (FNIP2), mRNA
4.52052

AB168218
gi|85060516|ref|NM_199321.2 Homo sapiens zona pellucida-binding protein 2 (ZPBP2), transcript variant 2, mRNA
4.20286

AB168199
gi|156523965|ref|NM_001102470.1 Homo sapiens alcohol dehydrogenase 6 (class V) (ADH6), transcript variant 1, mRNA
3.89514

AB172502
gi|50897849|ref|NM_001001936.1 Homo sapiens actin filament-associated protein 1-like 2 (AFAP1L2), transcript variant 1, mRNA
3.84241

CJ448047
gi|46909588|ref|NM_002731.2 Homo sapiens protein kinase, cAMP-dependent, catalytic, beta (PRKACB), transcript variant 2, mRNA
3.75324

DC857227
gi|239752603|ref|XM_002348257.1 PREDICTED: Homo sapiens similar to immunoglobulin lambda-like polypeptide 1 (LOC100294459), mRNA
3.66898

CJ449582
gi|9506614|ref|NM_019023.1 Homo sapiens protein arginine methyltransferase 7 (PRMT7), mRNA
3.64058

EF208813
gi|24797075|ref|NM_002121.4 Homo sapiens major histocompatibility complex, class II, DP beta 1 (HLA-DPB1), mRNA
3.57386

DQ417745
gi|194,248,050|ref|NM_000839.3 Homo sapiens glutamate receptor, metabotropic 2 (GRM2), transcript variant 1, mRNA
3.5509

AB049894
gi|66571326|ref|NM_020914.3 Homo sapiens ring finger protein 213 (RNF213), mRNA
3.51068

DC850932
gi|117676364|ref|NM_014350.2 Homo sapiens tumor necrosis factor, alpha-induced protein 8 (TNFAIP8), transcript variant 1, mRNA
3.47535

BB877436
gi|142360165|ref|NM_005123.2 Homo sapiens nuclear receptor subfamily 1, group H, member 4 (NR1H4), mRNA
3.46906

AB174726
gi|22208962|ref|NM_016150.3 Homo sapiens ankyrin repeat and SOCS box-containing 2 (ASB2), mRNA
3.30479

AB174122
gi|209862773|ref|NM_002483.4 Homo sapiens carcinoembryonic antigen-related cell adhesion molecule 6 (nonspecific cross-reacting antigen) (CEACAM6), mRNA
3.27904

AB173773
gi|38569483|ref|NM_017641.2 Homo sapiens kinesin family member 21A (KIF21A), mRNA
3.26679

DK578446
gi|15718677|ref|NM_033257.2 Homo sapiens DiGeorge syndrome critical region gene 6 like (DGCR6L), mRNA
3.26114

AB168950
gi|38261961|ref|NM_018179.3 Homo sapiens activating transcription factor 7-interacting protein (ATF7IP), mRNA
3.25607

CJ488707
gi|45333915|ref|NM_178456.2 Homo sapiens chromosome 20 open reading frame 85 (C20orf85), mRNA
3.24156

DC639327
gi|154800442|ref|NM_005074.3 Homo sapiens solute carrier family 17 (sodium phosphate), member 1 (SLC17A1), mRNA
3.23693

BB895966
gi|32483409|ref|NM_000583.2 Homo sapiens group-specific component (vitamin D-binding protein) (GC), mRNA
3.22662

DQ417744
gi|194248050|ref|NM_000839.3 Homo sapiens glutamate receptor, metabotropic 2 (GRM2), transcript variant 1, mRNA
3.1862

AB168486
gi|195972893|ref|NM_152764.2 Homo sapiens chromosome 16 open reading frame 73 (C16orf73), mRNA
3.17552

AB047624
gi|45446748|ref|NM_004984.2 Homo sapiens kinesin family member 5A (KIF5A), mRNA
3.1749

CJ446015
gi|187761371|ref|NM_004044.5 Homo sapiens 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase (ATIC), mRNA
3.17026

AB171508
gi|89276768|ref|NM_002747.3 Homo sapiens mitogen-activated protein kinase 4 (MAPK4), mRNA
3.12296

DC639656
gi|148596946|ref|NM_001098483.1 Homo sapiens chromosome 10 open reading frame 125 (C10orf125), transcript variant 1, mRNA
3.11887

AB048996
gi|211938419|ref|NM_002898.3 Homo sapiens RNA-binding motif, single stranded interacting protein 2 (RBMS2), mRNA
3.11444

DC630823
gi|215422360|ref|NM_004786.2 Homo sapiens thioredoxin-like 1 (TXNL1), transcript variant 1, mRNA
3.09601

AB173283
gi|197927150|ref|NM_006158.3 Homo sapiens neurofilament, light polypeptide (NEFL), mRNA
3.09117

DC633065
gi|34486089|ref|NM_004152.2 Homo sapiens ornithine decarboxylase antizyme 1 (OAZ1), mRNA
3.0599

AB174730
gi|115387109|ref|NM_017831.3 Homo sapiens ring finger protein 125 (RNF125), mRNA
3.03611

AB046044
gi|170650673|ref|NM_000440.2 Homo sapiens phosphodiesterase 6A, cGMP-specific, rod, alpha (PDE6A), mRNA
3.03568

AB179171
gi|82546851|ref|NM_175605.3 Homo sapiens intraflagellar transport 88 homolog (Chlamydomonas) (IFT88), transcript variant 1, mRNA
3.029

AB051155
gi|35493712|ref|NM_017890.3 Homo sapiens vacuolar protein sorting 13 homolog B (yeast) (VPS13B), transcript variant 5, mRNA
3.00764

AB072740
gi|155029549|ref|NM_178828.4 Homo sapiens chromosome 9 open reading frame 79 (C9orf79), mRNA
3.00753

BB881371
gi|162809333|ref|NM_002864.2 Homo sapiens pregnancy-zone protein (PZP), mRNA
2.99069

CJ463711
gi|215272411|ref|NM_001142334.1 Homo sapiens ataxin 2-binding protein 1 (A2BP1), transcript variant 6, mRNA
2.98758

AB170648
gi|5174424|ref|NM_006052.1 Homo sapiens Down syndrome critical region gene 3 (DSCR3), mRNA
2.9606

AB220465
gi|58331245|ref|NM_000817.2 Homo sapiens glutamate decarboxylase 1 (brain, 67 kDa) (GAD1), transcript variant GAD67, mRNA
2.95173

AB062990
gi|33149330|ref|NM_022463.3 Homo sapiens nucleoredoxin (NXN), mRNA
2.94787

AB220509
gi|139394620|ref|NM_006574.3 Homo sapiens chondroitin sulfate proteoglycan 5 (neuroglycan C) (CSPG5), mRNA
2.92569

AB070086
gi|22538813|ref|NM_002985.2 Homo sapiens chemokine (C-C motif) ligand 5 (CCL5), mRNA
2.92446

AB173147
gi|194294550|ref|NM_015989.4 Homo sapiens cysteine sulfinic acid decarboxylase (CSAD), mRNA
2.91509

AB051133
gi|28329444|ref|NM_014379.2 Homo sapiens potassium channel, subfamily V, member 1 (KCNV1), mRNA
2.87085

AB174705
gi|51477720|ref|NM_001003811.1 Homo sapiens testis-expressed 11 (TEX11), transcript variant 1, mRNA
2.86731

CJ469703
gi|169646771|ref|NM_002064.2 Homo sapiens glutaredoxin (thioltransferase) (GLRX), transcript variant 1, mRNA
2.85957

AB220438
gi|109633045|ref|NM_001042437.1 Homo sapiens ST3 beta-galactoside alpha-2,3-sialyltransferase 5 (ST3GAL5), transcript variant 2, mRNA
2.85364

BB881475
gi|170650673|ref|NM_000440.2 Homo sapiens phosphodiesterase 6A, cGMP-specific, rod, alpha (PDE6A), mRNA
2.84456

AB168610
gi|62632749|ref|NM_014616.1 Homo sapiens ATPase, class VI, type 11B (ATP11B), mRNA
2.84449

AB173806
gi|134031964|ref|NR_003491.1 Homo sapiens myocardial infarction associated transcript (nonprotein coding) (MIAT), noncoding RNA
2.82677

AB063045
gi|190570175|ref|NM_152906.4 Homo sapiens chromosome 22 open reading frame 25 (C22orf25), mRNA
2.80672

AB168446
gi|223278411|ref|NR_026782.1 Homo sapiens chromosome 1 open reading frame 175 (C1orf175), transcript variant 2, transcribed RNA
2.79215

CJ473171
gi|215490055|ref|NM_001142434.1 Homo sapiens meningioma-expressed antigen 5 (hyaluronidase) (MGEA5), transcript variant 2, mRNA
2.78342

CJ450383
gi|83641894|ref|NM_031157.2 Homo sapiens heterogeneous nuclear ribonucleoprotein A1 (HNRNPA1), transcript variant 2, mRNA
2.78321

DW528650
gi|58430810|ref|NM_148912.2 Homo sapiens abhydrolase domain-containing 11 (ABHD11), transcript variant 1, mRNA
2.77238

AB362499
gi|48255911|ref|NM_012338.3 Homo sapiens tetraspanin 12 (TSPAN12), mRNA
2.76878

AB173195
gi|196049386|ref|NM_002198.2 Homo sapiens interferon regulatory factor 1 (IRF1), mRNA
2.76544

AB168743
gi|188536107|ref|NM_001127458.1 Homo sapiens cardiolipin synthase 1 (CRLS1), transcript variant 2, mRNA
2.76042

CJ444006
gi|157151724|ref|NM_001004333.3 Homo sapiens ribonuclease, RNase K (RNASEK), mRNA
2.75719

DK583186
gi|156631002|ref|NM_006913.3 Homo sapiens ring finger protein 5 (RNF5), mRNA
2.75394

AB168582
gi|239757151|ref|XM_002345145.1 PREDICTED: Homo sapiens hypothetical protein LOC100292623 (LOC100292623), mRNA
2.75285

BB884235
gi|70906436|ref|NM_000509.4 Homo sapiens fibrinogen gamma chain (FGG), transcript variant gamma-A, mRNA
2.74831

AB070088
gi|148613875|ref|NM_144715.3 Homo sapiens EF-hand domain family, member B (EFHB), mRNA
2.74738

AB174502
gi|239757416|ref|XM_002345385.1 PREDICTED: Homo sapiens similar to hCG2019710 (LOC100294049), mRNA
2.73964

AB172306
gi|196162714|ref|NM_024786.2 Homo sapiens zinc finger, DHHC-type-containing 11 (ZDHHC11), mRNA
2.72049

BB878691
gi|19743563|ref|NM_000766.3 Homo sapiens cytochrome P450, family 2, subfamily A, polypeptide 13 (CYP2A13), mRNA
2.72031

AB174483
gi|55775474|ref|NM_194326.2 Homo sapiens ribosomal protein S19-binding protein 1 (RPS19BP1), mRNA
2.68579

DC632651
gi|22538474|ref|NM_018955.2 Homo sapiens ubiquitin B (UBB), mRNA
2.66418

AB168353
gi|197927266|ref|NM_004388.2 Homo sapiens chitobiase, di-N-acetyl (CTBS), mRNA
2.64444

AB169323
gi|156616291|ref|NM_018100.3 Homo sapiens EF-hand domain (C-terminal)-containing 1 (EFHC1), mRNA
2.6433

AB048961
gi|209413742|ref|NM_005458.6 Homo sapiens gamma-aminobutyric acid (GABA) B receptor, 2 (GABBR2), mRNA
2.63945

AB173162
gi|73747880|ref|NM_015113.3 Homo sapiens zinc finger, ZZ-type with EF-hand domain 1 (ZZEF1), mRNA
2.63631

AB179192
gi|237681201|ref|NM_019644.3 Homo sapiens ankyrin repeat domain 7 (ANKRD7), mRNA
2.62211

CJ469417
gi|169790802|ref|NM_005271.2 Homo sapiens glutamate dehydrogenase 1 (GLUD1), mRNA
2.62134

AB172772
gi|61835190|ref|NM_006578.3 Homo sapiens guanine nucleotide-binding protein (G protein), beta 5 (GNB5), transcript variant 1, mRNA
2.61592

DC857715
gi|169234652|ref|NM_007360.2 Homo sapiens killer cell lectin-like receptor subfamily K, member 1 (KLRK1), mRNA
2.6139

AB179131
gi|40807356|ref|NM_005094.2 Homo sapiens solute carrier family 27 (fatty acid transporter), member 4 (SLC27A4), mRNA
2.61058

DC647811
gi|92091576|ref|NM_015533.3 Homo sapiens dihydroxyacetone kinase 2 homolog (S. cerevisiae) (DAK), mRNA
2.60217

AB171456
gi|197927256|ref|NM_001134664.1 Homo sapiens sterile alpha motif domain-containing 13 (SAMD13), transcript variant 3, mRNA
2.59522

CJ490832
gi|169205007|ref|XM_001714899.1 PREDICTED: Homo sapiens hypothetical LOC100131988 (LOC100131988), mRNA
2.58063

DC635714
gi|57013275|ref|NM_006082.2 Homo sapiens tubulin, alpha 1b (TUBA1B), mRNA
2.57848

AB055358
gi|225735571|ref|NR_027416.1 Homo sapiens nuclear factor erythroid-derived 2-like 3 pseudogene (LOC100272146), noncoding RNA
2.57079

CJ458955
gi|197382308|ref|NM_183394.2 Homo sapiens Ca⁺⁺-dependent secretion activator (CADPS), transcript variant 2, mRNA
2.55777

