Inhibition of c-FLIPL 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-FLIPL 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-FLIPL expression. We found that MiRNA-708 and c-FLIPL expression were inversely correlated. While c-FLIPL expression was upregulated, miRNA-708 was rarely expressed in renal cancer cells. Luciferase reporter assays demonstrated that miRNA-708 negatively regulated c-FLIPL expression by binding to the miRNA-708 binding site in the 3' untranslated region (3'UTR) of c-FLIPL. 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-FLIPL without any change in c-FLIPs expression. In contrast, inhibition of endogenous miRNA-708 using antago-miRNAs resulted in an increase in c-FLIPL protein expression. The expression of c-FLIPL 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-FLIPL and regulates the sensitivity of renal cancer cells to various apoptotic stimuli.
Department of Anatomy, College of Medicine, Yeungnam University, Nam-gu, Daegu, Republic of Korea.
Eun-Ae
EA
Kim
Department of Biological Sciences, Pusan National University, Busan, Republic of Korea.
Sang-Woo
SW
Kim
Department of Biological Sciences, Pusan National University, Busan, Republic of Korea.
Jehyun
J
Nam
Department of Anatomy, College of Medicine, Yeungnam University, Nam-gu, Daegu, Republic of Korea.
Eon-Gi
EG
Sung
Department of Anatomy, College of Medicine, Yeungnam University, Nam-gu, Daegu, Republic of Korea.
In-Hwan
IH
Song
Department of Anatomy, College of Medicine, Yeungnam University, Nam-gu, Daegu, Republic of Korea.
Joo-Young
JY
Kim
Department of Immunology, School of Medicine, Keimyung University, Dalseo-Gu, Daegu, Republic of Korea.
Taeg Kyu
TK
Kwon
Department of Anatomy, College of Medicine, Yeungnam University, Nam-gu, Daegu, Republic of Korea.
Tae-Jin
TJ
Lee
D020413:3' Untranslated Regions; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D001665:Binding Sites; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein / Q000235:genetics / Q000378:metabolism*; D002292:Carcinoma, Renal Cell / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D053148:Caspase 3 / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D059585:G1 Phase Cell Cycle Checkpoints / Q000187:drug effects; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007680:Kidney Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D013997:Time Factors; D023041:Xenograft Model Antitumor Assays
D020413:3' Untranslated Regions; D000970:Antineoplastic Agents; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein; C506386:CFLAR protein, human; C568528:MIRN708 microRNA, human; D035683:MicroRNAs; D011065:Poly(ADP-ribose) Polymerases; C505407:CASP3 protein, human; D053148:Caspase 3
RCC; c-FLIP; doxorubicin; miR-708