Screening Of Aberrantly Expressed MicroRNAs In Gastric Cancer And MiR-155-5P Is Invovled In The Tumorigenesis Behavior Of Gastric Cancer

Objectives:To screen the down-regulated microRNAs in gastric cancer and to explore the relationship of gastric cancer tissues with clinical pathological features; and furtherly, this study was designed to explore proliferation, invasion and metastasis, apotosis of gastric cancer cell lines MKN-45, BGC-823and SGC-7901when overexpressed miR-155-5p in these cell lines. In addition, the target genes of miR-155-5p were screened by mRNA microarray analysis together with bioinformatics prediction analysis, and were verified by the method of qRT-PCR and WESTERN BLOT. And as a result, this study aimed to seek and find microRNAs with obvious clinical significance and the ability to change the biological behavior of gastric cancer cells, and to identify and provide some specific microRNA molecular markers for gasrric cancer diagnosis, treatment and prognostic analysis.Methods:The Human Cancer Pathway Finder miRNA PCR Array was applied to compare7gastric cancer cell lines AGS, SGC-7901, MKN-45, MKN-28, MGC-803, BGC-823, HGC-27with a immortalized normal gastric cell line, GES-1in cancer pathway related to miRNA expression profile, and followed by qRT-PCR verification, the clinical significance of down-regulated miRNAs and the Enriched KEGG pathways and GO terms of their target genes were analyzed. After screening above, Hsa-mir-155-5p was overexpressed in gastric cancer cells MKN-45, BGC-823and SGC-7901cell to observe proliferation, invasion and metastasis, apoptosis; in the other hand, combined with bioinformatics prediction of target genes, mRNA microarray was employed to screen aberrantly expressed target genes, and to validate aberrantly expressed target genes with qRT-PCR and WESTERN BLOT experiment. Results:(l)The Human Cancer Pathway Finder miRNA PCR Array combined with qRT-PCR verification was applied to compare7gastric cancer cell lines AGS, SGC-7901, MKN-45, MKN-28, MGC-803, BGC-823, HGC-27with a immortalized normal gastric cell line, GES-1to screen aberrantly expressed miRNAs, the results indicated that38miRNAs were up-regulated in7gastric cancer cell lines compared with GES-1cell line, which were mir-1, mir-7-5p, mir-10a-5p, mir-10b-5p, let-7b-5p, let-7g-5p, let-7c, let-7f-5p, mir-17-5p, mir-20a-5p, mir-20b-5p, mir-23b-3p, mir-25-3p, mir-27a-3p, mir-27b-3p, mir-34a, mir-92a-3p, mir-96-5p, mir-98, mir-106a-5p, mir-125a-5p, mir-133b, mir-135-5p, mir-136-5p, mir-143-3p, mir-148a-3p, mir-181d, mir-183-5p, mir-191-5p, mir-193-3p, mir-193a-5p, mir-196a-5p, mir-200c-3p, mir-203a, mir-215, mir-301a-3p, mir-214-3p, mir-373-3p and4miRNAs were down-regulated in7gastric cancer cell lines compared with GES-1cell line, which were mir-124-3p, mir-146a-5p, mir-155-5p and mir-335-5p. Except mir-25-3p, the expressions of most miRNAs were consistent with expression of that by Pathway Finder miRNA PCR Array expression profiles.(2) In the light of confusing reports about the expressions of mir-124-3p, mir-146a-5p, mir-155-5p and mir-335-5p in gastric tissues, the levels of these4miRNAs in58cancerous and corresponding non-cancerous tissues were detected by qRT-PCR method. Results showed that the numbers of mir-124-3p in high-expression group (T/N＞2) and low-expression group (T/N＜0.5) amounts to13and45respectively, according to the median cancer (T)/noncancerous (N) tissue ratio of mir-124-3p expression (Table1). The numbers of mir-146a-5p in high-expression group (T/N＞2and low-expression group (T/N＜0.5) amounts to35and20respectively, according to the median cancer (T)/noncancerous (N) tissue ratio of mir-146a-5p expression(Table2), the numbers of mir-155-5p in high-expression group (T/N＞2) and low-expression group (T/N＜0.5) amounts to26and26respectively, according to the median cancer (T)/noncancerous (N) tissue ratio of mir-155-5p expression, and the numbers of mir-335-5p in high-expression group (T/N＞2) and low-expression group (T/N＜0.5) amounts to30and25respectively, according to the median cancer (T)/noncancerous (N) tissue ratio of mir-335-5p expression. Clinical significances of4miRNAs including mir-124-3p, mir-146a-5p, mir-155-5p and mir-335-5p in gastric cancer tissue comparing with adjacent non tumor