微小核醣核酸miR-99a在人類膀胱癌細胞中標靶mTOR之抗癌作用
仇光宇1,4 #、林致凡2#、蔡德甫1,4、陳宏恩1、林宜佳 1,4、黃一勝1,3,4
1新光醫院 外科部 泌尿科、2新光醫院 中央研究室、3台北醫學大學 醫學院、4輔仁大學 醫學院
Mir-99a acts as tumor suppressor via targeting to mtor in human bladder cancer cells
Kuang-Yu Chou1,4#、Ji-Fan Lin2#、Te-Fu Tsai 1,4、Hung-En Chen1、Yi-Chia Lin1,4
Thomas I-Sheng Hwang1,3,4
1Division of Urology, Department of Surgery, Shin Kong Wu Ho-Su Memorial Hospital,
2Central Laboratory, Shin Kong Wu Ho-Su Memorial Hospital,
3Department of Urology, Taipei Medical University, Division of Urology,
 4School of Medicine, Fu-Jen Catholic University, Taipei, Taiwan.
# These authors contributed equally to this work.
Introduction: mTOR is recognized as an important target in many cancer types. In our previous studies, mTOR inhibitor Everolimus (RAD001) caused severed cell viability lost only when the induced-autophagy was coordinately inhibited in bladder cancer cells. 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 on Ser473. Therefore, novel inhibitors that inhibit both mTORC1 and mTORC2 are warrant to treat bladder cancer. 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 (5637 and T24) 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 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.
 
    位置
    資料夾名稱
    摘要
    發表人
    TUA秘書處
    單位
    台灣泌尿科醫學會
    標籤
    討論式海報
    建立
    2015-05-22 16:12:00
    最近修訂
    2015-05-22 16:12:38
    瀏覽
    1,547
    more