Vitro Mechanism Of MiRNA-21-mediated Autophagy Inhibition In Cisplatin Resistance Of Gastric Cancer

Objective: To validate the inhibitory effect of micro RNA-21(miR-21)on autophagy mediation in gastric cancer resistant cell lines,and to further explore the potential mechanism of miR-21-mediated gastric cancer resistance to cisplatin.Methods:(1)Acquisition and verification of cisplatin-resistant gastric cancer cell lines.The gastric cancer cell line SGC7901,which is more resistant to cisplatin than the parental cells,was selected to induce cell resistance by long-term increasing cisplatin concentration.The half-inhibitory concentration(IC50)of the cell line was determined by MTT assay,and the drug-resistant cells were finally obtained.Strain,called SGC7901/DDP,and obtained the optimal concentration of drug resistance in gastric cancer cell lines.Flow cytometry was used to measure the apoptosis rate of this cell line in different concentrations of cisplatin,and the validity of drug-resistant cell lines was verified.(2)Analysis of the expression of PI3K/Akt/m TOR signaling pathway in cisplatin-resistant gastric cancer cell lines.The expression of PI3 K,p Akt,m TOR in the PI3K/Akt/m TOR signaling pathway and the expression of the key regulator of autophagy Beclin-1 in the cisplatin-resistant cell line were studied by using the parental gastric cancer cell line as control.At the same time,the expression of p Akt,m TOR and the downstream regulatory factor Beclin-1in different cisplatin concentrations were studied to investigate the effect of cisplatin on PI3K/Akt/m TOR signaling pathway.(3)Analysis of autophagy activity in cisplatin-resistant gastric cancer cell lines.The drug-resistant cell line was used as the research object.The acridine orange staining was used to observe the staining by fluorescence microscopy.The ratio of LC3-II/LC3-I and the expression of P62 were detected by Western Blot method.The difference in autophagy activity of parental cell lines and the effect of different concentrations of cisplatin on autophagy in cell lines.The autophagy inhibitor3-methyladenine(3-MA)and the inducer rapamycin(RAP)were used to change the autophagy activity in the cells,and the sensitivity of the cell line to cisplatin was observed.(4)Analysis of the relationship between miR-21 and autophagy activity.The cisplatin-resistant human gastric cancer cell line was used as a research object,and the expression level of miRNA-21 in the cell strain was regulated by liposome transfection using miRNA-21 mimics and inhibitors.Furthermore,the parents and drug-resistant cells were stained with acridine orange.The staining was observed under fluorescence microscope.The ratio of Beclin-1 expression and LC3II/LC3 I in the two cell lines was detected by Western blot.The changes of intracellular autophagy were compared.(5)Analysis of the role of miR-21-mediated autophagy regulation in drug-resistant cells.MTT assay was used to detect the survival and death of drug-resistant cell lines transfected with miR-21 mimics and inhibitors.In addition,autophagy inhibitor 3-MA was added,and MTT assay was used to detect autophagy.The survival and death of the cells were evaluated to assess the regulation of miR-21-induced autophagy on cell survival and death.Results:(1)DDP-resistant GC cells are insensitive to DDP.(2)PI3K/Akt/m TOR pathway has been activated.(3)Inhibition of autophagy contributes to DDP resistance in GC cells.(4)miR-21 participates in DDP resistance and affects DDP-induced autophagy in GC cells.(5)miR-21 alters the effects of DDP on GC cells.Conclusion: miR-21 mediates autophagy inhibition by activating the PI3K/Akt/m TOR pathway,leading to resistance of gastric cancer cells to cisplatin.