tissues of58patients indicated that the low-expression group of mir-124-3p, mir-146a-5p, mir-155-5p and mir-335-5p showed more extensive lymph node metastasis, lymphatic invasion, venous invasion, high stage borrmann type, lymphatic invasion and poorly differentiation than that of the high-expression groups respectively. Enriched KEGG pathway analyses showed that most of the targeted genes of those4miRNAs concentrated on37signaling pathways, and involved in the same pathways related to cancer, such as apoptosis, toll-like receptor, chemokine, erbb signaling pathway, epithelial cell signaling in helicobacter pylori infection, cell cycle, NOD-like receptor, mTOR, MAPK and wnt signaling pathways. Enriched GO terms showed that targeted genes of those4miRNAs concentrated on339terms,24of339terms are associated with tumor proliferation, angiogenesis, cell-matrix adhesion, cell cycle, apoptosis and endoplasmic reticulum unfolded protein response.(3) Hsa-mir-155-5p mimics and Hsa-mir-155-5p micro-up lentiviral vector were transfected into gastric cancer cells lines MKN-45, BGC-823and SGC-790, and then, proliferation were detected by MTT experiments, invasion and metastasis were detected transwell chamber, cell cycle and apoptosis were detected by flow cytometry. The results showed that:overexpression of Hsa-mir-155-5p could inhibit proliferation of gastric cancer cell lines MKN-45, BGC-823and SGC-7901in vitro, and could inhibit invasion and metastasis of gastric cancer cell lines MKN-45, BGC-823and SGC-7901in vitro; in addition, overexpression of Hsa-mir-155-5p could induce apoptosis in gastric cancer cell lines MKN-45, BGC-823and SGC-7901in vitro.(4)The overlap genes of aberrantly expressed genes screened by mRNA microarray and predicted target genes by website database with miRWALK were calculated for further intersection analysis, The results showed that:the overlap genes of aberrantly expressed in MKN-45cells amounts to790; the overlap genes of aberrantly expressed in BGC-823cells amounts to1384; and then, SMAD1, STAT1, CAB39, CXCR4and CA9expression were validated down-regulated by qRT-PCR after overexpression of Hsa-mir-155-5p.in both gastric cancer cell lines MKN-45, BGC-823. At the same time, SMAD1, STAT1and CAB39expression were validated down-regulated by western blotting after overexpression of Hsa-mir-155-5p.in both gastric cancer cell lines MKN-45and BGC-823.Conclusion:(1) The Human Cancer Pathway Finder miRNA PCR Array combined with qRT-PCR verification discovered that38miRNAs were up-regulated in7gastric cancer cell lines compared with GES-1cell line, and4miRNAs were down-regulated in7gastric cancer cell lines compared with GES-1cell line, which provided the bases for further clinical significance analysis and functional study.(2) Clinical significances of4miRNAs including mir-124-3p, mir-146a-5p, mir-155-5p and mir-335-5p in gastric cancer tissue comparing with adjacent non tumor tissues of58patients indicated that the low-expression group of mir-124-3p, mir-146a-5p, mir-155-5p and mir-335-5p showed more extensive lymph node metastasis, lymphatic invasion, venous invasion, high stage borrmann type, lymphatic invasion and poorly differentiation than that of the high-expression groups respectively. Enriched KEGG pathway analyses showed that most of the targeted genes of4miRNAs concentrated on pathways associated with cancer which include cell cycle apoptosis, proliferation, invasion and metastasis.(3) Overexpression of Hsa-mir-155-5p could inhibit proliferation of gastric cancer cell lines MKN-45, BGC-823and SGC-7901in vitro, and could inhibit invasion and metastasis of gastric cancer cell lines MKN-45, BGC-823and SGC-7901in vitro; in addition, overexpression of Hsa-mir-155-5p could induce apoptosis in gastric cancer cell lines MKN-45, BGC-823and SGC-7901in vitro.(4)Based on the overlap genes of aberrantly expressed genes screened by mRNA microarray and predicted target genes by online miRWALK database, the results showed that qRT-PCR and WESTERN BLOT verification confirmed SMAD1, STAT1, CAB39, CXCR4and CA9were downregulated in gastric cancer cell lines MKN-45and BGC-823, which indicated that Hsa-mir-155-5p regulate the biological behavior of gastric cancer mediated by its target genes SMAD1, STAT1, CAB39, CXCR4and CA9.