Let-7b binding site polymorphism in the B-cell lymphoma-extra large 3'UTR is associated with fluorouracil resistance of hepatocellular carcinoma

Abstract

B-cell lymphoma.extra large (Bcl-xl) is an anti.apoptotic member of the B-cell lymphoma 2 (Bcl-2) family that is often found to be overexpressed in human hepatocellular carcinoma (HCC), therefore conferring a survival advantage to tumor cells. microRNA (miRNA) let.7b is downregulated in HCC and its expression correlates with multidrug resistance. Using computational programs, it was predicted that the 3' untranslated region (UTR) of the Bcl-xl gene contains a potential miRNA binding site for let.7b, and that a single nucleotide polymorphism (SNP) site rs3208684 (A or C allele) resides within this binding site. Luciferase assays and western blot analysis demonstrated that let.7b targeted Bcl-xl gene expression and negatively regulated the amount of Bcl-xl protein. SNP rs3208684 (A>C) variation enhanced the expression of Bcl-xl by disrupting the binding of let.7b to the 3'UTR of Bcl-xl. The effects of the two polymorphic variants on chemotherapeutic drug sensitivity were determined by cell counting kit 8 assays. Overexpression of the Bcl-xl mutated (C) allele in BEL.7402 HCC cells significantly decreased fluorouracil (5.FU) sensitivity, as compared with mock transfection and overexpression of the wild.type allele. From this, it was concluded that let.7b increased 5.FU sensitivity by repressing Bcl-xl expression in HCC cells. These results suggest that SNP (rs3208684) may be a potential marker for personalized treatment.