Function And Mechanism Of MiR-362-5p In Cisplatin Resistance Of Gastric Cancer Cells

Aim The present study aimed to identify function and mechanism of miR-362-5p in cisplatin resistance of gastric cancer cells.Methods Differentially expressed miRNAs were identified between the human cisplatin-sensitive gastric cancer cell line SGC7901 and the corresponding cisplatin-resistant cell line SGC7901/DDP by miRNA microarray analysis.The expression level of miR-362-5p was detected by quantitative real-time PCR(RT-PCR).Stable cell lines,knockdown or overexpression of miR-362-5p,were established by lentiviral transfection.Cell viability was examined by CCK-8 assay.The rates of cell apoptosis were determined by flow cytometry.The targets of mir-362 -5p could be predicted in gastric cancer through database micro RNA.org and miRDB.Knockdown of SUZ12 was complete by si RNA.The protein expression levels of SUZ12,NF-κB/p65,caspase-3 and cleaved-PARP were detected by western blotting.Results Results of miRNA microarray analysis shown that the expression level of miR-362-5p was found to be reduced in SGC7901/DDP cells compared with SGC7901 cells.Moreover,RT-PCR also confirmed that the expression of mir-362 -5p in SGC7901/DDP cells was lower than in SGC7901 cells.The expression of mir-362 -5p in SGC7901/DDP cells transfected with miR-362-5p mimic(SGC7901/DDP-miR-362-5p-OE)was higher than in SGC7901/DDP cells transfected with miR-362-5p control(SGC7901/DDP-miR-362-5p-NC)and in SGC7901/DDP cells.Cisplatin treated each group of cells for 48 hours.The IC50 for cisplatin in SGC7901/DDP-miR-362-5p-OE cells was significantly lower than in SGC7901/DDP-NC and SGC7901/DDP cells(IC50,1.609±0.332 vs.5.133±0.569 and 5.161±0.641 μg/m L,P<0.01).Meanwhile,The expression of mir-362 -5p in SGC7901 cells transfected with miR-362-5p inhibitor(SGC7901-miR-362-5p-KD)was lower than in SGC7901 cells transfected with miR-362-5p control(SGC7901-miR-362-5p-NC)and in SGC7901 cells.The IC50 for cisplatin in SGC7901-miR-362-5p-KD cells was distinctly higher than in SGC7901-NC and SGC7901 cells(IC50,0.676±0.042 vs.0.286±0.025 and 0.300±0.009 μg/m L,P<0.01).In addition,upregulation of miR-362-5p significantly increased cisplatin-induced apoptosis,whereas downregulation of miR-362-5p attenuated cisplatin-induced apoptosis.Databases predicted SUZ12 may as a target of miR-362-5p.Moreover,the expression level of SUZ12 protein and m RNA in SGC7901/DDP cells was markedly higher than that in SGC7901 cells.The protein expression of SUZ12 in SGC7901/DDP cells was knockdown studied by SUZ12-si RNA.Results shown that the IC50 for cisplatin in SGC7901/DDP-SUZ12-si RNA cells was clearly lower than that in the SGC7901/DDP-control cells(IC50,2.569±0.479 vs.5.097±0.629 μg/m L,P<0.01).Knockdown of SUZ12 decreased NF-κB/p65 protein levels in SGC7901/DDP cells.Upregulation of miR-362-5p in SGC7901/DDP cells decreased the protein expression level of SUZ12,whereas downregulation of miR-362-5p in SGC7901 cells increased the SUZ12 expression level.Conclusion Dysregulated miR-362-5p targets SUZ12 to reverse the resistance of cisplatin in gastric cancer cells.