The let-7 family of microRNAs inhibits Bcl-xL expression and potentiates sorafenib-induced apoptosis in human hepatocellular carcinoma

Background & Aims

Bcl-xL, an anti-apoptotic member of the Bcl-2 family, is over-expressed in human hepatocellular carcinoma, conferring a survival advantage to tumour cells. The mechanisms underlying its dysregulation have not been clarified. In the present study, we explored the involvement of microRNAs that act as endogenous sequence-specific suppressors of gene expression.

Methods

The expression profiles of microRNAs in Huh7 hepatoma cells and primary human hepatocytes were compared by microarray analysis. The effect of let-7 on Bcl-xL expression was examined by Western blot and a reporter assay. The involvement of let-7 microRNAs in human tissues was analysed by western blot and reverse transcription-PCR.

Results

Microarray analysis, followed by in silico target prediction, identified let-7 microRNAs as being downregulated in Huh7 hepatoma cells in comparison with primary human hepatocytes, as well as possessing a putative target site in the bcl-xl mRNA. Over-expression of let-7c or let-7g led to a clear decrease of Bcl-xL expression in Huh7 and HepG2 cell lines. Reporter assays revealed direct post-transcriptional regulation involving let-7c or let-7g and the 3′-untranslated region of bcl-xl mRNA. Human hepatocellular carcinoma tissues with low expression of let-7c displayed higher expression of Bcl-xL protein than those with high expression of let-7c, suggesting that low let-7 microRNA expression contributes to Bcl-xL over-expression. Finally, expression of let-7c enhanced apoptosis of hepatoma cells upon exposure to sorafenib, which downregulates expression of another anti-apoptotic Bcl-2 protein, Mcl-1.

Conclusions

let-7 microRNAs negatively regulate Bcl-xL expression in human hepatocellular carcinomas and induce apoptosis in cooperation with an anti-cancer drug targeting Mcl-1.

Keywords: Liver, Mcl-1, Bcl-2, Tumour, Epigenetic

Abbreviations: miRNA, microRNA, 3′UTR, 3′-untranslated region, mRNA, messenger RNA, HCC, hepatocellular carcinoma, CDK, cyclin-dependent kinase, DMEM, Dulbecco’s modified Eagle medium, RT, reverse transcription, PCR, polymerase chain reaction, 7-AAD, 7-amino-actinomycin D, DMSO, dimethyl sulfoxide