Hsa-miR-135b-5p Regulates The Proliferation And Apoptosis Of Gastric Cancer SGC-7901 Cells Via Suppressing ZNRF3

Objective: To clarify the effects of hsa-miR-135b-5p on the proliferation and apoptosis of gastric cancer(GC)SGC-7901 cells,and reveal the mechanism of hsa-miR-135b-5p targeting ZNRF3 to influence GC progress.Methods: 1.qRT-PCR was used to detect the expression of hsa-miR-135b-5p in tissues(including GC tissues and pan-cancer tissues)and cells(including normal gastric epithelial cell line GES-1,GC cell lines SGC-7901 and MGC-803).2.Bioinformatics analysis:(1)hsa-miR-135b-5p targeted m RNA was predicted using the online prediction sites,including Target Scan,miRDB and DIANA-micro T.(2)GO annotation and KEGG enrichment were applied to analyze the biological functions and signaling pathways of hsa-miR-135b-5p targeted genes.3.ZNRF3-wt and ZNRF3-mut vector were co-transfected with hsa-miR-135b-5p mimic into 293 T cells,respectively,and the relationship between ZNRF3 and hsa-miR-135b-5p was analyzed through the dual-luciferase reporter assay.4.hsamiR-135b-5p mimic,hsa-miR-135b-5p inhibitor and corresponding negative control(NC)were respectively transfected into SGC-7901 cells,then their effects on hsa-miR-135b-5p and ZNRF3 expression was confirmed by q RT-PCR.5.hsa-miR-135b-5p inhibitor and ZNRF3 si RNA were co-transfected into SGC-7901 cells,which were grouped as follows:hsa-miR-135b-5p inhibitor +ZNRF3 si RNA,hsa-miR-135b-5p inhibitor+si RNA NC,inhibitor NC+ ZNRF3 si RNA,inhibitor NC+ si RNA NC,then the CCK8 and Annexin V-APC/PI experiments were applied to detect SGC-7901 cell proliferation and apoptosis,respectively.Results: 1.Among 5 pairs of GC tissues,the average expression of hsa-miR-135b-5p in GC tissues was significantly higher than that in adjacent normal tissues(p<0.01).Compared with normal gastric epithelial cell line GES-1,hsa-miR-135b-5p was highly expressed in GC cell lines SGC-7901 and MGC-803,with higher expression level in SGC-7901 cells(p<0.001).2.Bioinformatics analysis showed that ZNRF3 is one of the targeted gene of hsa-miR-135b-5p,and 373 target genes of hsa-miR-135b-5p can be predicted in Target Scan,miRDB and DIANA-micro T;Meanwhile,GO annotation and KEGG analysis showed that these target genes can be involved in cancer related biological processes and signaling pathways,including cell apoptosis,protein phosphorylation and RNA transcription processes,as well as the Wnt,Hippo and c AMP signaling pathways,and so on.3.The dual-luciferase reporter assay confirmed that the overexpression of hsa-miR-135b-5p in 293 T cells suppresses the luciferase activity of wild-type ZNRF3 vector(p<0.01),while has no effect on mutant ZNRF3 vector,indicating that hsa-miR-135b-5p can directly bind with the 3’-UTR of ZNRF3.4.q RT-PCR indicated that hsamiR-135b-5p up-regulation suppresses the level of ZNRF3 in SGC-7901,while hsa-miR-135b-5p down-regulation exert a promote effect in the expression of ZNRF3(p<0.01).5.In CCK8 experiment,hsa-miR-135b-5p down-regulation inhibited the proliferation of SGC-7901 cells,while the suppression of ZNRF3 rescued the adverse effect of down-regulated hsa-miR-135b-5p in SGC-7901 proliferation(p<0.05).Apoptosis experiment showed that the apoptosis-promoting effect of hsa-miR-135b-5p downregulation in SGC-7901 cells can be relieved by the suppression of ZNRF3(p<0.001).Conclusion: 1.hsa-miR-135b-5p is highly expressed in GC tissues and cells.2.ZNRF3 can be directly targeted by hsa-miR-135b-5p.3.hsamiR-135b-5p is capable to exert proliferation-promoting and antiapoptosis effects on GC cells by directly suppressing the expression of antioncogene ZNRF3.