Aim:ABCB1 is a major player in cancer drug resistance.The purpose of this study was to functionally assess the regulation of ABCB1 activity in a doxorubicin-resistant breast cancer cell line by miR-200c and miR-203.Me...Aim:ABCB1 is a major player in cancer drug resistance.The purpose of this study was to functionally assess the regulation of ABCB1 activity in a doxorubicin-resistant breast cancer cell line by miR-200c and miR-203.Methods:Human breast carcinoma cell lines MCF-7(Doxorubicin-sensitive and not expressing ABCB1)and KCR(Doxorubicin-resistant and expressing ABCB1)were used to evaluate the expression levels of miR-200c and miR-203 by Real-time quantitative PCR(RT-qPCR).The effects of transient ectopic expression of miRNA-200c and miR-203 on the expression of ABCB1 in KCR and MCF-7 cells was verified by RT-qPCR and Western Blot.The extrusion activity of the ABCB1 pump was analyzed by fluorescence microscopy and flow cytometry through fluorescence substrate retention assays(DiOC2)in the presence and absence of the ABCB1 inhibitor verapamil.Results:RT-qPCR results indicated a 100,000-fold increase in ABCB1 mRNA expression levels in KCR cells compared to MCF-7 cells,and is inversely correlated with the expression of miR-203 and miR-200c.The insertion of miR-200c and miR-203 led to a higher retention of DiOC2 within KCR cells,and slightly reduced the protein levels of ABCB1 in KCR cells,although the high initial expression of ABCB1 masked the reduction in protein levels.The increased intracellular accumulation of the fluorescent due DiOC2 in the presence of the ABCB1 inhibitor verapamil correlated with the inhibition caused by miR-203 and miR-200c in transfected cells.Conclusion: The present study demonstrates that miR-200c and miR-203 exert a negative modulating effect on the activity of ABCB1 associated with doxorubicin resistance.展开更多
基金The work received financial support from Centre of Toxicogenomics and Human health and IHMTThis work was supported by Fundação da Ciência e Tecnologia(FCT,Portugal)through grants(UID/BIM/00009/2013),(UID/BIM/00009/2016),(GHTM-UID/Multi/04413/2013).
文摘Aim:ABCB1 is a major player in cancer drug resistance.The purpose of this study was to functionally assess the regulation of ABCB1 activity in a doxorubicin-resistant breast cancer cell line by miR-200c and miR-203.Methods:Human breast carcinoma cell lines MCF-7(Doxorubicin-sensitive and not expressing ABCB1)and KCR(Doxorubicin-resistant and expressing ABCB1)were used to evaluate the expression levels of miR-200c and miR-203 by Real-time quantitative PCR(RT-qPCR).The effects of transient ectopic expression of miRNA-200c and miR-203 on the expression of ABCB1 in KCR and MCF-7 cells was verified by RT-qPCR and Western Blot.The extrusion activity of the ABCB1 pump was analyzed by fluorescence microscopy and flow cytometry through fluorescence substrate retention assays(DiOC2)in the presence and absence of the ABCB1 inhibitor verapamil.Results:RT-qPCR results indicated a 100,000-fold increase in ABCB1 mRNA expression levels in KCR cells compared to MCF-7 cells,and is inversely correlated with the expression of miR-203 and miR-200c.The insertion of miR-200c and miR-203 led to a higher retention of DiOC2 within KCR cells,and slightly reduced the protein levels of ABCB1 in KCR cells,although the high initial expression of ABCB1 masked the reduction in protein levels.The increased intracellular accumulation of the fluorescent due DiOC2 in the presence of the ABCB1 inhibitor verapamil correlated with the inhibition caused by miR-203 and miR-200c in transfected cells.Conclusion: The present study demonstrates that miR-200c and miR-203 exert a negative modulating effect on the activity of ABCB1 associated with doxorubicin resistance.
