BRCA1 Promotes Cisplatin Resistance In Ovarian Cancer Stem Cells Through Regulating Autophagy And Apoptosis

Objective: In gynecological malignancies,the incidence of ovarian cancer(OC)is second only to cervical cancer and endometrial cancer,but mortality ranks first.Because of the anatomical location of the ovaries and atypical clinical symptoms,ovarian cancer lacks reliable early detection methods,and 70% of patients are already in advanced stages of the disease when they are discovered and diagnosed.At present,effective targeted therapy for ovarian cancer is still an urgent question for gynecological clinicians.The research on drug resistance mechanism has been the focus of research in recent years.This study focused on the relationship between BRCA1,autophagy and cisplatin resistance in ovarian cancer,the changes of autophagy related gene and protein expression in chemotherapy resistant ovarian cancer stem cells(EOCSCs)and the mechanism of autophagy and drug resistance after regulating BRCA1 expression,thereby enriching the etiology of ovarian cancer,providing new ideas for the treatment of this gynecological disease with high incidence,high malignancy and high mortality.Methods: 1.We used Realtime PCR and Western Blot to detect the expression of BRCA1 in human ovarian cancer cisplatin sensitive tissue and cisplatin resistant tissue samples,combining with the information of clinical pathological parameters analysis.We used Realtime PCR to detect the expression of BECN1,ATG5,ATG7,ABCB1,and ABCG2 Mrna in chemosensitive and chemoresistant ovarian cancer tissues.We used Western Blot to detect the protein levels of Beclin-1,ATG5,ATG7,LC3-II/I,POU5F1 and NANOG in chemosensitive and chemoresistant ovarian cancer tissues;we analyzed the correlation between BRCA1 and drug resistance related gene expression in ovarian cancer tissue samples.2.We cultured the human ovarian cancer cell line SKOV3 and enriched SKOV3-EOCSCs by serum-free suspension sphere method.We used flow cytometry to detect the expression of surface stemness markers CD44 and CD133 in EOCSCs.We used Realtime PCR to detect the expression of stemness genes POU5F1,NANOG,CD44 and drug resistance gene ABCG2.We used Western Blot to detect the expression of stemness factors POU5F1 and NANOG.We examined the cell viability and proliferative activity of EOCSCs by CCK-8 and Ed U assays.We used Realtime PCR and Western Blot to detect the expression of BRCA1 in EOCSCs.The expression of resistance related proteins P-gp, ABCG2 and LC3-II/I ratio were detected by Western Blot.We detected the autophagic flow intensity by m RFP-GFP-LC3 double-labeled adenovirus transfection.3.We constructed a BRCA1 overexpression vector,transfected it into EOCSCs and verified transfection efficiency by Western Blot.We used Western Blot to detect autophagy related factors ATG5,ATG7,Beclin-1 expression,LC3-II/I ratio and expression of resistance related proteins P-gp,ABCG2,TP53-BP1.We used Western Blot to detect the expression of stemness factors POU5F1 and NANOG.We detected the autophagic flow intensity by m RFP-GFP-LC3 double-labeled adenovirus transfection.We used flow cytometry to detect the level of apoptosis in cells after cisplatin treatment.We transfected the BRCA1 overexpression plasmid into differentially adherent human ovarian cancer cell line SKOV3 and used flow cytometry to detect the expression of the stemness factor CD44.We constructed si-BECN1 and si-ATG5 to knock down autophagy regulatory gene and autophagy related gene.We transfected each group of si RNA into EOCSCs and verified the transfection efficiency by Realtime PCR and Western Blot.We used flow cytometry to detect the level of apoptosis in each group of cells after cisplatin treatment.4.We constructed a silencing BRCA1 lentiviral vector,transfected the lentivirus into EOCSCs,verified the transfection efficiency by Western Blot and observed the transfection by fluorescence microscopy.Autophagy activation assay: 2.5?M Torkinib(PP242)for 24 h,100 n M Rapamycin for 36 h,after autophagy was activated,cisplatin intervention was initiated.Autophagy inhibition assay: 100 ?M Chloroquine for 48 h,100 m M 3-methyladenosine for 24 h,after autophagy was