摘要
BACKGROUND Breast cancer is a common malignant tumor that seriously threatens women’s health.Breast cancer stem cell(CSC)-like cell population may be the main factor for breast cancer metastasis.Therefore,targeted therapy for CSCs has great potential significance.Hypoxia-inducible factor is a transcription factor widely expressed in tumors.Studies have shown that down-regulation of the hypoxia signaling pathway inhibits tumor stem cell self-renewal and increases the sensitivity of stem cells to radiotherapy and chemotherapy mediated by hypoxiainducible factor-2α(HIF-2α).However,the specific mechanism remains unclear and further research is necessary.AIM To investigate the effect of HIF-2αdown-regulation on stem cell markers,microsphere formation,and apoptosis in breast cancer cell line MDA-MB-231 under hypoxia and its possible mechanism.METHODS Immunohistochemistry was used to detect the expression of HIF-2αand CD44 in triple-negative breast cancer(TNBC)and non-TNBC tissues.Double-labeling immunofluorescence was applied to detect the co-expression of HIF-2αand CD44 in MDA-MB-231 cells and MCF-7 cells.HIF-2αwas silenced by RNA interference,and the expression of CD44 and transfection efficiency were detected by real-time fluorescent quantitative PCR.Further,flow cytometry,TdT-mediated X-dUTP nick end labeling,and mammosphere formation assays were used to evaluate the effect of HIF-2αon CSCs and apoptosis.The possible mechanisms were analyzed by Western blot.RESULTS The results of immunohistochemistry showed that HIF-2αwas highly expressed in both TNBC and non-TNBC,while the expression of CD44 in different molecular types of breast cancer cells was different.In in vitro experiments,it was found that HIF-2αand CD44 were expressed almost in the same cell.Compared with hypoxia+negative-sequence control,HIF-2αsmall interfering ribonucleic acid transfection can lower the expression of HIF-2αand CD44 mRNA(P<0.05),increase the percentage of apoptotic cells(P<0.05),and resulted in a reduction of CD44+/CD24−population(P<0.05)and mammosphere formation(P<0.05)in hypoxic MDA-MB-231 cells.Western blot analysis revealed that phosphorylated protein-serine-threonine kinase(p-AKT)and phosphorylated mammalian target of rapamycin(p-mTOR)levels in MDA-MB-231 decreased significantly after HIF-2αsilencing(P<0.05).CONCLUSION Down-regulation of HIF-2αexpression can inhibit the stemness of human breast cancer MDA-MB-231 cells and promote apoptosis,and its mechanism may be related to the CD44/phosphoinosmde-3-kinase/AKT/mTOR signaling pathway.
BACKGROUND Breast cancer is a common malignant tumor that seriously threatens women’s health.Breast cancer stem cell(CSC)-like cell population may be the main factor for breast cancer metastasis.Therefore,targeted therapy for CSCs has great potential significance.Hypoxia-inducible factor is a transcription factor widely expressed in tumors.Studies have shown that down-regulation of the hypoxia signaling pathway inhibits tumor stem cell self-renewal and increases the sensitivity of stem cells to radiotherapy and chemotherapy mediated by hypoxiainducible factor-2α(HIF-2α).However,the specific mechanism remains unclear and further research is necessary.AIM To investigate the effect of HIF-2α down-regulation on stem cell markers,microsphere formation,and apoptosis in breast cancer cell line MDA-MB-231 under hypoxia and its possible mechanism.METHODS Immunohistochemistry was used to detect the expression of HIF-2α and CD44 in triple-negative breast cancer(TNBC) and non-TNBC tissues.Double-labeling immunofluorescence was applied to detect the co-expression of HIF-2α and CD44 in MDA-MB-231 cells and MCF-7 cells.HIF-2α was silenced by RNA interference,and the expression of CD44 and transfection efficiency were detected by real-time fluorescent quantitative PCR.Further,flow cytometry,Td T-mediated X-d UTP nick end labeling,and mammosphere formation assays were used to evaluate the effect of HIF-2α on CSCs and apoptosis.The possible mechanisms were analyzed by Western blot.RESULTS The results of immunohistochemistry showed that HIF-2α was highly expressed in both TNBC and non-TNBC,while the expression of CD44 in different molecular types of breast cancer cells was different.In in vitro experiments,it was found that HIF-2α and CD44 were expressed almost in the same cell.Compared with hypoxia + negative-sequence control,HIF-2α small interfering ribonucleic acid transfection can lower the expression of HIF-2α and CD44 m RNA(P < 0.05),increase the percentage of apoptotic cells(P < 0.05),and resulted in a reduction of CD44+/CD24-population(P < 0.05) and mammosphere formation(P < 0.05) in hypoxic MDA-MB-231 cells.Western blot analysis revealed that phosphorylated protein-serine-threonine kinase(p-AKT) and phosphorylated mammalian target of rapamycin(p-m TOR) levels in MDA-MB-231 decreased significantly after HIF-2α silencing(P < 0.05).CONCLUSION Down-regulation of HIF-2α expression can inhibit the stemness of human breast cancer MDA-MB-231 cells and promote apoptosis,and its mechanism may be related to the CD44/phosphoinosmde-3-kinase/AKT/mTOR signaling pathway.
