Let-7b Target Site SNP Regulates Bcl-xL Expression And Affects Chemosensitivity In Breast Cancer

Objective:This study aimed to investigate the effect of a SNP in a let-7b target sitein the3′UTR of Bcl-xL and its mechanism on chemosensitivity in breast cancer.Methods:Cross-referencing the miRNA target databases and the NCBI dbSNPsdatabase to identify SNPs within predicted miRNA binding sites in the3′UTR ofBcl-xL, we identified the SNP(rs3208684A＞C) as a candidate SNP. The wild-typeBcl-xL eukaryotic plasmid vectors (WT-Bcl-xL) and mutant Bcl-xL eukaryoticplasmid vectors (Mut-Bcl-xL) were constructed and transfected into MCF-7cells,respectively. Protein expressions were mesured by western blotting. Cell viability wasdetected by MTT assay. Usingdual-luciferase reporter assay to identify whether let-7bdirectly target the3′UTR of Bcl-xL and the effect of SNP(rs3208684A＞C) on theregulation.Results: After let-7b mimics were transfected into MCF-7cells, western blotanalysis demonstrated that let-7b down-regulated Bcl-xL proteins expression and hadno effect on Bax proteins expression compared with untreated cells or negative; MTTresults showed that let-7b increased the sensitivity of MCF-7cells to5-FU and ADM.WT-Bcl-xL and Mut-Bcl-xL were transfected into MCF-7cells, respectively. MTTresults showed that Mut-Bcl-xL decreased the sensitivity of MCF-7cells to5-FU andADM compared with WT-Bcl-xL. Western blot analysis demonstrated thatMut-Bcl-xL had no effect on Bcl-xL and Bax proteins expression compared withWT-Bcl-xL. WT-Bcl-xL or Mut-Bcl-xL was transfected with let-7b mimics intoMCF-7cells. Western blot indicated that Mut-Bcl-xL and let-7b co-transfected groupup-regulate Bcl-xL proteins but had no effect on Bax proteins expression comparedwith WT-Bcl-xL and let-7b co-transfected group. MTT results suggested thatMut-Bcl-xL and let-7b co-transfected group decreased the sensitivity of MCF-7cells to5-FU and ADM compared with WT-Bcl-xL and let-7b co-transfected group. Thedual-luciferase reporter assay analysis showed that, compared withpsiCHECK2-WT-Bcl-xL3′UTR and miRNA minics control co-transfection groupand blank group, the luciferase activity of psiCHECK2-WT-Bcl-xL3′UTR andlet-7b co-transfection group decreased significantly (P <0.001); compared withpsiCHECK2-Mut-Bcl-xL and miRNA minics controlco-transfection group and blankgroup, the luciferase activity of psiCHECK2-Mut-Bcl-xL and let-7b mimicsco-transfection group had no significantly change(P﹥0.05); compared withpsiCHECK2-Mut-Bcl-xL and let-7b mimics co-transfection group, the luciferaseactivity of psiCHECK2-WT-Bcl-xL3′UTR and let-7b co-transfection groupdecreased significantly (P <0.001).Conclusions:(1)let-7b increased the sensitivity of MCF-7cells to5-FU and ADMvia regulating Bcl-xLprotein expression.(2)SNP(rs3208684A＞C) variation mediatedBcl-xL up-regulation by directly disrupting the binding of let-7with Bcl-xL gene anddecreased5-FU and ADM sensitivity of MCF-7cells.