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

// Eun-Ae Kim 1, * , Sang-Woo Kim 2, * , Jehyun Nam 2 , Eon-Gi Sung 1 , In-Hwan Song 1 , Joo-Young Kim 1 , Taeg Kyu Kwon 3 , Tae-Jin Lee 1 1 Department of Anatomy, College of Medicine, Yeungnam University, Nam-gu, Daegu, Republic of Korea 2 Department of Biological Sciences, Pusan National University, Busan, Republic of Korea 3 Department of Immunology, School of Medicine, Keimyung University, Dalseo-Gu, Daegu, Republic of Korea * These authors have contributed equally to this work Correspondence to: Tae-Jin Lee, email: tjlee@med.yu.ac.kr Keywords: c-FLIP, miR-708, RCC, doxorubicin Received: May 04, 2015 Accepted: January 23, 2016 Published: February 03, 2016 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.