Gastric cancer (GC) is an invasive disease that continues to have a severe impact on global health [
Long noncoding RNAs (lncRNAs) are a group of noncoding RNAs to be defined as more than 200 bp in length with no protein-coding function [
In this study, real-time polymerase chain reaction (RT-PCR) was used to evaluate the expression level of RP11-357H14.17 in GC problems. We also assessed the relationship between RP11-357H14.17 levels and clinicopathological characteristics. Through bioinformatics analysis and experimental verification, its role in GC was predicted and analyzed. Our findings may contribute to a better understanding of the role of RP11-357H14.17 as a regulatory factor for GC and as a possible candidate target for new diagnosis and treatment.
The software R package limma (V.3.40.6) was used to calculate the fold-change of transcripts and to screen for differentially expressed genes (DEGs) in the RNA-seq cohort. A fold-change larger than two and an adjusted
GEPIA (
UCSC (
Total RNA in GC tissues was isolated using TRIzol reagent. The expression of genes of interest was detected using the syber-green-based real-time PCR. The primers for genes used in the study were listed in Table
Primer sequence for genes of interest.
Gene | Primer (forward/reverse) |
---|---|
RP11-357H14.17 | TCCGGCCATCTGGCGCT |
CAAAGGCGACGTGCCGG | |
RECQL4 | GCGCTCTACCGGGAATACC |
CAGCCCGATTCAGATGGGG | |
ATAD5 | GTGAAGGACTGCGAGATTGAG |
TGTCTCTAGTCTTCCCTAGTGGT | |
GINS1 | ACGAGGATGGACTCAGACAAG |
TGCAGCGTCGATTTCTTAACA | |
ORC6 | ACAAGGAGACATATCAGAGCTGT |
AGTGGCCTGGATAAGTCAAGAT | |
RAD51 | CCTCCTCTTTAACGCCTCCTG |
GGGGACAACTCCCAGACTTTTT | |
EME1 | TCTGAGGAGTTGCCAACATTTG |
GGCTTCACAATCTGAGATGTCAA | |
CCND1 | GCTGCGAAGTGGAAACCATC |
CCTCCTTCTGCACACATTTGAA | |
DBF4 | CCGGAAAGTCCTTTTACTTGGAT |
AACCCTCAATTACCCCACCCA | |
CDC25C | TCTACGGAACTCTTCTCATCCAC |
TCCAGGAGCAGGTTTAACATTTT | |
MCM2 | ATGGCGGAATCATCGGAATCC |
GGTGAGGGCATCAGTACGC | |
NASP | AGATTGGGAACCTAGAGCTTGC |
ACTTCTCCGAGTTTAAGATGTGC | |
EZH2 | AATCAGAGTACATGCGACTGAGA |
GCTGTATCCTTCGCTGTTTCC | |
PCNA | CCTGCTGGGATATTAGCTCCA |
CAGCGGTAGGTGTCGAAGC | |
CDC25A | GTGAAGGCGCTATTTGGCG |
TGGTTGCTCATAATCACTGCC | |
KRT8 | TGTGCCTACCTGCGGAAATC |
CTATGACCGAGGTGTCTGAGA | |
PARD6B | TTGGAGCTGAATTTCGTCGGT |
AGCCTACCAAAACGTCAACATT | |
THOC6 | TCCCAGAGCGTCTCACCAT |
CCACCGGCTTCTTACTTTCCT | |
WT1 | ACCAAAAATCGGTTCTTCATCCC |
TCACATCACTGGCAATCTTAGGA | |
ATF2 | CACCAGCAGCACGAGTCTC |
TGTGCGAGGCAAACAGGAG | |
FOXP3 | GTGGCCCGGATGTGAGAAG |
GGAGCCCTTGTCGGATGATG | |
HAT1 | AAGCCATTCGGAACCTTACTTC |
AGTGCCATCTTTCATCATCCAC | |
NUF2 | GGAAGGCTTCTTACCATTCAGC |
GACTTGTCCGTTTTGCTTTTGG | |
PLK1 | CAGTCACTCTCCGCGACAC |
GAGTAGCCGAATTGCTGCTG |
The hospital-based case-control study consists of 52 patients newly diagnosed with gastric cancer. All the subjects were recruited from the 1st People’s Hospital of Lianyungang and Suqian Hospital of Chinese traditional medicine, between February 2016 and August 2019. Patients with other hematological disorders, previous history of cancers, radiotherapy, and chemotherapy were excluded. The cancer-free control subjects from the same geographic area showed no evidence of a genetic relationship with the cases. The patients were classified according to World Health Organization classification. This study was approved by the Institutional Review Board of the 1st People’s Hospital of Lianyungang and Suqian Hospital of Chinese traditional medicine (LL-16-12 and SCY-17-15), and every patient had written informed consent. The clinical features of all the cases and controls were presented in Table
Frequency distributions of selected variables in gastric cancer patients.
