HIF-2? Transactivates BCRP Via A Hypoxia-response Element(HRE) And Mediates The Resistance Of Ovarian Cancer Stem Cells To ADR

Objective:In recent years,ovarian cancer is a serious threat to the health of women,and is one of the most difficult gynecological malignant tumors.mortality rate increased year by year,huge obsession for many women.The study found that the main reason for the poor treatment effect of ovarian cancer,poor prognosis and high mortality was the susceptibility to drug resistance during chemotherapy.Along this train of thought,then found the cancer stem cell?cancer stem cells,CSCs?,CSCs is present in the tumor cells to have strong self-renewal,unlimited proliferation and differentiation potential of a small group of special cells,is the root cause of tumorigenesis,metastasis and recurrence of resistant.Therefore,targeting Ovarian cancer stem cells?OCSCs?provides a direction for the study of the drug resistance mechanism of ovarian cancer,which is of great significance for improving the therapeutic effect of ovarian cancer.This article starts with the environmental aspects of the growth of CSCs and finds that the environment in which they live is mainly a niche microenvironment.Under this environment of low oxygen,CSCs are hard to come into contact with chemotherapeutic drugs,thus improving their ability to escape,this is the resistance mechanisms of CSCs.We also found that there are hypoxia-inducible factors?HIFs?,important transcription factors widely present in mammals under hypoxic conditions,and two biologically active functional subunits of HIFs are HIF-1?and HIF-2?.Although they have a highly homologous structure,they have specificity in the activation of transcription factors,tissue expression types,and biological functions mediated during hypoxia.At the same time,it has been reported that HIF-1?and HIF-2?are related to the regulation of stem cell stemness,especially HIF-2?.However,there are few studies on HIF-2?-induced stemness and drug resistance of OCSCs,and their specific mechanism of action It is unclear.Therefore,this article focuses on the regulation mechanism of HIF-2?on the stemness and drug resistance of OCSCs.Methods:The ovarian cancer balloon cells were induced by serum-free non-adherent suspension culture of OVCAR-3 and CAOV-3 ovarian cancer cells.OVCAR-3S and CAOV-3S ovarian cancer balloon cells with OCSCs-like properties were tested for different concentrations of adriamycin?ADR?and mitoxantrone?MX?,paclitaxel?PTX?,etoposide etoposide?VP-16?and Cisplatin Cisplatin?DDP?the changes of drug sensitivity;and the expression of BCRP protein in OVCAR-3S and CAOV-3S and their parental cells were detected by Western Blot.TCGA database was used to analyze the correlation of mRNA expression of HIF-2?and BCRP in 379 cases of ovarian cancer.The expression of these two proteins in 115 cases of ovarian cancer was detected by immunohistochemistry and the correlation was analyzed.Lentivirus transfection and screening were performed to construct OVCAR-3and CAOV-3 stable ovarian cancer cell lines overexpressing HIF-2?,and to stably transfect cells of OVCAR-3S and CAOV-3S silenced by HIF-2?.The expression of HIF-2?and BCRP was detected by qRT-PCR and Western Blot,respectively.Mass spectrometry and flow cytometry were used to examine the efflux ability of cells against doxorubicin.The effects of interference with HIF-2?on the drug sensitivity of ovarian cancer stem cells were investigated.Gene sequence analysis HRE element bound to HIF-2?within 2kb upstream of BCRP gene promoter,luciferase reporter gene assay and chip assay were used to detect the binding of HIF-2?to BCRP through HRE element.qRT-PCR detected changes in BCRP levels after interfering with HIF-2?.The OVCAR-3 MS cells stably transfected with HIF-2?silencing lentivirus were inoculated subcutaneously in nude mice to construct a HIF-2?silenced xenograft model in nude mice.To investigate the effect of HIF-2?silencing on the growth of transplanted tumor in nude mice and its sensitivity to ADR.The expression of HIF-2?and BCRP mRNA was detected by qRT-PCR in all groups.Immunohistochemistry and Western Blot were used to detect the expression of HIF-2?and BCRP in all groups.Mass spectrometry was used to detect the change of ADR content in tumor-bearing mice with silenced HIF-2?.Results:1.Compared with their parental OVCAR-3 and CAOV-3 cells,the IC₅₀0 values of ADR,MX,PTX,VP-16 and DDP were significantly higher in OVCAR-3 S and CAOV-3 S cells,ie,they had obvious resistance to these chemotherapeutic drugs.Among them,OVCAR-3 S and CAOV-3 S were the most resistant to ADR,and the IC₅₀ values were 103.6 and 24.7 times that of OVCAR-3 and CAOV-3 cells?resistance index RI?,respectively.In OVCAR-3 S and CAOV-3 S,the protein expression levels of BCRP were3.61 and 3.35 times that of parental cells OVCAR-3 and CAOV-3,respectively.2,Through TCGA database analysis,it was found that the expression of HIF-2?and BCRP mRNA was positively correlated with ovarian cancer.Immunohistochemistry and analysis showed that the expression of HIF-2?and BCRP protein in ovarian tissue was significantly positively correlated.Lentiviruses transfected shRNA-HIF-2?plasmid,can silence the expression of HIF-2?in OVCAR-3 MS and CAOV-3 MS cells,Compared with the sh-NC group,the protein expression of BCRP in OVCAR-3 S and CAOV-3 S cells transfected with sh-HIF-2?was significantly decreased,and the mean fluorescence intensity?MFI?in cells was significantly increased by flow cytometry.That is,ADR drug accumulation increased significantly;mass spectrometry analysis found that intracellular ADR content increased significantly,that is to reduce the ability of ADR efflux.Compared with NC-cDNA transfection group,the protein expression of BCRP in OVCAR-3 and CAOV-3 cells transfected with HIF-2?-cDNA lentivirus was significantly increased;Flow cytometry showed that the intracellular mean fluorescence intensity?MFI?was significantly reduced,that is,ADR drug accumulation was significantly reduced;mass spectrometry analysis revealed that intracellular ADR was significantly reduced,the efflux ability of ADR was increased.3.Gene sequence analysis revealed that there is a HRE element CACGTG that binds to HIF-2?in the 2-kb region within the 2kb region upstream of the BCRP gene promoter;The enzyme activity of 293T cells and OVCAR-3 cells co-transfected with HIF-2?-cDNA and BCRP promoter wild-type reporter plasmid was significantly increased,but the cell enzyme activity of the co-transfected BCRP promoter mutant reporter plasmid had no significant changes.The mRNA expression of BCRP in OVCAR-3 cells transfected with HIF-2?-cDNA lentivirus was significantly higher than that in NC-cDNA transfection group.Compared with sh-NC group,OVCAR-3 MS and CAOV-3 MS cells transfected with sh-HIF-2?lentivirus had significantly lower mRNA expression levels of BCRP.Conclusion:1,serum-free non-adherent suspension cultured OVCAR-3 S and CAOV-3S were resistant to chemotherapy such as ADR.2.HIF-2?mediates OCSCs resistance and is related to the regulation of BCRP expression and function.3,HIF-2?can bind to the HRE element in the promoter region of BCRP gene in ovarian cancer cells and exert a direct transcriptional activation effect on BCRP.