MP36-10 MIR-99A ACTS AS TUMOR SUPPRESSOR VIA TARGETING TO MTOR IN HUMAN BLADDER CANCER CELLS

Abstract

You have accessJournal of UrologyBladder Cancer: Basic Research I1 Apr 2015MP36-10 MIR-99A ACTS AS TUMOR SUPPRESSOR VIA TARGETING TO MTOR IN HUMAN BLADDER CANCER CELLS Te-Fu Tsai, Ji-Fan Lin, Yi-Chia Lin, Kuang-Yu Chou, Hung-En Chen, and Thomas I.S. Hwang Te-Fu TsaiTe-Fu Tsai More articles by this author , Ji-Fan LinJi-Fan Lin More articles by this author , Yi-Chia LinYi-Chia Lin More articles by this author , Kuang-Yu ChouKuang-Yu Chou More articles by this author , Hung-En ChenHung-En Chen More articles by this author , and Thomas I.S. HwangThomas I.S. Hwang More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.738AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES mTOR is recognized as an important target in many cancer types. The mTOR complex 2 (mTORC2) containing mTOR and rictor is thought to be insensitive to RAD001 and was shown to regulate the prosurvival kinase AKT by phosphrylation. We previously showed that the expression level of miR-99a was down-regulated in human bladder tumor tissues compared to their adjacent normal tissues. Since mTOR is predicted to be a potential target of miR-99a, we investigate the anti-cancer activity of miR-99a in bladder cancer cells. METHODS Vectors that express miR-99a were transfected into human bladder, prostate, embryonic kidney and cervical cancer cells. Expression level of miR-99a was monitored by miRNA Q-PCR. 3'-UTR of mTOR was constructed downstream of a luciferase gene, and luciferase reporter assays were performed to verify the direct binding of miR-99a to mTOR transcripts. The mRNA and protein expression level of mTOR were measured by Q-PCR and Western blot, respectively. Cell viability of miR-99a transfected cells was detected by WST-1 and colony formation assays. Inhibition of mTOR complex 1 and 2 (mTORC1 and mTORC2) signaling was monitored by detecting the phosphrylation of S6K and Akt using Western blot. Induction of autophagy was accessed by the expression of LC3-II marker protein. RESULTS Transfection of miR-99a expressing vector elevated the expression level of miR-99a up to 4.5-fold in cells compared to vector-only control. The function of matured miR-99a was confirmed by luciferase reporter assays. The level of mTOR RNA and protein were decreased in miR-99a transfected cells. Dual inhibition of mTORC1 and mTORC2 was confirmed by immunoprecipitation (IP) of mTOR associated Rictor and Raptor, and the decreased phosphrylation of S6K and Akt in miR-99a transfected cells. The LC3-II protein was accumulated in miR-99a transfected cells compared to monk transfected control, suggesting that inhibition of mTOR by miR-99a induces autophagy in bladder cancer cells. CONCLUSIONS This is the first study showed that miR-99a markedly inhibits bladder and prostate cancer cell growth via dual inhibition of mTORC1 and mTORC2. miR-99a treatment also induces autophagy through mTOR inhibition. However, the role of miR-99a induces autophagy remains further investigation. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e432 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Te-Fu Tsai More articles by this author Ji-Fan Lin More articles by this author Yi-Chia Lin More articles by this author Kuang-Yu Chou More articles by this author Hung-En Chen More articles by this author Thomas I.S. Hwang More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...