DC636940
gi|54112387|ref|NM_001005738.1 Homo sapiens formyl peptide receptor 2 (FPR2), transcript variant 2, mRNA
2.54881

CJ493104
gi|56676323|ref|NM_001001552.3 Homo sapiens LEM domain-containing 1 (LEMD1), mRNA
2.5487

AB172122
gi|239754513|ref|XM_001716238.2 PREDICTED: Homo sapiens hypothetical LOC100128081 (LOC100128081), mRNA
2.54837

CJ469678
gi|197304748|ref|NM_032727.3 Homo sapiens internexin neuronal intermediate filament protein, alpha (INA), mRNA
2.53491

AB070172
gi|32490571|ref|NM_012307.2 Homo sapiens erythrocyte membrane protein band 4.1-like 3 (EPB41L3), mRNA
2.52667

DW528013
gi|225735571|ref|NR_027416.1 Homo sapiens nuclear factor erythroid-derived 2-like 3 pseudogene (LOC100272146), noncoding RNA
2.52479

AB171587
gi|161169016|ref|NM_001111019.1 Homo sapiens neuron navigator 2 (NAV2), transcript variant 4, mRNA
2.5209

BB888693
gi|49574509|ref|NM_016013.2 Homo sapiens NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, assembly factor 1 (NDUFAF1), mRNA
2.51576

BB897871
gi|207113180|ref|NM_001097577.2 Homo sapiens angiogenin, ribonuclease, RNase A family, 5 (ANG), transcript variant 2, mRNA
2.50309

AB168153
gi|222352148|ref|NM_018665.2 Homo sapiens DEAD (Asp-Glu-Ala-Asp) box polypeptide 43 (DDX43), mRNA
2.50236

AB173823
gi|148225856|ref|NM_001097579.1 Homo sapiens G protein-coupled receptor 34 (GPR34), transcript variant 4, mRNA
2.49912

AB168907
gi|194688136|ref|NM_002358.3 Homo sapiens MAD2 mitotic arrest deficient-like 1 (yeast) (MAD2L1), mRNA
2.49825

DK583616
gi|18390348|ref|NM_000972.2 Homo sapiens ribosomal protein L7a (RPL7A), mRNA
2.49444

CJ471599
gi|197245445|ref|NM_024958.2 Homo sapiens neurensin 2 (NRSN2), mRNA
2.49281

AB179111
gi|118572587|ref|NM_001761.2 Homo sapiens cyclin F (CCNF), mRNA
2.49071

AB060886
gi|23199979|ref|NM_022470.2 Homo sapiens zinc finger, matrin type 3 (ZMAT3), transcript variant 1, mRNA
2.48362

AB056810
gi|126131101|ref|NM_138694.3 Homo sapiens polycystic kidney and hepatic disease 1 (autosomal recessive) (PKHD1), transcript variant 1, mRNA
2.48299

DC645529
gi|4501988|ref|NM_001134.1 Homo sapiens alpha-fetoprotein (AFP), mRNA
2.46808

AB050420
gi|189095267|ref|NM_000554.4 Homo sapiens cone-rod homeobox (CRX), mRNA
2.45627

AB243403
gi|116235483|ref|NM_002701.4 Homo sapiens POU class 5 homeobox 1 (POU5F1), transcript variant 1, mRNA
2.4534

AB173020
gi|94420687|ref|NM_004233.3 Homo sapiens CD83 molecule (CD83), transcript variant 1, mRNA
2.44967

DC643036
gi|50409862|ref|NM_017584.5 Homo sapiens myoinositol oxygenase (MIOX), mRNA
2.44791

BB897024
gi|142976728|ref|NM_016245.3 Homo sapiens hydroxysteroid (17-beta) dehydrogenase 11 (HSD17B11), mRNA
2.43894

DW522619
gi|34147617|ref|NM_138807.2 Homo sapiens chromosome 3 open reading frame 31 (C3orf31), mRNA
2.43416

DC645828
gi|16332359|ref|NM_033487.1 Homo sapiens cell division cycle 2-like 1 (PITSLRE proteins) (CDC2L1), transcript variant 3, mRNA
2.43092

AB050260
gi|203098333|ref|NM_032133.4 Homo sapiens MYCBP-associated protein (MYCBPAP), mRNA
2.41286

CJ436262
gi|150010638|ref|NM_015276.1 Homo sapiens ubiquitin specific peptidase 22 (USP22), mRNA
2.39886

AB056381
gi|225735571|ref|NR_027416.1 Homo sapiens nuclear factor erythroid-derived 2-like 3 pseudogene (LOC100272146), noncoding RNA
2.39471

CJ443349
gi|83367079|ref|NM_003801.3 Homo sapiens glycosylphosphatidylinositol anchor attachment protein 1 homolog (yeast) (GPAA1), mRNA
2.39333

AB171767
gi|126273571|ref|NM_144586.5 Homo sapiens LY6/PLAUR domain-containing 1 (LYPD1), transcript variant 1, mRNA
2.393

AB056817
gi|58535452|ref|NM_001011649.1 Homo sapiens CDK5 regulatory subunit-associated protein 2 (CDK5RAP2), transcript variant 2, mRNA
2.39285

AB174345
gi|145208007|ref|NM_173688.2 Homo sapiens Na⁺/K⁺-transporting ATPase interacting 3 (NKAIN3), mRNA
2.39236

DC648733
gi|134133239|ref|NM_032151.4 Homo sapiens pterin-4 alpha-carbinolamine dehydratase/dimerization cofactor of hepatocyte nuclear factor 1 alpha (TCF1) 2 (PCBD2), mRNA
2.39231

CJ435057
gi|189083855|ref|NM_000815.4 Homo sapiens gamma-aminobutyric acid (GABA) A receptor, delta (GABRD), mRNA
2.38598

AB172049
gi|41281366|ref|NM_001440.2 Homo sapiens exostoses (multiple)-like 3 (EXTL3), mRNA
2.37312

CJ489397
gi|71143136|ref|NM_005342.2 Homo sapiens high-mobility group box 3 (HMGB3), mRNA
2.36471

AB170096
gi|42741653|ref|NM_007375.3 Homo sapiens TAR DNA-binding protein (TARDBP), mRNA
2.36217

AB056391
gi|169216999|ref|XM_001720515.1 PREDICTED: Homo sapiens similar to pro-pol protein (LOC100129323), mRNA
2.3496

AB071089
gi|167900475|ref|NM_001080850.2 Homo sapiens coiled-coil domain-containing 30 (CCDC30), mRNA
2.34558

DK579603
gi|84626579|ref|NM_025108.2 Homo sapiens chromosome 16 open reading frame 59 (C16orf59), mRNA
2.34371

CJ431113
gi|226246632|ref|NR_027451.1 Homo sapiens hypothetical LOC647979 (LOC647979), noncoding RNA
2.34265

DK580610
gi|34335291|ref|NM_003312.4 Homo sapiens thiosulfate sulfurtransferase (rhodanese) (TST), nuclear gene encoding mitochondrial protein, mRNA
2.3426

AB168450
gi|81295815|ref|NM_012337.2 Homo sapiens coiled-coil domain-containing 19 (CCDC19), mRNA
2.34063

DQ159931
gi|163659857|ref|NM_000828.4 Homo sapiens glutamate receptor, ionotrophic, AMPA 3 (GRIA3), transcript variant 2, mRNA
2.33939

AB173516
gi|36287116|ref|NM_014319.3 Homo sapiens LEM domain-containing 3 (LEMD3), mRNA
2.33925

AB173575
gi|56550100|ref|NM_020978.3 Homo sapiens amylase, alpha 2B (pancreatic) (AMY2B), mRNA
2.33548

AB169015
gi|93277104|ref|NM_173812.4 Homo sapiens dpy-19-like 2 (C. elegans) (DPY19L2), mRNA
2.33006

BB898675
gi|70906438|ref|NM_021870.2 Homo sapiens fibrinogen gamma chain (FGG), transcript variant gamma-B, mRNA
2.32385

DK579646
gi|153791317|ref|NM_032332.3 Homo sapiens mitogen-activated protein kinase organizer 1 (MORG1), transcript variant 2, mRNA
2.32308

AB071115
gi|111548669|ref|NM_153376.2 Homo sapiens coiled-coil domain-containing 96 (CCDC96), mRNA
2.31559

DC632824
gi|23110926|ref|NM_002799.2 Homo sapiens proteasome (prosome, macropain) subunit, beta type, 7 (PSMB7), mRNA
2.31501

BB896362
gi|188595719|ref|NM_005141.3 Homo sapiens fibrinogen beta chain (FGB), mRNA
2.3131

AB292416
gi|143770740|ref|NM_001083899.1 Homo sapiens glycoprotein VI (platelet) (GP6), transcript variant 1, mRNA
2.311

AB055350
gi|67782353|ref|NM_001024844.1 Homo sapiens CD82 molecule (CD82), transcript variant 2, mRNA
2.31014

AB168962
gi|210147405|ref|NM_152621.5 Homo sapiens sphingomyelin synthase 2 (SGMS2), transcript variant 1, mRNA
2.30806

AB168166
gi|156415985|ref|NM_014579.2 Homo sapiens solute carrier family 39 (zinc transporter), member 2 (SLC39A2), mRNA
2.30287

AB172981
gi|73695942|ref|NM_001010927.2 Homo sapiens T-cell lymphoma invasion and metastasis 2 (TIAM2), transcript variant 2, mRNA
2.29903

CJ441025
gi|153252025|ref|NM_001830.3 Homo sapiens chloride channel 4 (CLCN4), mRNA
2.29786

CJ445440
gi|42764686|ref|NM_022652.2 Homo sapiens dual specificity phosphatase 6 (DUSP6), transcript variant 2, mRNA
2.29536

AB179072
gi|156119614|ref|NM_006901.2 Homo sapiens myosin IXA (MYO9A), mRNA
2.28584

AB060229
gi|239756940|ref|XM_001718053.2 PREDICTED: Homo sapiens similar to CD300C antigen (LOC100130520), mRNA
2.28415

CJ480802
gi|71772428|ref|NM_001021.3 Homo sapiens ribosomal protein S17 (RPS17), mRNA
2.28212

DK581053
gi|63054873|ref|NM_001615.3 Homo sapiens actin, gamma 2, smooth muscle, enteric (ACTG2), mRNA
2.27782

AB046030
gi|169210010|ref|XR_040492.1 PREDICTED: Homo sapiens hypothetical LOC440386 (LOC440386), miscRNA
2.27371

AB174638
gi|44680147|ref|NM_203327.1 Homo sapiens solute carrier family 23 (nucleobase transporters), member 2 (SLC23A2), transcript variant 2, mRNA
2.2635

CJ469779
gi|95147340|ref|NM_004603.2 Homo sapiens syntaxin 1A (brain) (STX1A), mRNA
2.25959

DC632108
gi|225637497|ref|NR_003286.2 Homo sapiens 18S ribosomal RNA (LOC100008588), noncoding RNA
2.25815

AB170956
gi|65506441|ref|NM_000282.2 Homo sapiens propionyl coenzyme A carboxylase, alpha polypeptide (PCCA), nuclear gene encoding mitochondrial protein, transcript variant 1, mRNA
2.25785

DC858184
gi|13569959|ref|NM_030980.1 Homo sapiens interferon stimulated exonuclease gene 20 kDa-like 2 (ISG20L2), mRNA
2.25755

CJ470090
gi|169790838|ref|NM_004172.4 Homo sapiens solute carrier family 1 (glial high affinity glutamate transporter), member 3 (SLC1A3), mRNA
2.24996

DW522847
gi|215983055|ref|NM_031471.5 Homo sapiens fermitin family homolog 3 (Drosophila) (FERMT3), transcript variant URP2SF, mRNA
2.23483

DK577957
gi|186910295|ref|NM_001126102.1 Homo sapiens haptoglobin (HP), transcript variant 2, mRNA
2.23107

AB168531
gi|162951880|ref|NM_001112707.1 Homo sapiens tousled-like kinase 2 (TLK2), transcript variant B, mRNA
2.22872

AB047603
gi|149363673|ref|NM_012194.1 Homo sapiens chromosome 11 open reading frame 41 (C11orf41), mRNA
2.22681

DC642489
gi|66392201|ref|NM_002512.2 Homo sapiens nonmetastatic cells 2, protein (NM23B) expressed in (NME2), transcript variant 1, mRNA
2.22176

AB072760
gi|91754184|ref|NM_152763.3 Homo sapiens chromosome 1 open reading frame 62 (C1orf62), mRNA
2.2183

AB168708
gi|36287109|ref|NM_194429.1 Homo sapiens FGFR1 oncogene partner (FGFR1OP), transcript variant 2, mRNA
2.21393

AB168136
gi|93004101|ref|NM_005730.3 Homo sapiens CTD (carboxy-terminal domain, RNA polymerase II, polypeptide A) small phosphatase 2 (CTDSP2), mRNA
2.20887

DC641033
gi|108389126|ref|NM_001042353.1 Homo sapiens family with sequence similarity 110, member A (FAM110A), transcript variant 3, mRNA
2.20687

CJ453010
gi|19743893|ref|NM_133480.1 Homo sapiens transcriptional adaptor 3 (NGG1 homolog, yeast)-like (TADA3L), transcript variant 2, mRNA
2.20608

AB168419
gi|32996736|ref|NM_173083.2 Homo sapiens lin-9 homolog (C. elegans) (LIN9), mRNA
2.20292

AB169251
gi|148727318|ref|NM_001098529.1 Homo sapiens thioredoxin domain-containing 2 (spermatozoa) (TXNDC2), transcript variant 2, mRNA
2.2019

