Increasing evidence suggests that
Transcriptomic profiling has been widely applied for studying the molecular mechanisms underpinning bacteria-host interactions. Fan et al. have employed a purpose built Bam4kOLI microarray to carry out transcriptome analysis of
The BeWo cell line has been shown to be a useful
Food-borne
Human choriocarcinoma cell line BeWo was purchased from Action-award Biotech Co., Ltd (Guangzhou, China). BeWo cells were cultured in DMEM/F12 medium supplemented with 10% (v/v) FBS (Gibco, Grand Island, NY) and grown in 25 mm2 cell culture flasks (NEST, Jiangsu, China) at 37°C under a 5% CO2 atmosphere, until approximately 80–90% cell confluence (as verified by inverted light microscopy). BeWo cells were then liberated from cell culture flasks using 0.25% trypsin-EDTA (Solarbio, Beijing, China), before being resuspended in fresh medium containing 10% FBS, and then transferred to new flasks.
For invasion experiments, the liberated BeWo cells were transferred into 24-well plates (Corning, NY) and cultured for 48 h until a confluent monolayer was obtained. Culture supernatant was then removed and 5
The total number of invaded bacteria associated with BeWo cells (including adherent and internalized
The numbers of internalized bacteria present were determined using minor modifications of an antibiotic survival assay that has been described previously [
BeWo cells were incubated in fresh medium for 4 h, with or without
Beijing EMTD Technology Development Co. Ltd. carried out all microarray analysis using Illumina Human HT-12 v4 Expression BeadChip microarrays (Illumina, Inc., San Diego, CA). RNA preparation and hybridization were performed according to the manufacturer’s instructions [
First strand cDNA was synthesized using a Quantscript RT Kit Quant cDNA first strand Synthesis Kit (TIANGEN, Beijing, China). Real-time PCR was carried out using a SYBR® Premix Ex Taq™ II (Tli RNaseH Plus) Kit (TaKaRa, Dalian, China) using the primer sets listed in Table
Unless specified, all experiments were performed in triplicate. All data was analyzed using statistics programs contained in SigmaPlot 11.0 (Systat Software, San Jose, CA).
The number of
DNA microarray techniques were used to compare the gene expression patterns of untreated BeWo cells with those infected by
The function of the DEGs that were affected by