inhibited,cisplatin intervention was initiated.We regulated autophagy activity in EOCSCs with different BRCA1 expression levels,and measured cell viability and proliferation activity of each group by CCK-8 method and Ed U assay.We used flow cytometry to detect the cell cycle of each group of cells.Finally,we used Western Blot to detect the changes of the expression of BRCA1,Beclin-1,POU5F1,NANOG,ABCG2,GSTP1,P-gp and LC3-II/I ratio in each group of EOCSCs.Results: 1.The expression of BRCA1 was significantly up-regulated in chemoresistant tissues of human ovarian cancer.The m RNA levels of BECN1,ATG5,ATG7,ABCG2 and ABCB1 were significantly higher in chemoresistant ovarian cancer tissues than in chemosensitive ovarian cancer tissues.The protein expression levels of Beclin-1,ATG5, POU5F1 and NANOG were significantly higher in chemoresistant ovarian cancer tissues than in chemosensitive ovarian cancer tissues,LC3-II/I ratio was significantly increased,the increase of ATG7 expression was not significant,and the change of p62 expression level had no statistically significant.There was a positive correlation between BRCA1 and ABCG2 gene expression levels in ovarian cancer tissues.2.We successfully isolated and enriched EOCSCs from human ovarian cancer cell line SKOV3 by serum-free suspension sphere method.Compared with SKOV3 cell line,EOCSCs possess stronger cell viability and proliferation ability,higher expression of stemness factors CD44,CD133,POU5F1,NANOG,higher expression of resistance related proteins P-gp,ABCG2 and stronger drug resistance,as well as higher expression of BRCA1 and stronger autophagy flux intensity.3.Using EOCSCs as a tool cell,overexpression of BRCA1 significantly promoted the occurrence and development of autophagy.ATG5 and Beclin-1 protein levels were significantly increased,the change of ATG7 was not significant,autophagic flow was significantly enhanced.Overexpression of BRCA1 up-regulated the expression of drug related proteins P-gp and ABCG2,and the regulation of TP53-BP1 was not obvious.With the increase of BRCA1 expression and autophagy levels,the expression of stemness marker POU5F1 and NANOG was significantly increased.After upregulation of BRCA1 in differentially adherent SKOV3 cells,the expression of stemness surface marker CD44 was significantly increased.Under the action of cisplatin,overexpression of BRCA1 can inhibit the apoptosis of EOCSCs,and knockdown of the autophagy regulatory gene BECN1 and autophagy related gene ATG5 can promote apoptosis.4.Silencing BRCA1 significantly reduced the cell viability of EOCSCs,and the cell cycle showed a G2/M phase arrest.After activation of autophagy,the cell viability increased significantly,and the degree of G2/M phase arrest was alleviated.After inhibiting autophagy activity,cell viability was significantly attenuated,G2/M phase blockade increased,and obvious apoptotic peak appeared.Silencing BRCA1 inhibits the transformation of LC3-I to LC3-II by inhibiting the expression of Beclin-1,inhibits the development of autophagy,inhibits the expression of P-gp,reduces the drug resistance of EOCSCs,and plays a negative regulatory role in the transformation of stem cell characteristics of ovarian cancer cells.BRCA1 expression is reduced after activation of autophagy.Conclusion: 1.BRCA1 is endogenously expressed in chemoresistant ovarian cancer tissues and EOCSCs,BRCA1 is highly expressed,and chemoresistant ovarian cancer tissues possess higher levels of basal autophagy.2.EOCSCs can be separated and enriched from human ovarian cancer cell line SKOV3 by serum-free suspension sphere method,which can express pluripotent stemness surface markers CD44,CD133 and stemness factors POU5F1,NANOG,higher cell viability and proliferative capacity,higher autophagy levels and drug resistance compared with parental cells.3.BRCA1 can positively regulate the development of autophagy,enhance the cell viability of EOCSCs and promote proliferation,inhibit cell apoptosis,alleviate cell cycle G2/M phase arrest,enhance drug resistance and promote stem cell transformation.Activation of autophagy can reduce the expression of BRCA1 to a certain extent,and there is a mutual regulation between the two.