AB173492
gi|31542657|ref|NM_018099.3 Homo sapiens fatty acyl CoA reductase 2 (FAR2), mRNA
2.20171

CJ443285
gi|62955828|ref|NM_033428.1 Homo sapiens chromosome 9 open reading frame 123 (C9orf123), mRNA
2.19894

AB169808
gi|20336295|ref|NM_018380.2 Homo sapiens DEAD (Asp-Glu-Ala-Asp) box polypeptide 28 (DDX28), nuclear gene encoding mitochondrial protein, mRNA
2.1907

AB169213
gi|125625349|ref|NM_001790.3 Homo sapiens cell division cycle 25 homolog C (S. pombe) (CDC25C), transcript variant 1, mRNA
2.18403

CJ491693
gi|21361889|ref|NM_021633.2 Homo sapiens Kelch-like 12 (Drosophila) (KLHL12), mRNA
2.1801

BB876451
gi|109389366|ref|NM_000312.2 Homo sapiens protein C (inactivator of coagulation factors Va and VIIIa) (PROC), mRNA
2.17421

AB171871
gi|153070253|ref|NM_001099680.1 Homo sapiens MAGI family member, X-linked (MAGIX), transcript variant 2, mRNA
2.17287

AB173097
gi|198041927|ref|NM_139241.2 Homo sapiens FYVE, RhoGEF and PH domain-containing 4 (FGD4), mRNA
2.16791

AB046632
gi|136255215|ref|NM_207351.3 Homo sapiens proline-rich transmembrane protein 3 (PRRT3), mRNA
2.16598

DK577943
gi|84872083|ref|NR_002798.1 Homo sapiens napsin B aspartic peptidase pseudogene (NAPSB), noncoding RNA
2.16578

AB168792
gi|141802709|ref|NM_145263.2 Homo sapiens spermatogenesis associated 18 homolog (rat) (SPATA18), mRNA
2.16491

BB881148
gi|189458840|ref|NM_005942.3 Homo sapiens molybdenum cofactor synthesis 1 (MOCS1), transcript variant 2, mRNA
2.16393

CJ472360
gi|24497456|ref|NM_139136.2 Homo sapiens potassium voltage-gated channel, Shaw-related subfamily, member 2 (KCNC2), transcript variant 1, mRNA
2.15994

CJ430900
gi|94721262|ref|NM_001040446.1 Homo sapiens myotubularin-related protein 12 (MTMR12), mRNA
2.15957

AB171550
gi|19913413|ref|NM_014203.2 Homo sapiens adaptor-related protein complex 2, alpha 1 subunit (AP2A1), transcript variant 1, mRNA
2.1589

AB173954
gi|188595678|ref|NM_014959.2 Homo sapiens caspase recruitment domain family, member 8 (CARD8), mRNA
2.13719

AB071125
gi|89903024|ref|NM_001031735.2 Homo sapiens chromosome 19 open reading frame 36 (C19orf36), transcript variant 1, mRNA
2.13665

AB063014
gi|170650671|ref|NM_001122769.1 Homo sapiens Leber congenital amaurosis 5 (LCA5), transcript variant 2, mRNA
2.13524

DC631520
gi|189163527|ref|NM_001127700.1 Homo sapiens serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 1 (SERPINA1), transcript variant 4, mRNA
2.1306

AY742821
gi|59806358|ref|NM_006011.3 Homo sapiens ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialyltransferase 2 (ST8SIA2), mRNA
2.12885

AK240628
gi|160298141|ref|NM_000668.4 Homo sapiens alcohol dehydrogenase 1B (class I), beta polypeptide (ADH1B), mRNA
2.12458

AB174195
gi|30794215|ref|NM_030961.1 Homo sapiens tripartite motif-containing 56 (TRIM56), mRNA
2.12446

DC646861
gi|91807120|ref|NM_033087.3 Homo sapiens asparagine-linked glycosylation 2, alpha-1,3-mannosyltransferase homolog (S. cerevisiae) (ALG2), transcript variant 1, mRNA
2.12291

AY650365
gi|27436932|ref|NM_172337.1 Homo sapiens orthodenticle homeobox 2 (OTX2), transcript variant 2, mRNA
2.11514

DW527197
gi|219555668|ref|NM_052855.3 Homo sapiens ankyrin repeat domain 40 (ANKRD40), mRNA
2.115

AB171287
gi|188497721|ref|NM_001127385.1 Homo sapiens cortexin 3 (CTXN3), transcript variant 2, mRNA
2.11438

AB173764
gi|219879811|ref|NM_005475.2 Homo sapiens SH2B adaptor protein 3 (SH2B3), mRNA
2.10791

DK582787
gi|221316657|ref|NM_004811.2 Homo sapiens leupaxin (LPXN), transcript variant 2, mRNA
2.10555

AB070128
gi|217416373|ref|NM_145038.2 Homo sapiens chromosome 2 open reading frame 39 (C2orf39), mRNA
2.10225

AB070165
gi|226491198|ref|NM_182496.2 Homo sapiens coiled-coil domain-containing 38 (CCDC38), mRNA
2.10075

DK577398
gi|52426772|ref|NM_002122.3 Homo sapiens major histocompatibility complex, class II, DQ alpha 1 (HLA-DQA1), mRNA
2.09004

AB169904
gi|34147601|ref|NM_004309.3 Homo sapiens rho GDP dissociation inhibitor (GDI) alpha (ARHGDIA), mRNA
2.08484

AB220503
gi|237681178|ref|NM_001160260.1 Homo sapiens cannabinoid receptor 1 (brain) (CNR1), transcript variant 6, mRNA
2.08434

AB173401
gi|239750034|ref|XR_039406.2 PREDICTED: Homo sapiens similar to yippee-like 5 (Drosophila) (LOC100132562), miscRNA
2.08407

AB171785
gi|253970447|ref|NM_014253.3 Homo sapiens odz, odd Oz/ten-m homolog 1(Drosophila) (ODZ1), transcript variant 3, mRNA
2.08184

AB171491
gi|117938287|ref|NM_004171.3 Homo sapiens solute carrier family 1 (glial high affinity glutamate transporter), member 2 (SLC1A2), mRNA
2.08087

AB174571
gi|182765446|ref|NM_001031711.2 Homo sapiens endoplasmic reticulum-Golgi intermediate compartment (ERGIC) 1 (ERGIC1), mRNA
2.07886

AB063092
gi|34577113|ref|NM_015576.1 Homo sapiens ELKS/RAB6-interacting/CAST family member 2 (ERC2), mRNA
2.07171

AB056378
gi|189163523|ref|NM_033064.4 Homo sapiens ataxia, cerebellar, Cayman type (ATCAY), mRNA
2.06758

AB055299
gi|163644324|ref|NM_001112732.1 Homo sapiens MCF.2 cell line derived transforming sequence-like (MCF2L), transcript variant 1, mRNA
2.06139

AB172748
gi|119220563|ref|NM_004852.2 Homo sapiens one cut homeobox 2 (ONECUT2), mRNA
2.05909

AB172478
gi|239746981|ref|XR_078603.1 PREDICTED: Homo sapiens similar to putative p150 (LOC100288106), miscRNA
2.05792

AB170807
gi|236459850|ref|NM_173569.3 Homo sapiens ubinuclein 2 (UBN2), mRNA
2.05471

EF208824
gi|239740919|ref|XM_002344047.1 PREDICTED: Homo sapiens similar to major histocompatibility complex, class II, DQ beta 1, transcript variant 2 (LOC100294318), mRNA
2.05031

AB169481
gi|150417992|ref|NM_033312.2 Homo sapiens CDC14 cell division cycle 14 homolog A (S. cerevisiae) (CDC14A), transcript variant 2, mRNA
2.04992

AB171520
gi|56243494|ref|NM_004586.2 Homo sapiens ribosomal protein S6 kinase, 90 kDa, polypeptide 3 (RPS6KA3), mRNA
2.0465

DC629151
gi|215982788|ref|NM_000477.5 Homo sapiens albumin (ALB), mRNA
2.04345

DC640591
gi|208609965|ref|NM_001135664.1 Homo sapiens RAB7, member RAS oncogene family-like 1 (RAB7L1), transcript variant 4, mRNA
2.04167

BB887273
gi|215,982,788|ref|NM_000477.5 Homo sapiens albumin (ALB), mRNA
2.0414

CJ435276
gi|75,812,975|ref|NM_001033574.1 Homo sapiens archaelysin family metallopeptidase 2 (AMZ2), transcript variant 6, mRNA
2.04064

DC643114
gi|33519462|ref|NM_004544.2 Homo sapiens NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 10, 42 kDa (NDUFA10), nuclear gene encoding mitochondrial protein, mRNA
2.03575

CJ436048
gi|62865867|ref|NM_004102.3 Homo sapiens fatty acid-binding protein 3, muscle and heart (mammary-derived growth inhibitor) (FABP3), mRNA
2.03528

AB179303
gi|196162694|ref|NM_003401.3 Homo sapiens X-ray repair complementing defective repair in Chinese hamster cells 4 (XRCC4), transcript variant 1, mRNA
2.03322

AB171313
gi|146219840|ref|NM_020709.1 Homo sapiens PNMA-like 2 (PNMAL2), mRNA
2.02925

AB173369
gi|149363694|ref|NM_001009984.1 Homo sapiens chromosome 20 open reading frame 194 (C20orf194), mRNA
2.01079

AB171481
gi|18496982|ref|NM_015526.1 Homo sapiens CAP-GLY domain-containing linker protein 3 (CLIP3), mRNA
2.00151

AB174068
gi|84872123|ref|NR_002833.1 Homo sapiens dpy-19-like 2 pseudogene 1 (C. elegans) (DPY19L2P1), noncoding RNA
1.99958

AB050434
gi|239753181|ref|XM_002345525.1 PREDICTED: Homo sapiens similar to hCG2041348 (LOC100293610), mRNA
1.99905

DK577438
gi|88999575|ref|NM_002622.4 Homo sapiens prefoldin subunit 1 (PFDN1), mRNA
1.99519

AB172315
gi|239753426|ref|XR_038411.2 PREDICTED: Homo sapiens similar to eukaryotic translation elongation factor 1 beta 2 (LOC646973), miscRNA
1.99372

BB895222
gi|38372939|ref|NM_001185.2 Homo sapiens alpha-2-glycoprotein 1, zinc-binding (AZGP1), mRNA
1.99214

AB173728
gi|111154086|ref|NM_020631.3 Homo sapiens pleckstrin homology domain-containing, family G (with RhoGef domain) member 5 (PLEKHG5), transcript variant 1, mRNA
1.98959

DC647709
gi|28416926|ref|NM_002560.2 Homo sapiens purinergic receptor P2X, ligand-gated ion channel, 4 (P2RX4), mRNA
1.98692

AB048919
gi|156766083|ref|NM_031418.2 Homo sapiens anoctamin 3 (ANO3), mRNA
1.98684

AB179103
gi|18860913|ref|NM_021818.2 Homo sapiens salvador homolog 1 (Drosophila) (SAV1), mRNA
1.97681

AB046073
gi|118498342|ref|NM_014861.2 Homo sapiens ATPase, Ca⁺⁺ transporting, type 2C, member 2 (ATP2C2), mRNA
1.97604

AB172144
gi|190341103|ref|NM_015163.5 Homo sapiens tripartite motif-containing 9 (TRIM9), transcript variant 1, mRNA
1.97599

DK583369
gi|171460955|ref|NM_005800.4 Homo sapiens ubiquitin-specific peptidase like 1 (USPL1), mRNA
1.9752

AB174098
gi|110815799|ref|NM_024345.3 Homo sapiens DDB1 and CUL4-associated factor 10 (DCAF10), mRNA
1.97309

DW526268
gi|31083173|ref|NM_181078.1 Homo sapiens interleukin 21 receptor (IL21R), transcript variant 2, mRNA
1.97258

AB171701
gi|20544144|ref|NM_139062.1 Homo sapiens casein kinase 1, delta (CSNK1D), transcript variant 2, mRNA
1.96537

CJ493302
gi|17136150|ref|NM_004724.2 Homo sapiens ZW10, kinetochore associated, homolog (Drosophila) (ZW10), mRNA
1.96534

DK577545
gi|24797075|ref|NM_002121.4 Homo sapiens major histocompatibility complex, class II, DP beta 1 (HLA-DPB1), mRNA
1.96346

CJ458429
gi|71067335|ref|NM_031462.2 Homo sapiens CD99 molecule-like 2 (CD99L2), transcript variant 1, mRNA
1.96208

AB172752
gi|32698785|ref|NM_182490.1 Homo sapiens zinc finger protein 227 (ZNF227), mRNA
1.95671

AB171668
gi|40385866|ref|NM_199227.1 Homo sapiens methionine aminopeptidase 1D (MAP1D), mRNA
1.95399

AB051117
gi|82617625|ref|NM_001037293.1 Homo sapiens paralemmin 2 (PALM2), transcript variant 2, mRNA
1.9528

AB169059
gi|91992151|ref|NM_000616.3 Homo sapiens CD4 molecule (CD4), mRNA
1.95226

CJ443230
gi|46094085|ref|NM_022758.4 Homo sapiens chromosome 6 open reading frame 106 (C6orf106), transcript variant 2, mRNA
1.95082

AB178987
gi|38045951|ref|NM_021030.2 Homo sapiens zinc finger protein 14 (ZNF14), mRNA
1.94816

AB172387
gi|163659919|ref|NM_052839.3 Homo sapiens pannexin 2 (PANX2), transcript variant 1, mRNA
1.94536