Features | RP11-357H14.17High | RP11-357H14.17High | |||
---|---|---|---|---|---|
% | % | ||||
Age (years) | 0.779 | ||||
≤50 | 10 | 19.23% | 12 | 23.08% | |
>50 | 16 | 30.77% | 14 | 26.92% | |
Gender | 0.999 | ||||
Male | 14 | 26.92% | 13 | 25.00% | |
Female | 12 | 23.08% | 13 | 25.00% | |
Differentiation | 0.0254 | ||||
G1-G2 | 17 | 32.69% | 8 | 15.38% | |
G3-G4 | 9 | 17.31% | 18 | 34.62% | |
Tumor size (cm) | 0.0145 | ||||
≤5 cm | 16 | 30.77% | 7 | 13.46% | |
>5 cm | 10 | 19.23% | 19 | 36.54% | |
TMN stage | 0.0118 | ||||
I-II | 18 | 34.62% | 8 | 23.08% | |
III-IV | 8 | 15.38% | 18 | 26.92% | |
0.999 | |||||
Positive | 16 | 30.77% | 15 | 28.85% | |
Negative | 10 | 19.23% | 11 | 21.15% |
Sections were stained according to the previous publication. The section was incubated within primary mouse anti-human Ab for ATF2(ab239361), WT1(ab89901), CD4(ab183685), Foxp3(ab215206), and TGF-
To fully screen the long noncoding RNA within human stomach cancer, we analyzed total 18036 lncRNAs within the Cancer Genome Atlas Stomach Adenocarcinoma (TCGA-STAD) data collection; we found 375 GC patients and 32 normal controls with the TCGA-STAD. All of the 18036 lncRNAs were compared within the GC patients and normal controls by using Limma R package; the results indicated that 3790 lncRNAs were significantly differently expressed (
RP11-357H14.17 is overexpressed in human stomach cancer. (a) Heatmap of the all significantly differently expression lncRNAs (
RP11-357H14.17 is located in chromesome 17 with a Ensembl ID of ENSG00000272763.1. We confirmed its expression in human GC tissues by using an online tool, Gepia [
To further investigate the oncogenic roles of RP11-357H14.17 in human GC, first, we performed KEGG and GO enrichment analysis using up-regulated DEGs obtained by two comparison patterns. One is the comparison between RP11-357H14.17 top-32 expression GC samples and normal GC control samples (T vs. N), and the other is the comparison between the GC samples with top 40 and bottom 40 RP11-357H14.17 expression (High40 vs. Low40). We figured out shared GO enrichment terms between two comparison described above including “DNA replication,” “nuclear DNA replication” in Biological Processes (BP) GO terms, “mitochondrial protein complex,” “respiratory chain” in the Cell Components (CC) GO terms, and “electron transfer activity” and “NADPH dehydrogenase activity” in Molecular Functions (MF) GO terms (Figures
Increased expression of RP11-357H14.17 is associated with increased DNA replication and metabolism in human gastric cancer. GO enrichment was performed using significantly up-regulated genes compared between cancer and normal controls (a) and RP11-357H14.17 high and low expression sample sets (b). KEGG enrichment was performed using significantly up-regulated genes compared between cancer and normal controls (c) and RP11-357H14.17 high and low expression samples from TCGA-STAD (d). Representative genes expression stands for “DNA replication” (e) and “NADPH dehydrogenase activity” (f). GO terms was determined by real-time PCR.