Of relevance to this study, 45 DEGs associated with placenta and embryonic development were identified, namely, “chordate embryonic development,” “embryonic development ending in birth or egg hatching,” “in utero embryonic development,” “placenta development,” and “embryonic placenta development” (Figure
Classification of DEGs according to their GO biological process categories.
Term | Genes |
---|---|
| RTN4, IER3, XRCC2, ZMAT3, TNFSF15, PRDX5, PRDX2, PRDX3, FOXO3, ITSN1, CITED2, MEN1, MAP3K7, CASP6, ZFP91, CDKN2A, FNTA, BAG1, BOK, AEN, ZNF443, NET1, IP6K2, ADAM9, EGFR, SOCS3, AIFM1, RELA, TP53, ARHGEF17, JMY, ARHGEF11, CARD11, GRM4, SERPINB9, KRT18, NME3, PSEN1, TXNDC5, JUN, TNFAIP8, VEGFA, MAP3K10, KCNH8, DCUN1D3, CDK5R1, YWHAZ, GSDMA, XIAP, ERBB3, ADAMTSL4, NFKBIA, COL2A1, BCL2L1, CHEK2, GCH1, DDX19B, PPP2CB, LHX4, CD2, BCL3, BCL6, TAF9, TRAF6, PCSK6, BMF, ERCC1, TERF1, PHLDA1, HIP1, IL6, TP53BP2, KLF10, CREB1, BAD, GAS1, YWHAE, C14ORF153, NAE1, CDKN1A, TNFSF10, SON, NUPR1, BNIP3L, ID3, HSPD1, DNAJB6, BARD1, E2F1, SGPP1, ZNF346, CTNNB1, PAK2, GSN, RHOB, LOC100128086, ACIN1, EBAG9, SGK1, STAG3L3, DDIT4, PPM1F, EP300, MAPK7, GADD45B, GADD45A, GULP1, GPR65, DIDO1, TSC22D3, ECE1, PTPN6, PDCD10, RYBP, DUSP22, TNFSF9, ASAH2C (117) |
| |
| XRCC2, PDGFB, PDGFA, RBM3, TIRAP, FOXO3, KIAA0652, CITED2, MUTYH, GP6, INSIG2, LTB4R, CTGF, COL4A3BP, ZNF443, MAP2K7, ADAM9, EGFR, F11R, ATG9B, SOCS3, USP1, RELA, NEIL1, GAGE12G, SERPING1, GRHL3, DDIT4, PLAUR, RFC3, DCLRE1A, EP300, GAGE5, JUN, GAGE12I, LILRB3, VEGFA, KIAA1715, RYR1, DSP, AMFR, COL1A1, PLA2G2D, CCL3, ACHE, BLM, ERBB3, ALDOB, MAP4K1, BCL2L1, CHEK2, PXK, ITGB1, ARNT, DUSP19, ECE1, TFEC, C7ORF68, PTPN6, MAFF, RECQL5, CFB, MAP2K4, MYO1F, AGER, DDX58, HDAC5, APOL2, HYOU1, CDKN1A, ATF4, CXCL16, POLD2, PCNA, LOC732360, HSPD1, ENG, DNAJB6, ELF3, HPS6, FAM175A, ZMAT3, PRDX5, PMS2L5, PRDX2, PRDX3, PMS2L1, OS9, PMS2L3, MEN1, HMOX2, FOS, ANKRD17, CASP6, MCM7, HSF1, GSN, SLC23A2, XYLT1, AEN, IL1RAP, ERRFI1, FANCC, NLRP7, NFKBIZ, SGK1, SP100, AIFM1, GEN1, DDB1, CNPY3, LYZ, TP53, MMP14, WAS, JMY, C8G, CHST1, EYA3, RIF1, ADM, PSEN1, HIST2H2BE, MAP3 K10, GADD45B, GADD45A, TTRAP, YWHAZ, XIAP, C17ORF70, WRNIP1, MKNK2, EGLN2, EGLN1, COMT, VARS, TPM1, GCH1, IGSF10, CD9, CYP27B1, IL10RB, POU2F3, PPP2CB, BCL3, ENO3, NFATC4, BCL6, ERCC1, FEN1, HSPA8, IL6, ATRN, SIRT7, STAT3, NAE1, CCL11, VWF, NUPR1, TFRC, CSNK1E, TDP1, BNIP3L, ALKBH1, ID3, BARD1, SYT1, CTNNB1, FNTA, CHRNA5, RARA, PRKACB, BAIAP2, LIFR, SOCS7, GRB10, SLC25A36, PDGFRB, STC1, NFKBIA, IRAK3, NDRG1, TAF9, SRP54, KLF10, ABCG1, NR1I2, TSC1, LDLR, LEPR, GPR81, RHOQ, ASAH1, VN1R2, AES, CYR61, ADAM10, UFD1L, SS18, G6PC, CTSC, GNB4, DGKD, LOC100128775, DDX19B, COL6A2, PIK3R3, PIK3R1, CREB1, CAPN3, LEP, ID2, ID1, GNG10, RSRC1, ANXA11, MAPK15, TJP2, SBNO2, MAP3K7, ZFP91, AP2B1, PVRL2, SPG21, TRAF6, ATP6V1H, DUSP22, TRIM24, FOXP1, NPY5R, CARD11, NDEL1, AP2A1, IRF4, NCOR1 (235) |