AB168775
gi|223468671|ref|NM_001145135.1 Homo sapiens carnitine palmitoyltransferase 1B (muscle) (CPT1B), nuclear gene encoding mitochondrial protein, transcript variant 6, mRNA
1.94062

DC633198
gi|206725531|ref|NM_001826.2 Homo sapiens CDC28 protein kinase regulatory subunit 1B (CKS1B), transcript variant 1, mRNA
1.93773

AB172044
gi|25168266|ref|NM_170709.1 Homo sapiens serum/glucocorticoid-regulated kinase family, member 3 (SGK3), transcript variant 2, mRNA
1.93543

DK578185
gi|239754745|ref|XM_002346052.1 PREDICTED: Homo sapiens hypothetical protein LOC100293771 (LOC100293771), mRNA
1.93421

AB171597
gi|113951732|ref|NM_012095.4 Homo sapiens adaptor-related protein complex 3, mu 1 subunit (AP3M1), transcript variant 2, mRNA
1.93313

AB179405
gi|31543301|ref|NM_032600.2 Homo sapiens coiled-coil domain-containing 54 (CCDC54), mRNA
1.93038

AB179267
gi|37594443|ref|NM_015896.2 Homo sapiens zinc finger, MYND-type-containing 10 (ZMYND10), mRNA
1.92859

DC640525
gi|35493837|ref|NM_004902.2 Homo sapiens RNA-binding motif protein 39 (RBM39), transcript variant 2, mRNA
1.92337

AB049869
gi|239753181|ref|XM_002345525.1 PREDICTED: Homo sapiens similar to hCG2041348 (LOC100293610), mRNA
1.92159

DW528250
gi|52487034|ref|NM_004618.3 Homo sapiens topoisomerase (DNA) III alpha (TOP3A), mRNA
1.91924

DC636463
gi|78214521|ref|NM_001035258.1 Homo sapiens ribosomal protein L38 (RPL38), transcript variant 2, mRNA
1.91826

AB179052
gi|115511031|ref|NM_004432.2 Homo sapiens ELAV- (embryonic lethal, abnormal vision, Drosophila-) like 2 (Hu antigen B) (ELAVL2), mRNA
1.91696

AB168809
gi|37622352|ref|NM_003551.2 Homo sapiens nonmetastatic cells 5, protein expressed in nucleoside-diphosphate kinase (NME5), mRNA
1.91336

CJ435007
gi|115527063|ref|NM_004859.3 Homo sapiens clathrin, heavy chain (Hc) (CLTC), mRNA
1.91318

AB171499
gi|50845406|ref|NM_031444.2 Homo sapiens chromosome 22 open reading frame 13 (C22orf13), mRNA
1.91088

DC647333
gi|118600974|ref|NM_007269.2 Homo sapiens syntaxin-binding protein 3 (STXBP3), mRNA
1.90575

AB172403
gi|142976637|ref|NM_017420.3 Homo sapiens SIX homeobox 4 (SIX4), mRNA
1.89362

AB174282
gi|31543080|ref|NM_016210.2 Homo sapiens chromosome 3 open reading frame 18 (C3orf18), mRNA
1.89271

DC648759
gi|6382072|ref|NM_005258.2 Homo sapiens GTP cyclohydrolase I feedback regulator (GCHFR), mRNA
1.88951

AB169033
gi|195927038|ref|NM_001786.3 Homo sapiens cell division cycle 2, G1 to S and G2 to M (CDC2), transcript variant 1, mRNA
1.88515

AB173309
gi|209447072|ref|NM_001135806.1 Homo sapiens synaptotagmin I (SYT1), transcript variant 3, mRNA
1.88194

AB063003
gi|116063563|ref|NM_018218.2 Homo sapiens ubiquitin-specific peptidase 40 (USP40), mRNA
1.88102

AB171041
gi|86787650|ref|NM_014800.9 Homo sapiens engulfment and cell motility 1 (ELMO1), transcript variant 1, mRNA
1.87945

CJ470094
gi|209364624|ref|NM_001822.4 Homo sapiens chimerin (chimaerin) 1 (CHN1), transcript variant 1, mRNA
1.87132

AB171236
gi|19743893|ref|NM_133480.1 Homo sapiens transcriptional adaptor 3 (NGG1 homolog, yeast)-like (TADA3L), transcript variant 2, mRNA
1.86897

BB885210
gi|32484974|ref|NM_006721.2 Homo sapiens adenosine kinase (ADK), transcript variant ADK-long, mRNA
1.86865

AB169067
gi|188528615|ref|NM_182911.3 Homo sapiens testis-specific, 10 (TSGA10), transcript variant 2, mRNA
1.86706

CJ464698
gi|221307560|ref|NR_026669.1 Homo sapiens synaptosomal-associated protein, 91 kDa homolog (mouse) (SNAP91), transcript variant 2, transcribed RNA
1.86542

AB179482
gi|51173716|ref|NM_006720.3 Homo sapiens actin-binding LIM protein 1 (ABLIM1), transcript variant 4, mRNA
1.85972

CJ442615
gi|239745120|ref|XR_015162.2 PREDICTED: Homo sapiens hypothetical protein LOC727880 (LOC727880), miscRNA
1.85021

CJ435208
gi|170650722|ref|NM_014236.3 Homo sapiens glyceronephosphate O-acyltransferase (GNPAT), mRNA
1.8499

AY650307
gi|51599155|ref|NM_001273.2 Homo sapiens chromodomain helicase DNA-binding protein 4 (CHD4), mRNA
1.84509

DW525872
gi|77404354|ref|NM_003908.3 Homo sapiens eukaryotic translation initiation factor 2, subunit 2 beta, 38 kDa (EIF2S2), mRNA
1.84501

DW529999
gi|78190459|ref|NM_000978.3 Homo sapiens ribosomal protein L23 (RPL23), mRNA
1.84409

AB174451
gi|223941821|ref|NM_014342.3 Homo sapiens mitochondrial carrier homolog 2 (C. elegans) (MTCH2), nuclear gene encoding mitochondrial protein, mRNA
1.84017

AB066534
gi|188528627|ref|NM_033109.3 Homo sapiens polyribonucleotide nucleotidyltransferase 1 (PNPT1), mRNA
1.83655

BB888999
gi|160298191|ref|NM_000507.3 Homo sapiens fructose-1,6-bisphosphatase 1 (FBP1), transcript variant 1, mRNA
1.8352

DC637318
gi|209977038|ref|NM_016074.3 Homo sapiens bolA homolog 1 (E. coli) (BOLA1), mRNA
1.83477

AB169205
gi|109948303|ref|NM_018225.2 Homo sapiens smu-1 suppressor of mec-8 and unc-52 homolog (C. elegans) (SMU1), mRNA
1.82627

CJ441961
gi|19913444|ref|NM_016257.2 Homo sapiens hippocalcin-like 4 (HPCAL4), mRNA
1.8159

AY650384
gi|141803509|ref|NM_058164.2 Homo sapiens olfactomedin 2 (OLFM2), mRNA
1.81587

DC647305
gi|38372918|ref|NM_001728.2 Homo sapiens basigin (Ok blood group) (BSG), transcript variant 1, mRNA
1.8093

AB172260
gi|112382251|ref|NM_178313.2 Homo sapiens spectrin, beta, nonerythrocytic 1 (SPTBN1), transcript variant 2, mRNA
1.80742

AB173850
gi|194097340|ref|NM_002616.2 Homo sapiens period homolog 1 (Drosophila) (PER1), mRNA
1.80415

AB168762
gi|242247096|ref|NM_001340.3 Homo sapiens cylicin, basic protein of sperm head cytoskeleton 2 (CYLC2), mRNA
1.80079

AB173856
gi|60302919|ref|NM_001752.2 Homo sapiens catalase (CAT), mRNA
1.79676

AB060862
gi|221219051|ref|NM_031924.4 Homo sapiens radial spoke 3 homolog (Chlamydomonas) (RSPH3), mRNA
1.79563

CJ470793
gi|224586819|ref|NR_027265.1 Homo sapiens Golgi apparatus protein 1 (GLG1), transcript variant 5, transcribed RNA
1.79405

DW528583
gi|239787833|ref|NM_015139.2 Homo sapiens solute carrier family 35 (UDP-glucuronic acid/UDP-N-acetylgalactosamine dual transporter), member D1 (SLC35D1), mRNA
1.79359

DK580881
gi|194394144|ref|NM_145870.2 Homo sapiens glutathione transferase zeta 1 (GSTZ1), transcript variant 1, mRNA
1.792

AB173997
gi|225543100|ref|NR_027378.1 Homo sapiens hypothetical LOC643763 (LOC643763), noncoding RNA
1.79131

AY650356
gi|223718142|ref|NM_173054.2 Homo sapiens reelin (RELN), transcript variant 2, mRNA
1.78729

DK584117
gi|15967154|ref|NM_016558.2 Homo sapiens SCAN domain-containing 1 (SCAND1), transcript variant 1, mRNA
1.78008

DC621384
gi|15431296|ref|NM_000977.2 Homo sapiens ribosomal protein L13 (RPL13), transcript variant 1, mRNA
1.77763

DK577712
gi|109148541|ref|NM_001605.2 Homo sapiens alanyl-tRNA synthetase (AARS), mRNA
1.77723

AB174251
gi|253314435|ref|NR_027995.1 Homo sapiens ankyrin repeat domain 20 family, member A2 pseudogene (LOC284232), noncoding RNA
1.77027

AB174247
gi|50897295|ref|NM_001002923.1 Homo sapiens IGF-like family member 4 (IGFL4), mRNA
1.76977

CJ490195
gi|78190459|ref|NM_000978.3 Homo sapiens ribosomal protein L23 (RPL23), mRNA
1.76786

AB171831
gi|167466275|ref|NM_152542.3 Homo sapiens protein phosphatase 1K (PP2C domain containing) (PPM1K), mRNA
1.76709

DK582810
gi|90652856|ref|NM_032818.2 Homo sapiens chromosome 9 open reading frame 100 (C9orf100), mRNA
1.765

AB170534
gi|108773786|ref|NM_000321.2 Homo sapiens retinoblastoma 1 (RB1), mRNA
1.76182

AB171096
gi|110347436|ref|NM_001042545.1 Homo sapiens latent transforming growth factor beta-binding protein 4 (LTBP4), transcript variant 3, mRNA
1.75594

AB168611
gi|21071068|ref|NM_004865.2 Homo sapiens TBP-like 1 (TBPL1), mRNA
1.74839

CJ492188
gi|30181234|ref|NM_003447.2 Homo sapiens zinc finger protein 165 (ZNF165), mRNA
1.74573

AB171700
gi|115527063|ref|NM_004859.3 Homo sapiens clathrin, heavy chain (Hc) (CLTC), mRNA
1.74566

AB171366
gi|22748942|ref|NM_152445.1 Homo sapiens family with sequence similarity 161, member B (FAM161B), mRNA
1.74405

AB168566
gi|148664196|ref|NM_017950.2 Homo sapiens coiled-coil domain-containing 40 (CCDC40), mRNA
1.74135

AB171657
gi|221316692|ref|NM_198449.2 Homo sapiens embigin homolog (mouse) (EMB), mRNA
1.73933

AB056808
gi|71772767|ref|NM_152826.2 Homo sapiens-sorting nexin 1 (SNX1), transcript variant 3, mRNA
1.73686

AB168849
gi|156766042|ref|NM_001103146.1 Homo sapiens GRB10-interacting GYF protein 2 (GIGYF2), transcript variant 3, mRNA
1.73412

AB172848
gi|95113665|ref|NM_018157.2 Homo sapiens resistance to inhibitors of cholinesterase 8 homolog B (C. elegans) (RIC8B), mRNA
1.72749

AB048894
gi|148727250|ref|NM_007137.2 Homo sapiens zinc finger protein 81 (ZNF81), mRNA
1.71845

DW524469
gi|239753181|ref|XM_002345525.1 PREDICTED: Homo sapiens similar to hCG2041348 (LOC100293610), mRNA
1.71582

AB173566
gi|89242130|ref|NM_014305.2 Homo sapiens TDP-glucose 4,6-dehydratase (TGDS), mRNA
1.71406

DC634783
gi|116812576|ref|NM_016019.2 Homo sapiens LUC7-like 2 (S. cerevisiae) (LUC7L2), mRNA
1.71163

AB168438
gi|64276485|ref|NM_005869.2 Homo sapiens serologically defined colon cancer antigen 10 (SDCCAG10), mRNA
1.71108

AB174725
gi|169194555|ref|XR_040716.1 PREDICTED: Homo sapiens hypothetical LOC439950 (LOC439950), miscRNA
1.70933

AB170786
gi|208879448|ref|NM_006265.2 Homo sapiens RAD21 homolog (S. pombe) (RAD21), mRNA
1.70551

CJ431422
gi|117938253|ref|NM_001077441.1 Homo sapiens BCL2-associated transcription factor 1 (BCLAF1), transcript variant 3, mRNA
1.70446

AB048954
gi|148596971|ref|NM_014951.2 Homo sapiens zinc finger protein 365 (ZNF365), transcript variant A, mRNA
1.70334

AB173447
gi|40288292|ref|NM_000361.2 Homo sapiens thrombomodulin (THBD), mRNA
1.70293

AB173287
gi|242117988|ref|NM_014702.4 Homo sapiens KIAA0408 (KIAA0408), mRNA
1.70162

