The Study Of Knocking Down Autophagic Receptor P62 On Reducing The Drug Resistance And Molecular Mechanism Of Ovarian Cancer Cells

Objective: Study on the effect on the expression of autophagic receptor p62 gene on the susceptibility of ovarian cancer resistant cell lines.Methods:The experiment was divided into three groups:control group,non-specific transfection group and specific transfection group.1.The p62 gene was knocked down by si RNA to constructed the p62 sh RNA expression vector:p LVX-sh RNA1-p62 si RNA,and established a SKOV3 / DDP cell model of silenced p62 gene.2.The effects of DDP on the proliferation of three groups of cells were detected by MTT assay.3.Detection of autophagy in three groups of cells after interfering p62 gene expression by immunofluorescence.4.Flow cytometry was used to detect the effects of three groups of cells on apoptosis and cell cycle after DDP treatment for 48 h.5.The changes of the expression of PI3 K,AKT and m TOR genes in three groups of cells after DDP treatment for 24,48 and 72 h were detected by RT-PCR.6.After the treatment of DDP for 24,48 and 72 h,the protein expression PI3 K,AKT,P-AKT,m TOR,p-m TOR and LC3 in three groups of cells was texted by Western blot.Results: 1.Successful build a cell model of silenced p62 gene.2.The results of MTT assay showed that DDP had a certain inhibitory effect on the proliferation of three groups of cells of the treatment of DDP.3.The results of immunofluorescence showed that the autophagy increased after DDP action,and the level of autophagy was decreased by knocking down the cells of the p62 gene.4.After the treatment of DDP for 24 h,48 h and 72 h.the results of PCR showed that the levels of PI3 K,AKT and m TOR gene were declined obviously,compared with the control group,the level of gene expression in the specific transfection group decreased more obviously(P<0.05,P<0.01).5.The results of western blot showed that the expression of PI3 K,AKT,p-AKT,m TOR and p-m TOR decreased significantly after cisplatin treatment.Compared with non-specific transfection group,the expression of LC3 in the specific transfection ?group was decreased.Compared with the control group,the level of gene expression in the specific transfection group decreased more significantly(P<0.05,P<0.01).Conclusion: 1.The successful construction of the cell model of the silent p62 gene can be used for the further experiment.2.Silencing of p62 gene can enhance the effect of DDP on the proliferation inhibition of ovarian cancer.3.Silencing of p62 gene can inhibit autophagy.4.Silencing the p62 gene can promote DDP-induced apoptosis in ovarian cancer.5.After DDP acts on the cell model of the silencing of the p62 gene,the expression level of PI3 K,AKT and m TOR were decreased,and the protein expression of PI3 K,AKT,p-AKT,m TOR and p-m TOR was decreased,too.It was suggested that the silencing of P62 gene can increase the sensitivity of ovarian cancer cells to the DDP,maybe relating to the PI3K/AKT/m TOR pathway.