| |
| XRCC2, COL2A1, EGLN1, BCL2L1, ITGB1, TPM1, CITED2, ARNT, MAP3K7, MEN1, OSR2, ECE1, HAND1, HSF1, COL4A3BP, PKD1, TRAF6, MAFF, ADAM10, MYO1E, GABPA, TP53, SPINT1, ICMT, MBNL1, GAS1, DVL1, HES1, ACVR2A, NDEL1, EP300, TSC1, PSMC4, PSEN1, MNX1, TGIF1, PDGFRB, LOC652826, COL1A1, RBPJ, VWF, CYP27B1, CCNF, PLCD3, PRDX3 (45) |
| |
| ALDOA, CCL3, SHROOM4, PDGFB, VPS54, CALD1, SIPA1, CETN3, NEDD9, RHOQ, ITGB1, TPM1, TTLL3, CTNNB1, SPC25, PTK2, PRR5, PACSIN1, PAK2, ANK3, GSN, FBXO5, TEKT2, BCL6, FGD5, ARHGAP8, SNHG3-RCC1, FGD6, TNXA, FSCN2, CKAP5, LOC100132288, ARHGEF17, CAPN3, WAS, ARHGEF11, NCK2, SS18, NDEL1, LOC399942, SDCBP, HAUS8, NEURL2, CRK, GADD45A, NCOR1, DNAJB6, CDKN2A, ADAM10, ERBB3, MYO1F, COL1A1, MMP14, PIK3R1, TSC1, CYR61, RELL2, NFKBIE, NFKBIB, G3BP2, NFKBIA, BARD1 (62) |
| |
| HIST1H4 K, FOXO3, CDKN2A, PVRL2, HIST1H4E, SPG21, RARA, ACIN1, CFB, KLF10, BAD, C8G, CDKN1A, ID2, JUN, HSPD1, RBM15, ATP6V1H, SERPING1, FOXP1, DDX58, AP2B1, AP2A1, YWHAZ, SBNO2, ITGB1, BCL3, NDRG1, ERCC1, PIK3R1, ADAM9, ADAM10, MYO1F, HDAC5, IRF1, KIR3DL1, BLM, TP53, WAS, PKNOX1, CD2, PATZ1, IRF4, KIF13B, STAT4, KRT18, SP100, LIFR, STAT3, MAST2, NFKBIB, TIRAP, G3BP2, IRAK1BP1, EDARADD, VAPA, SLC20A1, PDGFB, EFNA1, CHMP5, LEPR, RGL3, FSTL3, TNFSF15, PRDX2, ITSN1, CITED2, CTNNB1, MAP3K7, MEN1, GPR89B, ZFP91, SPRY1, TBC1D14, GIT2, GPR89A, NET1, EGFR, SOCS3, RELA, SOCS7, ARHGEF17, SOCS4, DDIT4, ARHGEF11, CARD11, SPAG9, GRM4, ACVR2A, RALGAPA2, NCK2, GRB10, NDEL1, PSEN1, VEGFA, MAP3K10, TGIF1, TBC1D3G, SNX6, ERBB3, SIPA1, PPM1A, MAP4K1, TRIM13, TFG, NFKBIA, TIMP2, ARNT, TBC1D3B, RASAL2, IRAK3, DUSP19, ECE1, CYP27B1, TSPYL2, SLC35B2, PPP2CB, BCL6, FGD5, TRAF6, PCSK6, FGD6, RASA2, IL6, TBC1D10A, PAK1IP1, DUSP22, ICMT, RGS17, GAS1, TRIM24, DVL1, TBC1D25, LEP, TNFSF10, LRP1, TSC1, GRTP1, C6ORF170, NCOR1, CRK, ENG, TOB1 |
DEGs were then mapped using the KEGG database for signal pathway analysis to gain further understanding of their biological function. These results reveal that these DEGs could be further categorized using 31 terms used by the KEGG database (Table
DEGs associated with signal pathways assigned by KEGG pathway analysis.