CJ489820
gi|218505834|ref|NM_001142782.1 Homo sapiens membrane-associated guanylate kinase, WW, and PDZ domain-containing 3 (MAGI3), transcript variant 1, mRNA
1.68381

AB173372
gi|78190481|ref|NM_025221.5 Homo sapiens Kv channel-interacting protein 4 (KCNIP4), transcript variant 1, mRNA
1.68058

AB172865
gi|31795545|ref|NM_012450.2 Homo sapiens solute carrier family 13 (sodium/sulfate symporters), member 4 (SLC13A4), mRNA
1.67878

AB168329
gi|223468562|ref|NM_005628.2 Homo sapiens solute carrier family 1 (neutral amino acid transporter), member 5 (SLC1A5), transcript variant 1, mRNA
1.67642

AB171546
gi|55956903|ref|NM_005922.2 Homo sapiens mitogen-activated protein kinase kinase kinase 4 (MAP3K4), transcript variant 1, mRNA
1.67151

AB063093
gi|194248055|ref|NM_002045.3 Homo sapiens growth-associated protein 43 (GAP43), transcript variant 2, mRNA
1.66805

AB220449
gi|23510394|ref|NM_138966.2 Homo sapiens neuropilin- (NRP-) and tolloid- (TLL-) like 1 (NETO1), transcript variant 3, mRNA
1.66789

AB169208
gi|22547155|ref|NM_002018.2 Homo sapiens flightless I homolog (Drosophila) (FLII), mRNA
1.66361

AB168324
gi|116014337|ref|NM_030981.2 Homo sapiens RAB1B, member RAS oncogene family (RAB1B), mRNA
1.66298

AB169835
gi|50726964|ref|NM_013392.2 Homo sapiens nuclear receptor-binding protein 1 (NRBP1), mRNA
1.65785

AB173501
gi|195539333|ref|NM_018176.3 Homo sapiens leucine-rich repeat LGI family, member 2 (LGI2), mRNA
1.6574

DC630946
gi|183227689|ref|NM_002049.3 Homo sapiens GATA-binding protein 1 (globin transcription factor 1) (GATA1), mRNA
1.65657

AB063075
gi|239743824|ref|XM_001128647.3 PREDICTED: Homo sapiens hypothetical LOC728701 (LOC728701), mRNA
1.65564

AB169782
gi|38261964|ref|NM_198399.1 Homo sapiens cyclic AMP-regulated phosphoprotein, 21 kD (ARPP-21), transcript variant 2, mRNA
1.65067

CJ477467
gi|133778911|ref|NM_003309.2 Homo sapiens TSPY-like 1 (TSPYL1), mRNA
1.65062

BB900725
gi|31542685|ref|NM_025125.2 Homo sapiens chromosome 10 open reading frame 57 (C10orf57), mRNA
1.64409

AB220555
gi|208973250|ref|NM_003702.3 Homo sapiens regulator of G-protein signaling 20 (RGS20), transcript variant 2, mRNA
1.64376

AB171804
gi|66932910|ref|NM_014676.2 Homo sapiens pumilio homolog 1 (Drosophila) (PUM1), transcript variant 2, mRNA
1.63724

DC625559
gi|4557320|ref|NM_000039.1 Homo sapiens apolipoprotein A-I (APOA1), mRNA
1.631

AB172266
gi|170932491|ref|NM_030770.2 Homo sapiens transmembrane protease, serine 5 (TMPRSS5), mRNA
1.62995

AB173763
gi|62953115|ref|NM_001017523.1 Homo sapiens BTB (POZ) domain-containing 11 (BTBD11), transcript variant b, mRNA
1.62499

AB172974
gi|111161293|ref|NM_005746.2 Homo sapiens nicotinamide phosphoribosyltransferase (NAMPT), mRNA
1.62078

AB179155
gi|187608347|ref|NM_145046.3 Homo sapiens calreticulin 3 (CALR3), mRNA
1.6111

AB169148
gi|153792481|ref|NM_033048.4 Homo sapiens CPX chromosome region, candidate 1 (CPXCR1), mRNA
1.60985

AB171264
gi|130977817|ref|NM_024549.4 Homo sapiens tectonic family member 1 (TCTN1), transcript variant 3, mRNA
1.60704

AB172446
gi|193083128|ref|NM_001128920.1 Homo sapiens MAP/microtubule affinity-regulating kinase 3 (MARK3), transcript variant 4, mRNA
1.60471

DC852298
gi|195972796|ref|NM_001130917.1 Homo sapiens leukocyte immunoglobulin-like receptor, subfamily A (with TM domain), member 2 (LILRA2), transcript variant 1, mRNA
1.59852

AB046637
gi|209571546|ref|NM_018095.4 Homo sapiens Kelch repeat and BTB (POZ) domain-containing 4 (KBTBD4), transcript variant 1, mRNA
1.58107

CJ445723
gi|90903230|ref|NM_002111.6 Homo sapiens huntingtin (HTT), mRNA
1.57689

DC630899
gi|83641894|ref|NM_031157.2 Homo sapiens heterogeneous nuclear ribonucleoprotein A1 (HNRNPA1), transcript variant 2, mRNA
1.5766

AB168476
gi|219555742|ref|NM_015335.3 Homo sapiens mediator complex subunit 13-like (MED13L), mRNA
1.57355

DC642541
gi|20357546|ref|NM_004231.2 Homo sapiens ATPase, H+ transporting, lysosomal 14 kDa, V1 subunit F (ATP6V1F), mRNA
1.57215

AB170370
gi|224177554|ref|NM_002340.5 Homo sapiens lanosterol synthase (2,3-oxidosqualene-lanosterol cyclase) (LSS), transcript variant 1, mRNA
1.57006

DC636538
gi|71164876|ref|NM_001014.3 Homo sapiens ribosomal protein S10 (RPS10), mRNA
1.56413

DC648258
gi|4557818|ref|NM_000277.1 Homo sapiens phenylalanine hydroxylase (PAH), mRNA
1.56396

AB168688
gi|75709218|ref|NM_001324.2 Homo sapiens cleavage stimulation factor, 3′ pre-RNA, subunit 1, 50 kDa (CSTF1), transcript variant 2, mRNA
1.56349

CJ486539
gi|194018543|ref|NM_031451.4 Homo sapiens testis expressed 101 (TEX101), transcript variant 1, mRNA
1.55338

AB173591
gi|56699472|ref|NM_006298.2 Homo sapiens zinc finger protein 192 (ZNF192), mRNA
1.54892

AB168460
gi|56090619|ref|NM_001007531.1 Homo sapiens NFKB-activating protein-like (NKAPL), mRNA
1.54807

AB046102
gi|215272394|ref|NM_001080475.2 Homo sapiens pleckstrin homology domain containing, family M, member 3 (PLEKHM3), mRNA
1.53926

AB097526
gi|46409303|ref|NM_207332.1 Homo sapiens glutamate-rich 1 (ERICH1), mRNA
1.53642

AB052134
gi|227430412|ref|NM_024827.3 Homo sapiens histone deacetylase 11 (HDAC11), transcript variant 1, mRNA
1.53543

AB170181
gi|21265090|ref|NM_007208.2 Homo sapiens mitochondrial ribosomal protein L3 (MRPL3), nuclear gene encoding mitochondrial protein, mRNA
1.52903

AB171241
gi|116235443|ref|NM_138421.2 Homo sapiens serum amyloid A-like 1 (SAAL1), mRNA
1.52713

AB171237
gi|48675815|ref|NM_015723.2 Homo sapiens patatin-like phospholipase domain-containing 8 (PNPLA8), mRNA
1.52587

DC625517
gi|47578120|ref|NM_177947.2 Homo sapiens armadillo repeat containing, X-linked 3 (ARMCX3), transcript variant 2, mRNA
1.52547

AB168964
gi|87159814|ref|NM_001696.3 Homo sapiens ATPase, H+ transporting, lysosomal 31 kDa, V1 subunit E1 (ATP6V1E1), transcript variant 1, mRNA
1.52424

DC631115
gi|239752151|ref|XM_002348112.1 PREDICTED: Homo sapiens similar to immunoglobulin lambda locus (LOC100290481), mRNA
1.51711

DC640134
gi|208609986|ref|NM_014655.2 Homo sapiens solute carrier family 25, member 44 (SLC25A44), transcript variant 1, mRNA
1.51672

AB173691
gi|94536855|ref|NM_013301.2 Homo sapiens coiled-coil domain-containing 106 (CCDC106), mRNA
1.50477

AB168370
gi|154744869|ref|NM_022752.5 Homo sapiens zinc finger protein 574 (ZNF574), mRNA
1.50452

Table 2: Genes that are downregulated in TM cells.

Accession number
Human RefSeq description
Fold change

DC624859
gi|215982788|ref|NM_000477.5 Homo sapiens albumin (ALB), mRNA
0.11952

AB171761
gi|148271103|ref|NM_173495.2 Homo sapiens patched domain-containing 1 (PTCHD1), mRNA
0.13543

AB047615
gi|70780382|ref|NM_004285.3 Homo sapiens hexose-6-phosphate dehydrogenase (glucose 1-dehydrogenase) (H6PD), mRNA
0.17649

DC621007
gi|38016905|ref|NR_001578.1 Homo sapiens L-threonine dehydrogenase (TDH), noncoding RNA
0.20061

CJ443677
gi|38327038|ref|NM_002154.3 Homo sapiens heat shock 70 kDa protein 4 (HSPA4), mRNA
0.2086

DC622138
gi|145386530|ref|NM_001084392.1 Homo sapiens D-dopachrome tautomerase (DDT), transcript variant 2, mRNA
0.21345

BB891761
gi|33519462|ref|NM_004544.2 Homo sapiens NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 10, 42 kDa (NDUFA10), nuclear gene encoding mitochondrial protein, mRNA
0.23147

CJ444181
gi|226437566|ref|NM_001018060.2 Homo sapiens apoptosis-inducing factor, mitochondrion-associated 3 (AIFM3), nuclear gene encoding mitochondrial protein, transcript variant 2, mRNA
0.24045

AB171890
gi|118572602|ref|NM_001079514.1 Homo sapiens ubinuclein 1 (UBN1), transcript variant 2, mRNA
0.24676

AB174511
gi|153792041|ref|NM_020823.1 Homo sapiens transmembrane protein 181 (TMEM181), mRNA
0.29472

AB168319
gi|116256484|ref|NM_006781.3 Homo sapiens chromosome 6 open reading frame 10 (C6orf10), mRNA
0.3105

DW526909
gi|20302159|ref|NM_005999.2 Homo sapiens translin-associated factor X (TSNAX), mRNA
0.32949

AB173471
gi|154354995|ref|NM_002222.4 Homo sapiens inositol 1,4,5-triphosphate receptor, type 1 (ITPR1), transcript variant 2, mRNA
0.33892

BB898986
gi|167003944|ref|NM_000204.3 Homo sapiens complement factor I (CFI), mRNA
0.34234

DK578390
gi|56788350|ref|NM_001008695.1 Homo sapiens THAP domain-containing 7 (THAP7), transcript variant 2, mRNA
0.35502

CJ444326
gi|209413724|ref|NM_003692.3 Homo sapiens transmembrane protein with EGF-like and two follistatin-like domains 1 (TMEFF1), mRNA
0.3719

AB048874
gi|239753181|ref|XM_002345525.1 PREDICTED: Homo sapiens similar to hCG2041348 (LOC100293610), mRNA
0.38524

DC635743
gi|239750740|ref|XM_002347480.1 PREDICTED: Homo sapiens similar to hCG2038941 (LOC100290006), mRNA
0.3882

CJ442045
gi|96975096|ref|NM_016577.3 Homo sapiens RAB6B, member RAS oncogene family (RAB6B), mRNA
0.39034

BB897881
gi|31542685|ref|NM_025125.2 Homo sapiens chromosome 10 open reading frame 57 (C10orf57), mRNA
0.39308

AB168422
gi|194306536|ref|NM_144594.2 Homo sapiens gametocyte-specific factor 1 (GTSF1), mRNA
0.43061

DW524779
gi|226342870|ref|NR_027449.1 Homo sapiens TBC1 domain family, member 15 (TBC1D15), transcript variant 4, transcribed RNA
0.44075

DC630545
gi|39812105|ref|NM_198941.1 Homo sapiens serine incorporator 3 (SERINC3), transcript variant 2, mRNA
0.44508

AB172901
gi|42544225|ref|NM_020857.2 Homo sapiens vacuolar protein sorting 18 homolog (S. cerevisiae) (VPS18), mRNA
0.44872

DW528888
gi|40068463|ref|NM_020732.2 Homo sapiens AT-rich interactive domain 1B (SWI1-like) (ARID1B), transcript variant 2, mRNA
0.44985

DC636880
gi|17738314|ref|NM_006835.2 Homo sapiens cyclin I (CCNI), mRNA
0.45196

AB220379
gi|185134767|ref|NM_002524.3 Homo sapiens neuroblastoma RAS viral (v-ras) oncogene homolog (NRAS), mRNA
0.46121

AB055316
gi|226437631|ref|NM_001004339.2 Homo sapiens zyg-11 homolog A (C. elegans) (ZYG11A), mRNA
0.46399

DC641070
gi|58331227|ref|NM_005223.3 Homo sapiens deoxyribonuclease I (DNASE1), mRNA
0.46575

AB056428
gi|49574533|ref|NM_032782.3 Homo sapiens hepatitis A virus cellular receptor 2 (HAVCR2), mRNA
0.46705

AB168577
gi|146260272|ref|NM_001085451.1 Homo sapiens leukemia NUP98 fusion partner 1 (LNP1), mRNA
0.46891

