Inhibition of c-FLIPL expression by miRNA-708 increases the sensitivity of renal cancer cells to anti-cancer drugs

Abstract

Dysregulation of the anti-apoptotic protein, cellular FLICE-like inhibitory protein (c-FLIP), has been associated with tumorigenesis and chemoresistance in various human cancers. Therefore, c-FLIP is an excellent target for therapeutic intervention. MicroRNAs (miRNAs) are small non-coding RNAs that are involved in tumorigenesis, tumor suppression, and resistance or sensitivity to anti-cancer drugs. However, whether miRNAs can suppress c-FLIP_L expression in cancer cells is unclear. The aim of this study was to identify miRNAs that could inhibit the growth of renal cancer cells and induce cell death by inhibiting c-FLIP_L expression. We found that MiRNA-708 and c-FLIP_L expression were inversely correlated. While c-FLIP_L expression was upregulated, miRNA-708 was rarely expressed in renal cancer cells. Luciferase reporter assays demonstrated that miRNA-708 negatively regulated c-FLIP_L expression by binding to the miRNA-708 binding site in the 3’ untranslated region (3’UTR) of c-FLIP_L. Ectopic expression of miRNA-708 increased the accumulation of sub-G1 populations and cleavage of procaspase-3 and PARP, which could be prevented by pretreatment with the pan-caspase inhibitor, Z-VAD. Ectopic expression of miRNA-708 also increased the sensitivity to various apoptotic stimuli such as tumor necrosis factor-related apoptosis-inducing ligand, doxorubicin (Dox), and thapsigargin in Caki cells. Interestingly, miRNA-708 specifically repressed c-FLIP_L without any change in c-FLIP_s expression. In contrast, inhibition of endogenous miRNA-708 using antago-miRNAs resulted in an increase in c-FLIP_L protein expression. The expression of c-FLIP_L was upregulated in renal cell carcinoma (RCC) tissues compared to normal tissues. In contrast, miRNA-708 expression was reduced in RCC tissues. Finally, miRNA-708 enhanced the tumor-suppressive effect of Dox in a xenograft model of human RCC. In conclusion, miRNA-708 acts as a tumor suppressor because it negatively regulates the anti-apoptotic protein c-FLIP_L and regulates the sensitivity of renal cancer cells to various apoptotic stimuli.