No. | Term | Count | Genes |
---|---|---|---|
1 | hsa04310:Wnt signaling pathway | 18 | CTBP1, CTBP2, TP53, CTNNB1, DVL1, MAP3K7, EP300, PSEN1, PRICKLE1, CSNK1E, JUN, PPP2CB, NFAT5, FRAT1, CAMK2B, NFATC4, PRKACB, NFATC1 |
| |||
2 | hsa04010:MAPK signaling pathway | 31 | FGFR4, PDGFB, PDGFA, PPM1A, MKNK2, MAP4K1, PPM1B, NFKB2, MAP3K7, MAX, FOS, PAK2, PLA2G12A, NFATC4, PRKACB, TRAF6, MAP2K7, HSPA8, RASA2, EGFR, RELA, MAP2K4, TP53, ATF4, JUN, PDGFRB, MAPK7, GADD45B, CRK, PLA2G2D, GADD45A |
| |||
3 | hsa04662:B cell receptor signaling pathway | 15 | PTPN6, IFITM1, NFKBIE, RELA, NFKBIB, NFKBIA, FOS, CARD11, LILRB3, JUN, NFAT5, NFATC4, PIK3R3, PIK3R1, NFATC1 |
| |||
4 | hsa04660:T cell receptor signaling pathway | 18 | PTPN6, NFKBIE, RELA, NFKBIB, NFKBIA, MAP3K7, FOS, NCK2, CARD11, PAK2, JUN, PAK4, NFAT5, NFATC4, PIK3R3, MAP2K7, PIK3R1, NFATC1 |
| |||
5 | hsa04620:Toll-like receptor signaling pathway | 13 | CCL3, IL6, RELA, MAP2K4, TIRAP, NFKBIA, MAP3K7, FOS, JUN, PIK3R3, TRAF6, MAP2K7, PIK3R1 |
| |||
6 | hsa04630:Jak-STAT signaling pathway | 20 | PTPN6, IL6, SOCS3, LEPR, LIFR, SOCS7, SOCS4, BCL2L1, STAT3, LEP, ZFP91, TSLP, STAT4, SPRY1, EP300, IL10RB, IL5RA, MPL, PIK3R3, PIK3R1 |
| |||
7 | hsa04510:focal adhesion | 25 | PDGFB, XIAP, PDGFA, COL2A1, ITGB1, CTNNB1, MYL9, VCL, PTK2, PAK2, PAK4, COL6A2, PDGFC, PIK3R3, PIK3R1, EGFR, TNXA, BAD, VWF, JUN, ITGA7, VEGFA, PDGFRB, COL1A1, CRK |
| |||
8 | hsa04520:adherens junction | 12 | MAP3K7, EGFR, PTPN6, PVRL4, EP300, PVRL1, BAIAP2, PVRL2, CTNND1, WAS, CTNNB1, VCL |
| |||
9 | hsa04810:regulation of actin cytoskeleton | 23 | EGFR, FGFR4, PDGFB, LIMK2, PDGFA, BAIAP2, ITGB1, WAS, MYL9, TTLL3, VCL, PTK2, ARPC3, PAK2, GSN, CFL2, PAK4, ITGA7, PDGFRB, PDGFC, PIK3R3, CRK, PIK3R1 |
| |||
10 | hsa05130:pathogenic | 9 | NCK2, YWHAZ, KRT18, ARPC3, LOC399942, WAS, ITGB1, CTNNB1, TTLL3 |
| |||
11 | hsa04115:p53 signaling pathway | 10 | CDKN1A, CDKN2A, ZMAT3, BAI1, RPRM, TP53, CHEK2, GADD45B, CCNG2, GADD45A |
| |||
12 | hsa04210:apoptosis | 14 | XIAP, AIFM1, RELA, TP53, NFKBIA, BAD, BCL2L1, IRAK3, CASP6, TNFSF10, IL1RAP, PRKACB, PIK3R3, PIK3R1 |
| |||
13 | hsa04150:mTOR signaling pathway | 8 | CAB39L, EIF4E, TSC1, STK11, VEGFA, PIK3R3, PIK3R1, DDIT4 |
Primers used for real-time PCR gene expression assays.