AB048999
gi|225735571|ref|NR_027416.1 Homo sapiens nuclear factor erythroid-derived 2-like 3 pseudogene (LOC100272146), noncoding RNA
0.47824

AB174085
gi|142360382|ref|NM_176815.3 Homo sapiens dihydrofolate reductase-like 1 (DHFRL1), mRNA
0.49049

DC642335
gi|148491081|ref|NM_001343.2 Homo sapiens-disabled homolog 2, mitogen-responsive phosphoprotein (Drosophila) (DAB2), mRNA
0.4971

AB173771
gi|38176290|ref|NM_001233.3 Homo sapiens caveolin 2 (CAV2), transcript variant 1, mRNA
0.49863

DW523198
gi|145312264|ref|NM_033266.3 Homo sapiens endoplasmic reticulum to nucleus signaling 2 (ERN2), mRNA
0.50158

AF492282
gi|52630343|ref|NM_021983.4 Homo sapiens major histocompatibility complex, class II, DR beta 4 (HLA-DRB4), mRNA
0.50533

AB047937
gi|194097480|ref|NM_020412.4 Homo sapiens chromatin-modifying protein 1B (CHMP1B), mRNA
0.51198

AB179165
gi|118136291|ref|NM_006465.2 Homo sapiens AT-rich interactive domain 3B (bright-like) (ARID3B), mRNA
0.51349

AJ585530
gi|75709168|ref|NM_002260.3 Homo sapiens killer cell lectin-like receptor subfamily C, member 2 (KLRC2), mRNA
0.52335

AB170944
gi|40255250|ref|NM_144635.3 Homo sapiens family with sequence similarity 131, member A (FAM131A), mRNA
0.55244

AB171281
gi|154759258|ref|NM_003127.2 Homo sapiens spectrin, alpha, nonerythrocytic 1 (alpha-fodrin) (SPTAN1), transcript variant 2, mRNA
0.555

AB172429
gi|224994204|ref|NM_001145853.1 Homo sapiens Wolfram syndrome 1 (wolframin) (WFS1), transcript variant 2, mRNA
0.57755

AB171421
gi|110611175|ref|NM_000843.3 Homo sapiens glutamate receptor, metabotropic 6 (GRM6), mRNA
0.58226

AB096987
gi|239750853|ref|XR_079356.1 PREDICTED: Homo sapiens hypothetical protein LOC100130855 (LOC100130855), miscRNA
0.59997

AB049000
gi|209977116|ref|NM_080872.2 Homo sapiens unc-5 homolog D (C. elegans) (UNC5D), mRNA
0.60066

BB893759
gi|194018407|ref|NM_178148.2 Homo sapiens solute carrier family 35, member B2 (SLC35B2), mRNA
0.61849

AB056791
gi|162951883|ref|NM_014925.3 Homo sapiens R3H domain-containing 2 (R3HDM2), mRNA
0.64652

AB070176
gi|221307501|ref|NM_001143976.1 Homo sapiens WEE1 homolog (S. pombe) (WEE1), transcript variant 2, mRNA
0.64704

AB048962
gi|154146186|ref|NM_152634.2 Homo sapiens transcription elongation factor A (SII) N-terminal and central domain containing (TCEANC), mRNA
0.65673

DK578501
gi|24797073|ref|NM_033554.2 Homo sapiens major histocompatibility complex, class II, DP alpha 1 (HLA-DPA1), mRNA
0.65963

Table 3: Gene ontology of upregulated genes in Y-27632-treated TM cells.

Ontology
Term
Changed genes
Total genes
value

Cellular component
Cell projection
31 (1)
331 (38)
0.0000306

Cellular component
Neuron projection
20 (6)
180 (28)
0.0000901

Cellular component
Cell projection part
17 (0)
142 (0)
0.000135

Biological process
Regulation of neurotransmitter levels
8 (0)
47 (2)
0.00148

Molecular function
Calcium channel regulator activity
4 (3)
11 (9)
0.00225

Cellular component
Presynaptic membrane
5 (5)
20 (20)
0.0024

Cellular component
Plasma membrane part
45 (0)
706 (2)
0.00259

Biological process
Synaptic transmission
16 (10)
170 (66)
0.00374

Molecular function
Channel regulator activity
5 (0)
23 (0)
0.00421

Biological process
Cellular nitrogen compound biosynthetic process
21 (0)
255 (0)
0.00443

Biological process
L-Glutamate import
3 (3)
6 (4)
0.00456

Cellular component
Platelet alpha granule
5 (1)
24 (2)
0.00472

Biological process
Transmission of nerve impulse
17 (0)
189 (3)
0.00563

Molecular function
Anion:cation symporter activity
4 (0)
15 (0)
0.00565

Molecular function
Sodium:dicarboxylate symporter activity
3 (3)
7 (7)
0.00585

Biological process
L-Amino acid import
3 (0)
7 (0)
0.00632

Biological process
Amino acid import
3 (0)
7 (0)
0.00632

Cellular component
Axon part
6 (0)
38 (2)
0.00665

Biological process
Regulation of mitotic cell cycle
8 (1)
62 (6)
0.00683

Molecular function
Anion transmembrane transporter activity
8 (0)
66 (5)
0.00813

Biological process
Cell-cell signaling
21 (4)
268 (74)
0.00841

Molecular function
High-affinity glutamate transmembrane transporter activity
2 (2)
2 (2)
0.00875

Cellular component
Cytoplasmic vesicle part
9 (0)
83 (2)
0.00918

Biological process
Deoxyribonucleoside triphosphate biosynthetic process
2 (0)
2 (0)
0.00923

Biological process
Response to calcium ion
5 (4)
28 (25)
0.00969

Molecular function
Rho guanyl-nucleotide exchange factor activity
5 (5)
29 (29)
0.00984

Biological process
Carboxylic acid transport
9 (0)
81 (1)
0.01005

Molecular function
Phosphatidylinositol binding
3 (3)
9 (9)
0.01012

Molecular function
Dicarboxylic acid transmembrane transporter activity
3 (0)
9 (0)
0.01012

Biological process
Organic acid transport
9 (0)
82 (0)
0.01077

Biological process
Glutamate metabolic process
3 (0)
9 (4)
0.01091

Biological process
Dicarboxylic acid transport
3 (3)
9 (7)
0.01091

Cellular component
Axoneme
4 (2)
19 (5)
0.01104

Molecular function
Structural constituent of cytoskeleton
5 (5)
31 (31)
0.01251

Cellular component
Cytoplasmic membrane-bounded vesicle lumen
4 (0)
20 (0)
0.01285

Cellular component
Platelet alpha granule lumen
4 (4)
20 (20)
0.01285

Cellular component
Dendritic spine
4 (4)
20 (20)
0.01285

Cellular component
Neuron spine
4 (0)
20 (0)
0.01285

Cellular component
Axon
9 (6)
89 (62)
0.01361

Cellular component
Neurofilament
2 (2)
3 (3)
0.01394

Molecular function
Antioxidant activity
5 (2)
32 (12)
0.01401

Biological process
Rho protein signal transduction
7 (1)
57 (15)
0.01403

Molecular function
Phenylalanine 4-monooxygenase activity
2 (2)
3 (3)
0.0142

Biological process
ER to Golgi vesicle-mediated transport
4 (4)
20 (20)
0.01457

Cellular component
Vesicle lumen
4 (0)
21 (1)
0.01484

Biological process
D-Amino acid transport
2 (0)
3 (0)
0.01497

Biological process
D-Aspartate import
2 (2)
3 (3)
0.01497

Biological process
D-Aspartate transport
2 (0)
3 (0)
0.01497

Biological process
Glutamate biosynthetic process
2 (2)
3 (3)
0.01497

Biological process
2′-Deoxyribonucleotide biosynthetic process
2 (0)
3 (0)
0.01497

Biological process
Fatty acid transport
4 (1)
21 (6)
0.01681

Molecular function
Ras guanyl-nucleotide exchange factor activity
5 (0)
34 (3)
0.01737

Biological process
Regulation of cell cycle process
6 (0)
47 (0)
0.01912

Biological process
Nucleoside triphosphate biosynthetic process
8 (1)
76 (3)
0.0195

Molecular function
Transporter activity
34 (8)
556 (147)
0.01993

Molecular function
Monocarboxylic acid binding
4 (0)
23 (0)
0.02002

Cellular component
Neurofilament cytoskeleton
2 (0)
4 (1)
0.02037

Biological process
Pyrimidine nucleoside triphosphate biosynthetic process
3 (0)
12 (0)
0.02063

Biological process
Pyrimidine nucleoside triphosphate metabolic process
3 (0)
12 (0)
0.02063

Molecular function
Oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen, reduced pteridine as one donor, and incorporation of one atom of oxygen
2 (1)
4 (2)
0.02075

Molecular function
Thioredoxin-disulfide reductase activity
2 (2)
4 (4)
0.02075

Biological process
Regulation of secretion
8 (0)
77 (0)
0.0208

Biological process
Neurotransmitter biosynthetic process
2 (2)
4 (4)
0.02186

Biological process
Tetrahydrobiopterin metabolic process
2 (1)
4 (1)
0.02186

Biological process
Deoxyribonucleoside triphosphate metabolic process
2 (0)
4 (1)
0.02186

Cellular component
Clathrin coat
4 (1)
24 (1)
0.02193

Biological process
Response to metal ion
8 (0)
78 (3)
0.02216

Cellular component
Transport vesicle membrane
3 (0)
13 (0)
0.02236

Biological process
Neurotransmitter metabolic process
3 (1)
13 (3)
0.02465

Biological process
Long-chain fatty acid transport
3 (2)
13 (4)
0.02465

Molecular function
Oxidoreductase activity, acting on sulfur group of donors
4 (0)
25 (0)
0.02549

Molecular function
Organic acid:sodium symporter activity
3 (0)
14 (0)
0.02707

Molecular function
Phosphoinositide binding
6 (2)
53 (30)
0.02738

Molecular function
Carboxylic acid binding
8 (0)
84 (5)
0.02754

Cellular component
MHC protein complex
4 (0)
26 (0)
0.02764

Molecular function
DNA topoisomerase type I activity
2 (2)
5 (5)
0.02831

Molecular function
Solute:sodium symporter activity
4 (0)
26 (0)
0.02853

Cellular component
Synapse
13 (10)
167 (116)
0.0289

Biological process
L-Amino acid transport
3 (0)
14 (1)
0.02906

Biological process
Cilium morphogenesis
3 (1)
14 (3)
0.02906

Cellular component
Secretory granule
8 (3)
86 (27)
0.02929

Biological process
Positive regulation of myeloid leukocyte differentiation
2 (0)
5 (0)
0.0298

Biological process
Glutamate catabolic process
2 (0)
5 (0)
0.0298

Biological process
Sulfate transport
2 (2)
5 (5)
0.0298

Biological process
Deoxyribonucleotide biosynthetic process
2 (0)
5 (2)
0.0298

Cellular component
Endomembrane system
26 (1)
416 (14)
0.03019

Cellular component
External side of plasma membrane
6 (5)
55 (49)
0.03035

Cellular component
Plasma membrane
64 (48)
1224 (935)
0.03044

Cellular component
Endocytic vesicle membrane
3 (3)
15 (12)
0.03078

Cellular component
Clathrin coated vesicle membrane
4 (0)
27 (3)
0.0308

Cellular component
Intrinsic to organelle membrane
8 (0)
87 (0)
0.03095

Biological process
Regulation of rho protein signal transduction
5 (5)
39 (29)
0.03101

Biological process
Neurotransmitter secretion
4 (3)
26 (11)
0.03114

Cellular component
Synapse part
10 (0)
117 (2)
0.03187

Biological process
Vitamin transport
3 (1)
15 (1)
0.03386

Molecular function
Symporter activity
6 (6)
57 (44)
0.03631

Molecular function
Syntaxin-1 binding
2 (2)
6 (6)
0.03678

Molecular function
Ion channel inhibitor activity
2 (1)
6 (2)
0.03678

Biological process
Cell communication
34 (4)
562 (40)
0.03717

Cellular component
Endocytic vesicle
4 (1)
29 (10)
0.03772

Biological process
Regulation of mitosis
4 (2)
28 (7)
0.03839

Biological process
Regulation of nuclear division
4 (0)
28 (0)
0.03839

Biological process
Response to inorganic substance
10 (1)
118 (8)
0.03843

Biological process
Phosphatidylcholine biosynthetic process
2 (2)
6 (5)
0.03869

Biological process
Interleukin-1 beta secretion
2 (0)
6 (0)
0.03869

Biological process
Interleukin-1 secretion
2 (0)
6 (0)
0.03869

Biological process
Regulation of interleukin-1 beta secretion
2 (0)
6 (0)
0.03869

Biological process
Regulation of interleukin-1 secretion
2 (0)
6 (0)
0.03869

Biological process
Cdc42 protein signal transduction
2 (1)
6 (3)
0.03869

Biological process
L-Phenylalanine catabolic process
2 (2)
6 (6)
0.03869

Biological process
L-Phenylalanine metabolic process
2 (0)
6 (0)
0.03869

Biological process
Tyrosine metabolic process
2 (0)
6 (1)
0.03869

Biological process
Multicellular organismal aging
2 (1)
6 (2)
0.03869

Biological process
Aspartate transport
2 (0)
6 (3)
0.03869

Biological process
Negative regulation of transforming growth factor beta receptor signaling pathway
2 (2)
6 (6)
0.03869

