Effect Of OPTN Gene Expression On Drug Resistance Of Ovarian And Cervical Cancer Cells

Objective: SKOV3 and Hela cell lines were constructed by knocking down,knocking out and over-expressing the OPTN gene to study the sensitivity of the OPTN gene to SKOV3 and Hela cell anticancer drugs DDP.Method:Design sh RNA,sg RNA,LV-OPTN sequences for OPTN genes.Construction of a lentiviral expression vector.Transfect 293 T cells to obtain lentiviral supernatant,and infect SKOV3 and Hela cells.Screening for stable knockdown,knockout,and overexpression of SKOV3 and Hela cell lines.RT-PCR detection of OPTN gene expression before and after knockdown and overexpression.Detection of genomic DNA at OPTN gene knockout by PCR.Western blot detection of OPTN protein expression in SKOV3 and Hela cells that knocked down,knocked out,and overexpressed the OPTN gene.Effect of DDP on the proliferation of SKOV3 and Hela cells with knockdown,knockout and overexpression of OPTN by MTT assay.Effects of OPTN expression on apoptosis of SKOV3 and Hela cells by flow cytometry.Detection of caspase3,Bax,Bcl-2,and PI3K-AKT-m TOR signaling pathways and resistance-related genes MDR1,ERCC1,and BRCA1 by q RT-PCR.Western blot was used to detect the expression of apoptosis-related genes caspase3,Bax,Bcl-2,and PI3K-AKT-m TOR signaling pathways,as well as the expression of resistance-related genes MDR1,ERCC1,and BRCA1.Result: 1.Successfully constructed SKOV3 and Hela cell lines with knockout,knockout,and overexpression of OPTN gene.2.The MTT results showed that the cell proliferation rate of the knockdown and knockout groups increased compared with the control group,and the cell proliferation rate of the overexpression group decreased compared with the control group.3.Flow cytometry results showed that the apoptotic rate of the knockdown and knockout groups decreased compared with the control group,and the apoptosis rate of the overexpression group increased compared with the control group.4.The q RT-PCR results showed that the expression levels of caspase3 and Bax genes in the knockdown and knockout groups were decreased compared to the control group,and the expression levels of Bcl-2,PI3 K,AKT,m TOR,MDR1,ERCC1,and BRCA1 genes were compared with those in the control group.The expression levels of caspase3 and Bax genes in the overexpression group were increased compared to the control group,and the expression levels of Bcl-2,PI3 K,AKT,m TOR,MDR1,ERCC1,and BRCA1 genes were decreased compared to the control group.5.Western blot results showed that the protein expressions of Cleavedcaspase3,Procaspase3,and Bax in the knockdown and knockout groups decreased compared to the control group,and the proteins of Bcl-2,PI3K-AKT-m TOR signaling pathway related proteins,MDR1,ERCC1,and BRCA1 proteins Increased expression compared to control.The protein expression levels of Cleavedcaspase3,Procaspase3,and Bax in the overexpression group increased compared to the control group,while the protein expression levels of Bcl-2,PI3K-AKT-m TOR signaling pathway related proteins,MDR1,ERCC1,and BRCA1 decreased compared to the control group.Conclusion:The expression of OPTN gene promotes the apoptosis of SKOV3 and Hela cells and reduces the sensitivity of SKOV3 and Hela cells to the anti-cancer drug DDP.The possible mechanism is to regulate the expression of Bax / Bcl-2 apoptotic genes by regulating the PI3K-AKT-m TOR signaling pathway.The expression of OPTN gene also down-regulates the expression of MDR1,ERCC1,BRCA1,and other resistance-related genes,thereby promoting the decline of cell resistance.