Experimental Study On The Role Of MicroRNA-22 In Regulating Autophagy/Proliferation Of Osteosarcoma Cells In Chemosensitivity To Cisplatin

Objective:To investigate the proliferation of osteosarcoma(OS)cells and the ro le of MTDH-mediated autophagy in the chemosensitivity of cisplatin,and to fu rther study the mechanism of microRNA-22 regulation,so as to provide new i deas for targeted therapy of osteosarcoma.Method: The osteoblastoma cell line MG-63 was cultured in vitro,and the cul tured cells were divided into ctrl group,NC group,cisplatin group and cisplati n+miR-22 group.MTT assay and cell cloning assay were used to detect the pr oliferation of MG-63 cells at 6,12,and 24 h,respectively.The degree of auto phagy in MG-63 cells was detected by MDC staining.The LC3 flow assay wa s used to detect the changes of autophagy-related protein LC3 expression.The effect of combined treatment of cisplatin and miR-22 on autophagy of osteosar coma cells by transmission electron microscopy(TEM).The mRNA expression of Beclin1,MAP1LC3,ATG5 and MTDH was detected by quantitative PCR.The protein expression of Beclin1,MAP1LC3,ATG5 and MTDH was detected by Western blotting.The Luciferase assay(luciferase)detects whether the auto phagy gene MTDH is a direct target of miR-22,and the expression of MTDH is detected by Western blotting and quantitative PCR when miR-22 is overexp ressed and interfered.Results:(1)The results of MTT assay showed that cisplatin inhibited the proli feration of MG-63 cells(0.723±0.017),while the addition of miR-22 and cispl atin inhibited the proliferation of MG-63 cells(0.515±0.012).And with the pr olongation of time,the proliferation ability of MG-63 cells in cisplatin+miR-22 group also gradually decreased.Cell cloning experiments showed that compare d with the ctrl group(174.7±19.85)and the NC group(186.7±5.033),the cloning ability of MG-63 cells in the cisplatin group was decreased(129.7±4.163),when miR-22 was transfected into MG-63.When cells were used,the cloni ng ability of MG-63 cells was further reduced(101.0±10.58).(2)MDC staini ng results indicated that cisplatin promoted autophagy expression in MG-63 cell s,while miR-22 inhibited autophagy induced by cisplatin.The LC3 flow assay showed that compared with the control group(5.673±0.145),cisplatin promot ed the expression of autophagy-associated protein LC3 in MG-63 cells(15.23±0.153),while miR-22 inhibited MG-63 cells.The expression of LC3 was inc reased by cisplatin(12.56±0.306,P<0.05).TEM results showed that autophagi c lysosomes existed in each group of cells.Autophagosomes gradually produce d over time,and cisplatin+miR-22 co-processing group had autophagic treatmen t compared with cisplatin alone.Reduced.(3)Western blotting and quantitative PCR showed that cisplatin promoted the expression of autophagy-related genes Beclin1,MAP1LC3,ATG5 and MTDH in MG-63 cells.Overexpression of mi R-22 inhibited MG-63 cells.Phage related gene expression.(4)The Luciferase assay showed that miR-22 has two binding sites with the MTDH 3’-UTR.In MG-63 cells,miR-22 overexpression inhibited MTDH expression,whereas when miR-22 expression was down-regulated,MTDH expression was up-regulated.Conclusion: Autophagy and proliferation are associated with cisplatin chemothe rapy sensitivity of osteosarcoma.miRNA-22 further inhibits autophagy by regul ating autophagy-related genes,thereby increasing the sensitivity of cisplatin che motherapy and further resisting the resistance of cisplatin chemotherapy to oste osarcoma.These findings suggest that the miR-22/MTDH/autophagy regulatory pathway plays a key role in chemoresistance in osteosarcoma.