Target gene | Sequence (5′-3′) |
---|---|
IL1 | ACCTTCAAGGAGAGCATGGTG |
GGTGCTGACCTAGGCTTGATG | |
IL4 | GCTATGACTAAAGCAGGGACAA |
CACTCTCCTCAGCAAACACAGA | |
IL6 | TGCTCCTGGTGTTGCCTG |
GCCGTCGAGGATGTACCG | |
IL8 | GGCTCTCTTGGCAGCCTTC |
GCAGTTGGTCCACTCTCAATC | |
IL10 | CCAGGCAACCTGCCTAACA |
TCTCAGACAAGGCTTGGCAAC | |
IL12 | GTCCTCCTGGACCACCTCAG |
TCAATCTCTTCAGAAGTGCAAGG | |
TNF- | CACCACGCTCTTCTGCCTG |
GTTCAGCCACTGGAGCTGC | |
IFN- | GCAGGTCATTCAGATGTAGCG |
TTCCTTGATGGTCTCCACACTC | |
TLR1 | CAGTGTCTGGTACACGCATGGT |
TTTCAAAAACCGTGTCTGTTAGAGA | |
TLR2 | CACTCAGGAGCAGCAAGCAC |
GCAGGAACAGAGCACAGCAC | |
TLR3 | CCTGGTTTGTTAATTGGATTAACGA |
TGAGGTGGAGTGTTGCAAAGG | |
TLR4 | CAGAGTTTCCTGCAATGGATCA |
GCTTATCTGAAGGTGTTGCACAT | |
TLR6 | GAAGAAGAACAACCCTTTAGGATAGC |
AGGCCAAACAAAATGGAAGCTT | |
Claudin 1 | TGCTGGAATTGAACTACTCCAC |
TACTCTTTCCGGTTGGTGTGA | |
E-cadherin | GAATGATGATGGTGGACAAT |
TACCACATTCGTCACTGCTA | |
Occludin | GGCACCTGCATACTCACCC |
CTGGGAGAGCAACTCATCCTC | |
ZO-1 | CAACATACAGTGACGCTTCACA |
CACTATTGACGTTTCCCCACTC | |
ICAM | TTGTCCTGCCAGCGACTC |
AAGGAGTCGTGCCATAGGTG | |
madCAM | GCAGGAGGAGGAGGAGGAGGAG |
TCCAAGCCAGGCAGCC | |
GGCTATCCAGCGTACTCCAAA | |
CGGCAGGCATACTCATCTTTTT |
According to descriptions used in the KEGG database (
Importantly, these KEGG analysis results were largely in accord with results obtained from bioinformatic analyses (Figure
Changes to the transcriptional level of selected genes were studied using real-time PCR. As shown in Figure
Previously, we have shown that
According to the DNA microarray results, 45 DEGs were associated with placenta and embryonic development, which belonged to 5 terms within the GO “biological process” category. It is proposed that these changes to gene expression in placental trophoblasts in response to
Other genes were identified, which control three signaling pathways involved in “focal adhesion,” “regulation of actin cytoskeleton,” and “adherens junction.” These pathways are associated with changes to the cell-extracellular matrix, cell-cell adhesion properties, and the maintenance of normal cellular structure and function (Table
Large numbers of DEGs and signaling pathways that are closely related to the immune system response were also affected. Whilst changes in gene expression may activate or suppress host immune defense systems, disruption of the immune system in pregnancy can cause trophoblast death, resulting in placental insufficiency, spontaneous abortion, preterm labor, or stillbirth [
The authors declare that they have no conflicts of interest.
This research project was supported by the National Natural Science Foundation of China (NSF31570075, 31170091, 31000048), Academic and Technical Leaders Training Program for Major Subjects of Jiangxi Province (2009), Research Program of the State Key Laboratory of Food Science and Technology of Nanchang University (SKLF-MB-201002), Foundation for Program of Department of Education from Fujian Province (JA15813), and Research Program of Xiamen Medical College (K2015-07).
Table S1: differential gene expression profile of BeWo cell in response to