| Objective: To study differentially expressed micro RNAs(miRNAs)after treatment of gastric cancer cells with tetrandrine(Tet)and explore the function and mechanisms of Tet.The effects of miR-500a-5p on proliferation,cell cycle,apoptosis,migration and invasion of gastric cancer cells were further studied.The candidate target genes of miR-500a-5p were predicted by bioinformatics,combined with high-throughput sequencing of RNA-seq results,and validated by RT-q PCR,aiming at providing reference for the diagnosis and treatment of gastric cancer.Method: High-throughput sequencing technology was used to screen differentially expressed miRNAs in Tet-treated gastric cancer cells HGC-27.After literature search,four tumor-associated miRNAs were screened,and their target genes were predicted by bioinformatics methods.GO analysis and KEGG signal pathway analysis were performed on the target genes.Then,miR-500a-5p with reduced expression level after Tet-treated gastric cancer cells was selected as the research object.MTT assay,flow cytometry and transwell chamber assay were used to detect the effects of miR-500a-5p inhibitor on proliferation,cell cycle,apoptosis,migration and invasion of gastric cancer cells.The target genes were predicted by bioinformatics combined with high-throughput sequencing of RNA-seq results,and were checked by RT-q PCR to verify the regulation of miR-500a-5p on its target genes from m RNA levels.Result:(1)Four tumor-associated miRNAs were selected.The expression levels of miR-500a-5p and miR-122-5p were decreased after Tet treatment,and the expression levels of miR-7641 and miR-149-3p were increased.The target genes of the four miRNAs were predicted using bioinformatics software.The predicted results showed that the numbers of the target genes of miR-500a-5p,miR-122-5p,miR-7641 and miR-149-3p were 1,050,773,1105 and 1758,respectively.Gene function annotation and pathway enrichment analysis of these target genes revealed that the target genes of four miRNAs were involved in tumor-related signaling pathways such as cell proliferation,cell migration,cell cycle,apoptosis and angiogenesis.(2)In HGC-27 and AGS gastric cancer cell lines,after transfection of miR-500a-5p inhibitor for 48 h,the cell viability of miR-500a-5p inhibitor group were significantly reduced than that of inhibitor NC(inhibitor negative control)group;After transfection of miR-500a-5p inhibitor for 48 h and 72 h,the cell cycle of gastric cancer cells were arrested in S phase and the apoptotic rate of gastric cancer cells were increased significantly.After transfection with miR-500a-5p inhibitor,the number of transmembrane cells of gastric cancer cells decreased significantly,and the cell migration and invasion abilities were weakened.(3)Bioinformatics software predicted that miR-500a-5p had 1050 target genes and it had 139 up-regulated genes in RNA-seq.The two were used for intersection analysis and found that there were 8 genes,namely DNAJA1,ABCA1,CBWD6.VPS41,ANO5,ZNF326,PCDH10 and ROR? may be the target genes of miR-500a-5p.Combined with literature reports,5 genes including VPS41,ANO5,ZNF326,PCDH10 and ROR? involved in tumorigenesis were selected for RT-q PCR verification.The results showed that after transfection of miR-500a-5p inhibitor,the m RNA expression levels of VPS41 and ANO5 in miR-500a-5p inhibitor group were significantly up-regulated compared with the inhibitor NC group;ZNF326 m RNA was up-regulated in HGC-27 cells but the expression was significantly down-regulated in AGS cells;the m RNA expression of ROR? was decreased in both cells;the m RNA of PCDH10 was not expressed in the two gastric cancer cells.Conclusions:(1)Bioinformatics analysis:the target genes of miR-500a-5p,miR-122-5p,miR-7641 and miR-149-3p were involved in tumor-associated signaling pathways such as cell proliferation,cell migration,cell cycle,apoptosis and angiogenesis.(2)Inhibiting the expression of miR-500a-5p in gastric cancer cells,could inhibit the proliferation of HGC-27 and AGS cells,arrest the cell cycle of HGC-27 and AGS in S phase,inhibit the migration and invasion of gastric cancer cells,and promote apoptosis of gastric cancer cells.(3)The possible downstream target genes of miR-500a-5p were predicted bioinformatics combined with the results of RNA-seq,including VPS41,ANO5,ZNF326,PCDH10 and ROR?.Among them,VPS41 and ANO5 were verified by RT-q PCR to be consistent with the sequencing results.The regulation functions of miR-500a-5p in gastric cancer cells may be related to the up-regulation of these two genes. |
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