The Mechanisms And Effects Of MiR-375 In Combined Treatment With Imatinib And Lapatinib Suppresses The Growth Of Fulvestrant-resistant Breast Cancer Cells

Objective:(1)To establish fulvestrant-resistant breast cancer(FRBC)cell line models and to detect its biological characteristics.(2)To explore the effect of dual treatment with inhibitors of c-Abl(imatinib)and EGFR(lapatinib)to inhibit growth of FRBC cells.(3)To explore the roles of miR-375 in combined treatment with imatinib and lapatinib suppresses the growth of FRBC cells.(4)To elucidate the regulation and molecular mechanism of miR-375 in the autophagy of FRBC cells.Methods:(1)FRBC cells were developed from human breast cancer parental cell line MCF-7,by exposing MCF-7 to high dose for shorts periods of time in vitro;MTT assay was used to detect the sensitivity of FRBC cells and parental cells to fulvestrant;MTT assay was also used to detect the growth of FRBC cells compared with parental cells;Wound healing assay was performed to assess the cells’ migration ability of FRBC cells compared with parental cells.(2)Different expression of ER,PR,HER2,EGFR and c-Abl between FRBC cells and parental cells were examined by Western blot assay;MTT assay was used to explore the effect of dual treatment with imatinib and lapatinib to inhibit FRBC cells compared with single treatment.(3)Microarray analysis was carried out to identify micro RNA which is significantly changed between parental and FRBC cells,and the miRNAs with significantly changed were validated by qRT-PCR and miR-375 was predicted by Target Scan,miRDB and miRanda as the target;MiR-375-overexpressed lentiviral vector and miR-375-inhibitor lentiviral vector were transfected into FRBC cells,and the transfection effect was verified by qRT-PCR;MTT assay was used to detect the sensitivity of dual treatment to FRBC cells after miR-375 overexpression or downregulation.(4)Autophagy level between FRBC and parental cells was analyzed by acridine orange(AO)staining and Western blot assay;AO staining and Western blot assay were also performed to analyze the proteins that related to miR-375.Results:(1)After 12 months inducement,the established FRBC cells could grow stably in the medium containing 0.5 μM fulvestrant.MTT assay showed that FRBC cells were much less sensitive to fulvestrant compared with parental cells,indicating the fulvestrant-resistant breast cancer cell model was successfully built;Compared with parental cells,the growth ability of FRBC cells decreased while the migration ability of FRBC cells increased.(2)The expression of ER,PR and HER2 was abolished in FRBC cells,these FRBC phenotypes were similar to triple negative breast cancer(TNBC)cells;MTT assay was used and cell viability significantly decreased after combined treatment than any single treatment along.(3)MiR-375 was selected as the research object by microarray screening and qRT-PCR verification;Dual treatment significantly inhibited growth of FRBC cells and upregulated miR-375expression;Overexpression of miR-375 increase sensitivity of FRBC cells to dual treatment,while downregulation of miR-375 reduced sensitivity of FRBC cells to dual treatment.(4)Compared with parental cells,autophagy increased in FRBC cells;ATG7/LC3-Ⅱ were identified as potential target candidates of miR-375 according to Target Scan,miRTarbase and miRanda programs;MiR-375 downregulates ATG7 and LC3-Ⅱ and suppresses autophagy in FRBC cells.Conclusion: Combined treatment with imatinib and lapatinib effectively inhibits the growth of fulvestrant-resistant breast cancer cells by upregulating miR-375;MiR-375 could inhibit the growth of fulvestrant-resistant breast cancer cells by downregulating autophagy.Because most cancer-relevant deaths arise from resistance to therapy,reexpression of miR-375 might be a potential therapeutic approach for treating fulvestrant-resistant breast cancer with imatinib and lapatinib.