PARP Inhibitor Increases Chemosensitivity By Upregulating MiR-664b-5p In BRCA1-mutated Triple-negative Breast Cancer

Background:Emerging evidence has shown that adding poly(ADP-ribose)polymerase(PARP)inhibitors to chemotherapy regimens is superior to the control regimens alone in BRCA1-mutated triple-negative breast cancer(TNBC)patients.Many clinical trials on platinum-based chemotherapy have confirmed that platinum compounds have a relevant role in the treatment of TNBC patients,especially those harbouring BRCA1/2 mutations.MicroRNAs(miRNAs)down-regulated target gene expression post-transcriptionally by binding to the 3' untranslated region(3'UTR)of mRNA,and functioned in numerous important pathophysiological processes.Dysregulation of miRNAs is reported to be involved in the chemotherapy sensitivity of breast cancer.But their underlying mechanisms have not been fully elucidated.Methods:We screened the differentially expressed miRNAs in two BRCA1-mutated TNBC cell lines,which were exposed to carboplatin(CBP)plus gemcitabine(GEM)with or without the PARP inhibitor Olaparib using a miRNA microarray.After verification by quantitative RT-PCR,miR-664b-5p was selected for further study.We detected cell growth by MTT assays,the cell cycle and apoptosis by flow cytometry,the abilities of cell migration and invasion by wound-healing and transwell assays and the expression of CCNE2 by quantitative RT-PCR(qRT-PCR),western blotting and immunofluorescence assays after treatment with drugs or transfection in vitro.The luciferase reporter assay was performed to assess the effect of miR-664b-5p on CCNE2.Tumor growth was evaluated in vivo using mice xenograft tumor model.To form tumour xenografts in male six-week-old BALB/c nude mice,empty or lentiviral miR-664b-5p vector transfected-HCC1937 cells(lx 107)were injected subcutaneously in the back under aseptic conditions,and measured the tumour volume regularly.Lastly,immunohistochemical staining was performed for Cyclin E2 expression,and fluorescent in situ hybridization for miR-664b-5p expression in TNBC tissues,then the correlation between them was analyzed by chi-square test.Results:we found that miR-664b-5p expression was increased after adding a PARP inhibitor,Olaparib,to a carboplatin(CBP)plus gemcitabine(GEM)therapy regimen in two BRCA1-mutated TNBC cell lines,MDA-MB-436 and HCC1937,not in non-BRCA1-mutated TNBC cell line,MDA-MB-231.Functional assays showed that miR-664b-5p overexpression inhibited cell proliferation,blocked G1-to-S-phase transition of cell cycle,promoted cell apoptosis,and suppressed migration and invasion in BRCA1-mutated TNBC cells,but miR-664b-5p suppression acted the opposite effects.CCNE2 was identified as a novel functional target of miR-664b-5p,and the protein expression and mRNA levels of CCNE2 were down-regulated when miR-664b-5p was overexpressed compared with the control cells.CCNE2 knockdown revealed effects similar to those observed with miR-664b-5p overexpression in BRCA1-mutated TNBC cells.Both CCNE2 knockdown and miR-664b-5p overexpression significantly increased the chemosensitivity of BRCA1-mutated TNBC cells to CBP plus GEM,miR-664b-5p inhibition weakened the growth inhibition caused by CBP plus GEM combined with the PARP inhibitor Olaparib in BRCA1-mutated TNBC cells.In addition,in vivo studies indicated that miR-664b-5p inhibited tumour growth compared with the control in tumour xenograft models,and the results of qRT-PCR and immunohistochemistry(IHC)displayed lower CCNE2 mRNA and protein levels in miR-664b-5p-overexpressing tumour xenografts.We also found that of the 90 TNBC tissues,54 cases(60.0%)expressed cyclin E2 at high level,while 40 cases(44.4%)expressed miR-664b-5p at high level.CCNE2 expression was inversely correlated with miR-664b-5p expression.Conclusion:miR-664b-5p functions as a tumour suppressor and has an important role in the regulation of PARP inhibitors to increase chemosensitivity by targeting CCNE2.This may be one of the possible mechanisms by which PARP inhibitors increase chemosensitivity in BRCA1-mutated TNBC.