A Let-7b MicroRNA-binding Site Polymorphism In The Bcl-xl3’UTR Is Associated With5-FU Sensitivity In BEL-7402Cells

Objective: To study the effects of SNP rs3208684(A＞C) on regulation of let-7b inhuman hepatocellular carcinoma (HCC) BEL-7402cells, and the effects of HCC cellsto5-FU.Methods: Bioinformatics were used to predict the miRNA binding sites of Bcl-xlgene3′UTR, and search the SNP which is located in miRNA binding sites combinedwith PUBMED SNP database. HCC cell line (BEL-7402cell) was cultured in vitro.MiRNA mimics, wild type and mutant (contains SNP) pcDNA3.1-Bcl-xl expressionvector were transfected into BEL-7402cells. The drug sensitivity of BEL-7402cellsto5-FU was detected by MTT assay. The protein expressions of Bcl-xl and Bax weredetected by Western blot. The targeting effect between miRNA and Bcl-xl and theregulation effect of SNP rs3208684(A＞C) were validated by the dual-luciferasereporter system.Results: Bioinformatics prediction programs identified a common binding site forthe let-7b, SNP rs3208684(A＞C) was located in let-7b binding site on Bcl-xl gene3′UTR combined with PUBMED SNP database.MTT and Western blot analysis showed that, compared with let-7b negativecontrol group and normal control group, let-7b mimics transfection group significantlyincreased the drug sensitivity of BEL-7402cells to5-FU (P <0.05), and the amount ofBcl-xl protein significantly decreased (P <0.05), While, the Bax peotein level had nosignificantly changed.MTT and Western blot analysis showed that, compared with let-7b mimics+wildtype-Bcl-xl vector co-transfection group and negative control group, let-7b mimics+mutant-Bcl-xl vector co-transfection group decreased the drug sensitivity ofBEL-7402cells to5-FU (P <0.05), and the amount of Bcl-xl protein significantlyincreased (P <0.05), While, the Bax peotein level had no significantly changed.MTT and Western blot analysis showed that, compared with negative controlgroup and no transfection group, wild type and mutant-Bcl-xl vector transfection group decreased the drug sensitivity of BEL-7402cells to5-FU (P <0.05), and theamount of Bcl-xl protein significantly increased (P <0.05), While, the Bax peoteinlevel had no significantly changed. Compared with wild type-Bcl-xl vectortransfection group, mutant-Bcl-xl vector transfection group decreased the drugsensitivity of BEL-7402cells to5-FU (P <0.05), but the Bcl-xl and Bax peoteinleveles had no significantly changed (P﹥0.05).Dual-luciferase reporter system analysis showed that, compared with negativecontrol+wild type-Bcl-xl vector co-transfection group, the luciferase activity of let-7bmimics+wild type-Bcl-xl vector co-transfection group decreased significantly (P <0.01); Compared with negative control+mutant-Bcl-xl vector co-transfection group,the luciferase activity of let-7b mimics+mutant-Bcl-xl vector co-transfection grouphad no significantly changed (P﹥0.05).Conclusion: Let-7b increased the sensitivity of BEL-7402cells to5-FU bydown-regulated expression of Bcl-xl protein; SNP rs3208684(A＞C) decreased thesensitivity of BEL-7402cells to5-FU maybe by disrupted the binding of let-7b andBcl-xl.