Biological process
Neurofilament cytoskeleton organization
2 (1)
6 (5)
0.03869

Molecular function
Chloride ion binding
4 (4)
29 (29)
0.0389

Biological process
L-Glutamate transport
3 (0)
16 (7)
0.03904

Biological process
Platelet activation
3 (2)
16 (11)
0.03904

Biological process
Positive regulation of secretion
5 (0)
42 (1)
0.03976

Molecular function
Substrate-specific transporter activity
28 (0)
463 (0)
0.04034

Biological process
Nucleotide biosynthetic process
12 (0)
151 (7)
0.04052

Cellular component
Microtubule basal body
3 (3)
17 (17)
0.04062

Molecular function
Calcium-dependent protein binding
3 (3)
17 (17)
0.04164

Biological process
Anion transport
7 (0)
73 (10)
0.04185

Molecular function
Solute:cation symporter activity
5 (0)
44 (0)
0.04201

Biological process
Purine nucleoside triphosphate biosynthetic process
7 (0)
74 (0)
0.0443

Biological process
Acidic amino acid transport
3 (0)
17 (0)
0.04461

Biological process
Pyrimidine nucleoside metabolic process
3 (0)
17 (1)
0.04461

Biological process
Pyrimidine nucleotide biosynthetic process
3 (0)
17 (4)
0.04461

Cellular component
MHC class II protein complex
3 (3)
18 (18)
0.04607

Molecular function
Oxidoreductase activity, acting on sulfur group of donors, NAD, or NADP as acceptor
2 (0)
7 (2)
0.04608

Molecular function
Fatty acid transporter activity
2 (1)
7 (3)
0.04608

Molecular function
Channel inhibitor activity
2 (0)
7 (1)
0.04608

Molecular function
DNA topoisomerase activity
2 (1)
7 (2)
0.04608

Biological process
Regulation of cytokine production
7 (0)
75 (0)
0.04684

Molecular function
NADP or NADPH binding
3 (3)
18 (17)
0.04721

Biological process
Nucleobase, nucleoside, and Nucleotide biosynthetic process
12 (0)
157 (0)
0.04725

Biological process
Nucleobase, nucleoside, nucleotide, and nucleic acid biosynthetic process
12 (0)
157 (0)
0.04725

Biological process
G1 phase
2 (1)
7 (1)
0.04845

Cellular component
Lamellipodium
4 (4)
32 (32)
0.04965

Cellular component
Transport vesicle
4 (1)
32 (11)
0.04965

Table 4: Gene ontology of downregulated genes in Y-27632-treated TM cells.

Ontology
Term
Changed genes
Total genes
value

Molecular function
Endonuclease activity, active with either ribo- or deoxyribonucleic acids and producing 5′-phosphomonoesters
2 (0)
13 (0)
0.00279

Biological process
Antigen processing and presentation of peptide or polysaccharide antigen via MHC class II
2 (2)
16 (14)
0.00421

Cellular component
MHC class II protein complex
2 (2)
18 (18)
0.00606

Molecular function
D-Dopachrome decarboxylase activity
1 (1)
1 (1)
0.01065

Molecular function
Glucose 1-dehydrogenase activity
1 (1)
1 (1)
0.01065

Molecular function
Glucose-6-phosphate dehydrogenase activity
1 (1)
1 (1)
0.01065

Molecular function
Deoxyribonuclease I activity
1 (1)
1 (1)
0.01065

Molecular function
3′-Phosphoadenosine 5′-phosphosulfate transmembrane transporter activity
1 (1)
1 (1)
0.01065

Biological process
Olfactory behavior
1 (1)
1 (1)
0.01091

Biological process
Positive regulation of dopamine receptor signaling pathway
1 (1)
1 (1)
0.01091

Biological process
Regulation of dopamine receptor signaling pathway
1 (0)
1 (0)
0.01091

Biological process
3′-Phosphoadenosine 5′-phosphosulfate transport
1 (1)
1 (1)
0.01091

Biological process
Negative regulation of neuron apoptosis
2 (2)
28 (28)
0.01148

Cellular component
MHC protein complex
2(0)
26(0)
0.01171

Molecular function
Toxin binding
1 (1)
2 (2)
0.01594

Molecular function
C2H2 zinc finger domain binding
1 (1)
2 (2)
0.01594

Molecular function
Dopachrome isomerase activity
1 (1)
2 (2)
0.01594

Molecular function
6-Phosphogluconolactonase activity
1 (1)
2 (2)
0.01594

Molecular function
Hedgehog receptor activity
1 (1)
2 (2)
0.01594

Molecular function
Dihydrofolate reductase activity
1 (1)
2 (2)
0.01594

Molecular function
Purine nucleoside transmembrane transporter activity
1 (0)
2 (1)
0.01594

Biological process
Cytolysis by symbiont of host cells
1 (0)
2 (0)
0.01632

Biological process
Cytolysis of cells in other organism during symbiotic interaction
1 (0)
2 (0)
0.01632

Biological process
Cytolysis of cells of another organism
1 (0)
2 (0)
0.01632

Biological process
Disruption by symbiont of host cells
1 (0)
2 (0)
0.01632

Biological process
Hemolysis by symbiont of host erythrocytes
1 (1)
2 (2)
0.01632

Biological process
Hemolysis of cells in other organism
1 (0)
2 (0)
0.01632

Biological process
Hemolysis of cells in other organism during symbiotic interaction
1 (0)
2 (0)
0.01632

Biological process
Killing by symbiont of host cells
1 (0)
2 (0)
0.01632

Biological process
Maintenance of mitochondrion location
1 (1)
2 (2)
0.01632

Biological process
Modification by organism of cell membrane in other organism during symbiotic interaction
1 (0)
2 (0)
0.01632

Biological process
Modification by symbiont of host cell membrane
1 (0)
2 (0)
0.01632

Biological process
Modification by symbiont of host cellular component
1 (0)
2 (0)
0.01632

Biological process
Modification by symbiont of host structure
1 (0)
2 (0)
0.01632

Biological process
Modification of cellular component in other organism during symbiotic interaction
1 (0)
2 (0)
0.01632

Biological process
Modification of structure of other organism during symbiotic interaction
1 (0)
2 (0)
0.01632

Biological process
Caveola assembly
1 (1)
2 (2)
0.01632

Biological process
Membrane raft assembly
1 (0)
2 (0)
0.01632

Biological process
Positive regulation of G-protein coupled receptor protein signaling pathway
1 (0)
2 (1)
0.01632

Biological process
Chromatin-mediated maintenance of transcription
1 (1)
2 (2)
0.01632

Biological process
Positive regulation of gene expression, epigenetic
1 (0)
2 (0)
0.01632

Biological process
ncRNA catabolic process
1 (0)
2 (0)
0.01632

Biological process
rRNA catabolic process
1 (1)
2 (2)
0.01632

Biological process
Purine nucleoside transport
1 (0)
2 (1)
0.01632

Biological process
Striated muscle cell differentiation
2 (1)
38 (5)
0.0199

Biological process
Antigen processing and presentation
2 (2)
38 (32)
0.0199

Molecular function
Copper ion binding
2 (2)
39 (38)
0.01996

Molecular function
Endodeoxyribonuclease activity, producing 5′-phosphomonoesters
1 (0)
3 (1)
0.0212

Cellular component
Membrane
24 (17)
2769 (1768)
0.02168

Biological process
Maintenance of organelle location
1 (0)
3 (0)
0.02171

Biological process
Melanin biosynthetic process
1 (1)
3 (3)
0.02171

Biological process
Melanin metabolic process
1 (0)
3 (0)
0.02171

Biological process
Endoplasmic reticulum calcium ion homeostasis
1 (1)
3 (1)
0.02171

Biological process
Multicellular organismal water homeostasis
1 (0)
3 (0)
0.02171

Biological process
Renal water homeostasis
1 (1)
3 (1)
0.02171

Biological process
Positive regulation of Rac protein signal transduction
1 (1)
3 (3)
0.02171

Biological process
Apoptosis
7 (3)
522 (189)
0.0221

Biological process
Programmed cell death
7 (0)
525 (2)
0.02273

Cellular component
Extrinsic to internal side of plasma membrane
1 (1)
3 (3)
0.02347

Cellular component
Spectrin
1 (1)
3 (3)
0.02347

Molecular function
Endonuclease activity
2 (2)
43 (30)
0.02378

Molecular function
Nucleoside transmembrane transporter activity
1 (0)
4 (1)
0.02643

Biological process
Regulation of neuron apoptosis
2 (0)
45 (2)
0.02693

Biological process
Detection of visible light
1 (1)
4 (1)
0.02706

Biological process
Chemosensory behavior
1 (0)
4 (3)
0.02706

Biological process
Protein maturation by protein folding
1 (1)
4 (4)
0.02706

Biological process
Cellular chaperone-mediated protein complex assembly
1 (1)
4 (2)
0.02706

Biological process
Mitochondrial outer membrane translocase complex assembly
1 (1)
4 (4)
0.02706

Biological process
Outer mitochondrial membrane organization
1 (0)
4 (0)
0.02706

Biological process
Glycine biosynthetic process
1 (1)
4 (2)
0.02706

Cellular component
Membrane part
21 (0)
2325 (1)
0.02771

Biological process
Cellular membrane organization
4 (0)
212 (30)
0.02909

Biological process
Membrane organization
4 (0)
212 (0)
0.02909

Biological process
Neuron apoptosis
2 (0)
48 (3)
0.03021

Biological process
Neuron death
2 (0)
48 (0)
0.03021

Biological process
Muscle cell differentiation
2 (0)
49 (1)
0.03134

Biological process
Disruption of cells of other organism during symbiotic interaction
1 (0)
5 (0)
0.03239

Biological process
Killing of cells in other organism during symbiotic interaction
1 (0)
5 (0)
0.03239

Biological process
Water homeostasis
1 (0)
5 (1)
0.03239

Biological process
Endoplasmic reticulum organization
1 (1)
5 (5)
0.03239

Biological process
Membrane raft organization
1 (0)
5 (1)
0.03239

Biological process
Pinocytosis
1 (1)
5 (2)
0.03239

Biological process
Nucleoside transport
1 (0)
5 (2)
0.03239

Biological process
Response to light stimulus
2 (0)
51 (8)
0.03364

Biological process
Cell death
7 (0)
578 (68)
0.03597

Biological process
Vesicle-mediated transport
5 (2)
336 (120)
0.03622

Biological process
Death
7 (0)
579 (0)
0.03626

Biological process
Positive regulation of signaling pathway
3 (0)
133 (0)
0.03743

Biological process
Modification by host of symbiont morphology or physiology
1 (0)
6 (0)
0.03769

Biological process
ER overload response
1 (1)
6 (5)
0.03769

Biological process
Regulation of Rac protein signal transduction
1 (0)
6 (1)
0.03769

Molecular function
Actin binding
3 (3)
139 (121)
0.0394

Cellular component
HOPS complex
1 (1)
6 (6)
0.04072

Molecular function
Intramolecular oxidoreductase activity, transposing C=C bonds
1 (0)
7 (0)
0.04196

Biological process
Detection of light stimulus
1 (0)
7 (0)
0.04296

Biological process
Metabotropic glutamate receptor signaling pathway
1 (1)
7 (4)
0.04296

Biological process
Regulation of synaptic transmission, GABAergic
1 (1)
7 (2)
0.04296

Cellular component
Internal side of plasma membrane
1 (0)
7 (4)
0.04641

Molecular function
Scavenger receptor activity
1 (1)
8 (8)
0.04708

Biological process
Interaction with symbiont
1 (0)
8 (1)
0.0482

Biological process
Modification by symbiont of host morphology or physiology
1 (0)
8 (0)
0.0482

Biological process
Chaperone-mediated protein complex assembly
1 (0)
8 (4)
0.0482

Biological process
Positive regulation of Ras protein signal transduction
1 (0)
8 (4)
0.0482

Biological process
Positive regulation of small GTPase-mediated signal transduction
1 (0)
8 (0)
0.0482

Biological process
Synaptic transmission, GABAergic
1 (0)
8 (1)
0.0482

Biological process
Actin filament capping
1 (1)
8 (6)
0.0482

Biological process
Pentose-phosphate shunt
1 (1)
8 (7)
0.0482

Biological process
Calcium ion transport
2 (1)
63 (41)
0.04876

Cellular component
Intrinsic to membrane
17 (0)
1867 (20)
0.0491

(a)

(b)

Figure 1: (a) Quantitative PCR analysis of catalase mRNA. The TM cells were treated with 25 μM Y-27632 for 30 min. The relative expression level of catalase of samples treated with Y-27632 was compared to that of the control sample using the comparative CT method (ΔΔCT method). The 18S ribosomal RNA was used as an endogenous control. Data are shown as mean ± SE from six independent experiments. compared with control by Wilcoxon rank sum test. (b) The effects of Y-27632 on the intracellular production of reactive oxygen species (ROS). The TM cells were treated with or without 25 μM Y-27632 for 30 min, followed by 100 μM menadione stimulated for 1 h. ROS were detected by CellROX reagent, and the fluorescence of the TM cells were measured by cell sorter SH800. Data are shown as mean ± SE from five independent experiments. and compared with control by the Wilcoxon rank sum test (a) and Tukey Kramer HSD test (b).

3.2. Effects of Y-27632 on the Production of Reactive Oxygen Species in TM Cells

To assess the effects of Y-27632 on the production of ROS in TM cells, we utilized a fluorogenic probe that exhibits bright fluorescence upon oxidation by ROS. In the absence of an oxidative reagent, the fluorescence intensity was not significantly different in TM cells treated with Y-27632 compared to control (3673.2 ± 452.3 versus 5104.5 ± 735.0; Figure 1(b)). In the presence of 100 μM menadione, the fluorescence intensity was significantly elevated (16097.7 ± 1133.0; ); this elevation was partly suppressed by treatment with Y-27632 (11443.6 ± 1332.2; ), suggesting that Y-27632 reduces ROS production in TM cells under oxidative stress.

