Inhibition of c-FLIP(L) expression by miRNA-708 increases the sensitivity of renal cancer cells to anti-cancer drugs

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.