In order to further clarify the possible mechanism for RP11-357H14.17-associated malignancy, we did single sample GSEA (ssGSEA) analysis using oncogenic signatures (c6) gene sets using “N vs. T” and “High 40 vs. Low40”. We found that high expression of RP11-357H14.17 in stomach cancer tissues were associated with ATF2-related gene sets including “ATF2_s_UP v1_up,” “ATF2_up. V1_up,” and “ATF2_up.V1_DN” (Figures
RP11-357H14.17 promotes the malignancy by promoting ATF2 signaling in human gastric cancer. Heatmap of all significantly different gene sets of c6 ssGSEA analysis for comparison between cancer and normal control samples (a) and RP11-357H14.17 high and low expression samples (b) from TCGA-STAD. ATF2-related gene sets were highlighted with red font (c). Heatmap of up-regulated genes in “ATF2_up.v1_DN” gene set derived from comparison between cancer and normal control samples and RP11-357H14.17 high and low expression samples from TCGA-STAD. The representative genes stand for “ATF2_up.v1_DN” gene set were highlighted in bold. (d) Representative gene expression stands for “ATF2_up.v1_DN” gene set was compared between cancer and normal control samples, RP11-357H14.17 high and low expression samples from TCGA-STAD,
To study the role of RP11-357H14.17 in tumor microenvironment (TME), we analyzed the immune-related roles of RP11-357H14.17 by ssGSEA analysis using immunologic signatures (c7) including 4872 gene sets for two sample sets described above. The results from both comparisons indicated that RP11-357H14.17 was significantly associated with Treg cell percentage within human GC by sharing gene sets “GSE37532_TREG_VS_TCONV_CD4_TCELL_FROM_LN_UP,” “GSE24634_TREG_VS_TCONV_POST_DAY5_IL4_CONVERSION_UP,” “GSE18893_TCONV_VS_TREG_24H_TNF_STIM_UP,” “GSE14415_TCONV_VS_FOXP3_KO_INDUCED_TREG_DN,” and “GSE14415_NATURAL_TREG_VS_TCONV_DN” (Figures
RP11-357H14.17 promotes the immunosuppression in human gastric cancer tissues by increasing Treg cell percentage. Heatmap of all significantly different gene sets of c7 ssGSEA analysis for comparison between cancer and normal control samples (a) and RP11-357H14.17 high and low expression samples (b) from TCGA-STAD. The “Treg” cell-related gene sets were highlighted in red font. (c) Heatmap of up-regulated genes in “GSE37532_TREG_VS_TCONV_CD4_TCELL_FROM_UP” gene set derived from comparison between cancer and normal control samples and RP11-357H14.17 high and low expression samples from TCGA-STAD, and the representative genes were highlighted in bold. (d) Representative gene expression stands for “GSE37532_TREG_VS_TCONV_CD4_TCELL_FROM_UP” gene set was compared between cancer and normal control samples, RP11-357H14.17 high and low expression samples from TCGA-STAD,
LncRNA significantly regulates gene expression in both nucleus and cytoplasm [
By using ssGSEA on c6 gene sets, we found that RP11-357H14.17 was associated with enhanced ATF2 signaling. Activated transcription factor 2 (ATF2) is a member of the activated protein 1 (AP-1) transcription factor family that contains DNA-binding proteins in basic Leucine Zipper (bZIP) [
By using ssGSEA on c7 gene sets, we found the high expression of RP11-357H14.17 was also associated with Treg cell-related immunosuppression, which can also be the other reason for RP11-357H14.17-related tumorigenesis. Regulatory T cells (Tregs) are a specific subset of T cells whose role is to suppress the immune response, thereby maintaining homeostasis and self-tolerance [
In summary, by using bioinformatic analysis, we found that RP11-357H14.17 was overexpressed within human gastric cancer tissues. High expression of RP11-357H14.17 was associated with higher TMN stage, poor prognosis, and poor differentiation by promoting DNA replication. Deeper analysis revealed that RP11-357H14.17 was associated through ATF2 signaling and enhanced immunosuppression by promoting Treg cell percentage within human GC tissues.
Full name
Homo sapiens RecQ protein-like 4
Homo sapiens ATPase family, AAA domain containing 5
Homo sapiens GINS complex subunit 1 (Psf1 homolog)
Homo sapiens origin recognition complex, subunit 6
Homo sapiens RAD51-associated protein 2 (RAD51AP2)
Homo sapiens essential meiotic endonuclease 1
Homo sapiens cyclin D1 (CCND1)
Homo sapiens DBF4 homolog B (
Homo sapiens minichromosome maintenance complex component 2
Homo sapiens nuclear autoantigenic sperm protein (histone-binding)
Homo sapiens enhancer of zeste homolog 2 (Drosophila) (EZH2)
Homo sapiens proliferating cell nuclear antigen
Homo sapiens cell division cycle 25 homolog A
Homo sapiens keratin 85
Homo sapiens THO complex 6 homolog (Drosophila)
Homo sapiens SWT1 RNA endoribonuclease homolog
Homo sapiens basic leucine zipper transcription factor, ATF-like 2
Homo sapiens forkhead box P3 (FOXP3)
Homo sapiens histone acetyltransferase 1
Homo sapiens NUF2, NDC80 kinetochore complex component, homolog
Homo sapiens M-phase specific PLK1 interacting protein.
The RNA-seq data supporting the findings of this study have been deposited in the Cancer Genome Atlas (TCGA) repository (TCGA-STAD). The other data are included within the article.
The authors declare that they have no conflicts of interest.
Tang Xiaoli, Wang Wenting, and Zhang Meixiang contributed equally to this work.