3.3. Effects of Y-27632 on the Viability of TM Cells under Oxidative Stress

Finally, we investigated the effects of Y-27632 on the viability of TM cells under oxidative stress. As shown in Figure 2(a), menadione reduced TM cell viability in a dose-dependent manner. At a lower dose of menadione, Y-27632-stimulated TM cells regained significant viability against menadione treatment compared to control cells (). In contrast, the effects of Y-27632 on cell viability were not significant at a higher dose of menadione.

(a)

(b)

Figure 2: (a) The effect of Y-27632 on oxidative stress-induced cell death. The TM cells were treated with or without 25 μM Y-27632 for 30 min, followed by menadione stimulation of the cells for 24 h. Cell viabilities were shown as relative value compared with the control. Data are shown as the mean ± SE from six independent experiments. compared with control by Wilcoxon rank sum test. (b) The effect of Y-27632 on extracellular antioxidative activity. The xanthine-oxidase-induced superoxide production was assessed using a superoxide-sensitive luminescent reagent. Data are shown as the mean ± SE from six independent experiments. compared with the control by Dunnett’s test. Nac: n-acetyl cysteine.

3.4. Direct Antioxidant Activity of Y-27632

To confirm the extracellular antioxidant activity of Y-27632, we assessed xanthine oxidase-induced superoxide production using a luminescent reagent. As shown in Figure 2(b), there was no significant difference in ROS production between the control and Y-27632 treatment. Thus, Y-27632 does not appear to affect extracellular oxidants.

4. Discussion

In the present study, we have identified the antioxidative effect of Y-27632 in TM cells by microarray analysis, an exhaustive investigation of gene expression, and shown that Y-27632 partially suppresses ROS production and cell death induced by menadione. To the best of our knowledge, this is the first report to show the antioxidant effect of ROCK inhibitor on TM cells. Previously, we presented depolymerization of F-actin before morphometric recovery from oxidative stress in TM cells [24], suggesting a correlation between oxidative stress and regulation of the actin cytoskeleton in TM cells. In other tissues, rho-kinase was identified as a mediator of various diseases associated with inflammation and oxidative stress, and inhibition of rho-kinase has been drawing attention as a promising therapeutic strategy. For instance, activation of the rho/rho-kinase pathway is related to the pathophysiology of chronic renal injury, and long-term fasudil treatment has renoprotective effects in this malignant hypertension model. The mechanism of the renoprotective effect of fasudil, a nonspecific ROCK inhibitor, was suggested to involve a combination of factors, including inhibition of the TGF-β-collagen cascade, control of inflammation, reduction of oxidative stress, and upregulation of eNOS [18]. Clinical studies with fasudil have suggested that it may be useful for the treatment of a wide range of cardiovascular diseases [19]. Importantly, rho-kinase inhibitors block ROS production by suppressing CyPA secretion from vascular smooth muscle cells [25], suggesting the beneficial effect of rho-kinase inhibitors against cardiovascular diseases.

Recently, Yamamoto and colleagues demonstrated the neuroprotective effect of the ROCK inhibitor K-115, a novel IOP-lowering drug, using the mouse optic crush model [20]. They showed the effect was at least partially dependent on suppression of ROS production via inhibition of Nox1 expression in retinal ganglion cells. We also showed that ROCK inhibitors’ antioxidant effects are indirect using monkey TM cells. However, in the present study using microarray analysis, Nox family genes were not identified as affected, but catalase was upregulated after treatment with Y-27632. This disagreement might be caused by differences in species and/or tissues. Thus, the precise molecular mechanisms of the antioxidative effect of ROCK inhibitors have not been clarified completely. On the other hand, a recent study reported that Y-27632 induced p-53-mediated apoptosis in hemangioma [26]. In the present study, we indicated that ROCK inhibitor effected cell survival in TM cells. This is interesting point since ROCK inhibitor-induced effects such as cell death or cell protection were changed by differences of cell types.

TM has a critical role in the maintenance of aqueous outflow resistance through the regulation of extracellular matrix metabolism, phagocytosis of debris, and empty space associated with tissue contraction [27, 28]. Indeed, the number of TM cells is decreased in glaucomatous eyes [29], suggesting that functional TM cells are essential in controlling IOP. In this context, oxidative stress is a potential cause of cellular dysregulation in TM, both functionally and numerally, because it has been suggested that the TM of glaucomatous eyes is continuously exposed to oxidative stress [2–7]. Thus, an antioxidant drug might reduce oxidative stress in TM cells, slowing progression of glaucomatous damage in outflow tissues. Though it remains unknown whether clinically used eye-drops containing ripasudil have significant antioxidative effects on TM cells in vivo, the present study’s findings may be clinically relevant.

The effect of Y-27632 on cell survival under oxidative stress was significant, but limited. Since glaucoma progresses chronically in the majority of the patients, the acute oxidative damage in the present study may not reflect pathological conditions in glaucomatous TM cells, which is one of the limitations of the present study. Another limitation is that the antioxidative effects of ROCK inhibition were not corroborated in vivo. Further studies are required to acquire more clinically relevant evidence of the effects of ROCK inhibitor on oxidative stress in TM.

5. Conclusion

Microarray analysis reveals that Y-27632 upregulates antioxidative genes including catalase and partially reduces the ROS production and cell death by oxidative stress induced by menadione.

Conflicts of Interest

Dr. Hidenobu Tanihara has received consulting fees from Kowa and MSD and board membership fees from Senju Pharmaceutical, Santen Pharmaceutical, Alcon Japan, and Pfizer Japan.

Acknowledgments

This work was supported by the JSPS KAKENHI Grant nos. 26293375, 15K15636, and 26462664.

References

M. A. Babizhayev, K. S. Vishnyakova, and Y. E. Yegorov, “Oxidative damage impact on aging and age-related diseases: drug targeting of telomere attrition and dynamic telomerase activity flirting with imidazole-containing dipeptides,” Recent Patents on Drug Delivery & Formulation, vol. 8, no. 3, pp. 163–192, 2014.

S. M. Ferreira, S. F. Lerner, R. Brunzini, P. A. Evelson, and S. F. Llesuy, “Oxidative stress markers in aqueous humor of glaucoma patients,” American Journal of Ophthalmology, vol. 137, no. 1, pp. 62–69, 2004.

A. A. Ghanem, L. F. Arafa, and A. El-Baz, “Oxidative stress markers in patients with primary open-angle glaucoma,” Current Eye Research, vol. 35, no. 4, pp. 295–301, 2010.

V. Zanon-Moreno, P. Marco-Ventura, A. Lleo-Perez et al., “Oxidative stress in primary open-angle glaucoma,” Journal of Glaucoma, vol. 17, no. 4, pp. 263–268, 2008.

A. Goyal, A. Srivastava, R. Sihota, and J. Kaur, “Evaluation of oxidative stress markers in aqueous humor of primary open angle glaucoma and primary angle closure glaucoma patients,” Current eye Research, vol. 39, no. 8, pp. 823–829, 2014.

A. Izzotti, S. C. Sacca, C. Cartiglia, and S. De Flora, “Oxidative deoxyribonucleic acid damage in the eyes of glaucoma patients,” American Journal of Medicine, vol. 114, no. 8, pp. 638–646, 2003.

S. C. Sacca, A. Pascotto, P. Camicione, P. Capris, and A. Izzotti, “Oxidative DNA damage in the human trabecular meshwork: clinical correlation in patients with primary open-angle glaucoma,” Archives of Ophthalmology, vol. 123, no. 4, pp. 458–463, 2005.

M. Tanito, S. Kaidzu, Y. Takai, and A. Ohira, “Correlation between systemic oxidative stress and intraocular pressure level,” PLoS One, vol. 10, no. 7, article e0133582, 2015.

Y. Tang, E. A. Scheef, S. Wang et al., “CYP1B1 expression promotes the proangiogenic phenotype of endothelium through decreased intracellular oxidative stress and thrombospondin-2 expression,” Blood, vol. 113, no. 3, pp. 744–754, 2009.

Y. Zhao, S. Wang, C. M. Sorenson et al., “Cyp1b1 mediates periostin regulation of trabecular meshwork development by suppression of oxidative stress,” Molecular and Cellular Biology, vol. 33, no. 21, pp. 4225–4240, 2013.

Y. A. Ito, I. S. Goping, F. Berry, and M. A. Walter, “Dysfunction of the stress-responsive FOXC1 transcription factor contributes to the earlier-onset glaucoma observed in Axenfeld-Rieger syndrome patients,” Cell Death & Disease, vol. 5, article e1069, 2014.

F. B. Berry, J. M. Skarie, F. Mirzayans et al., “FOXC1 is required for cell viability and resistance to oxidative stress in the eye through the transcriptional regulation of FOXO1A,” Human Molecular Genetics, vol. 17, no. 4, pp. 490–505, 2008.

M. Honjo, H. Tanihara, M. Inatani et al., “Effects of rho-associated protein kinase inhibitor Y-27632 on intraocular pressure and outflow facility,” Investigative Ophthalmology and Visual Sciences, vol. 42, no. 1, pp. 137–144, 2001.

P. V. Rao, P. F. Deng, J. Kumar, and D. L. Epstein, “Modulation of aqueous humor outflow facility by the rho kinase-specific inhibitor Y-27632,” Investigative Ophthalmology and Visual Sciences, vol. 42, no. 5, pp. 1029–1037, 2001.

K. P. Garnock-Jones, “Ripasudil: first global approval,” Drugs, vol. 74, no. 18, pp. 2211–2215, 2014.

H. Tanihara, T. Inoue, T. Yamamoto et al., “Phase 2 randomized clinical study of a rho kinase inhibitor, K-115, in primary open-angle glaucoma and ocular hypertension,” American Journal of Ophthalmology, vol. 156, no. 4, pp. 731–736, 2013.

H. Tanihara, T. Inoue, T. Yamamoto et al., “Additive intraocular pressure-lowering effects of the rho kinase inhibitor ripasudil (K-115) combined with timolol or latanoprost: a report of 2 randomized clinical trials,” JAMA Ophthalmology, vol. 133, no. 7, pp. 755–761, 2015.

T. Nishikimi and H. Matsuoka, “Molecular mechanisms and therapeutic strategies of chronic renal injury: renoprotective effect of rho-kinase inhibitor in hypertensive glomerulosclerosis,” Journal of Pharmacological Sciences, vol. 100, no. 1, pp. 22–28, 2006.

K. Satoh, Y. Fukumoto, and H. Shimokawa, “Rho-kinase: important new therapeutic target in cardiovascular diseases,” American Journal of Physiology Heart and Circulatory Physiology, vol. 301, no. 2, pp. H287–H296, 2011.

K. Yamamoto, K. Maruyama, N. Himori et al., “The novel rho kinase (ROCK) inhibitor K-115: a new candidate drug for neuroprotective treatment in glaucoma,” Investigative Ophthalmology and Visual Sciencies, vol. 55, no. 11, pp. 7126–7136, 2014.

T. Fujimoto, T. Inoue, T. Kameda et al., “Involvement of RhoA/Rho-associated kinase signal transduction pathway in dexamethasone-induced alterations in aqueous outflow,” Investigative Ophthalmology and Visual Sciences, vol. 53, no. 11, pp. 7097–7108, 2012.

M. Inoue-Mochita, T. Inoue, T. Fujimoto et al., “p38 MAP kinase inhibitor suppresses transforming growth factor-beta2-induced type 1 collagen production in trabecular meshwork cells,” PLoS One, vol. 10, no. 3, article e0120774, 2015.

T. Kameda, T. Inoue, M. Inatani et al., “The effect of rho-associated protein kinase inhibitor on monkey Schlemm’s canal endothelial cells,” Investigative Ophthalmology and Visual Sciences, vol. 53, no. 6, pp. 3092–3103, 2012.

N. Awai-Kasaoka, T. Inoue, T. Kameda, T. Fujimoto, M. Inoue-Mochita, and H. Tanihara, “Oxidative stress response signaling pathways in trabecular meshwork cells and their effects on cell viability,” Molecular Vision, vol. 19, pp. 1332–1340, 2013.

K. Satoh, P. Nigro, T. Matoba et al., “Cyclophilin A enhances vascular oxidative stress and the development of angiotensin II-induced aortic aneurysms,” Nature Medicine, vol. 15, no. 6, pp. 649–656, 2009.

M. K. Qiu, S. Q. Wang, C. Pan et al., “ROCK inhibition as a potential therapeutic target involved in apoptosis in hemangioma,” Oncology Reports, vol. 37, no. 5, pp. 2987–2993, 2017.

U. Roy Chowdhury, C. R. Hann, W. D. Stamer, and M. P. Fautsch, “Aqueous humor outflow: dynamics and disease,” Investigative Ophthalmology and Visual Sciences, vol. 56, no. 5, pp. 2993–3003, 2015.

J. A. Vranka, M. J. Kelley, T. S. Acott, and K. E. Keller, “Extracellular matrix in the trabecular meshwork: intraocular pressure regulation and dysregulation in glaucoma,” Experimental eye Research, vol. 133, pp. 112–125, 2015.

J. Alvarado, C. Murphy, and R. Juster, “Trabecular meshwork cellularity in primary open-angle glaucoma and nonglaucomatous normals,” Ophthalmology, vol. 91, no. 6, pp. 564–579, 1984.