探討小分子核糖核酸-34a對膀胱癌細胞週期、移動和保護性自噬的抑癌功能
何肇晏1、張安辰2、仇光宇1、蔡德甫1、黃一勝1,3,4
1新光醫院 泌尿科，2轉譯醫學中心；3台北醫學大學 醫學院；4輔仁大學 醫學院
Tumor suppressive functions of hsa-miR-34a on cell cycle, migration, and protective autophagy in bladder cancer
Chao-Yen Ho1, An-Chen Chang2, Kuang-Yu Chou1, Te-Fu Tsai1 and Thomas I-Sheng Hwang1,3,4
1Department of Urology and Translational 2Medicine Center, 3Shin Kong Wu Ho-Su Memorial Hospital; Department of Urology, Taipei Medical University; 4Division of Urology, School of Medicine, Fu-Jen Catholic University, Taipei, Taiwan.
Purpose:
Bladder cancer (BC) cells exhibit a high basal level of autophagy activity, which contributes to the development of a protective mechanism for cellular survival against current treatments. Hsa-miR-34a (miR-34a) is described to display anti-tumor activity in several types of cancer. However, the functional mechanism of miR-34a in regulating tumor aggressiveness and protective autophagy of BC remains largely unknown. In this study, we aim to investigate whether miR-34a can block autophagic flux in BC and thus advances clinical treatment.
Materials and Methods:
Human BC cell lines 5637 and T24 are obtained from the Bioresource Collection and Research Center (BCRC; Hsinchu, Taiwan). Both cell lines are cultured in RPMI-1640 medium. The miRanda database is used to screen the potential targets of miR-34a. Viromer RED (Lipocalyx, Saale, Germany), a plasmid transfection reagent, was used to transfect miR-34a mimic. Western blot and qPCR were used to detect indicated protein and mRNA expression, respectively. Immunofluorescent staining was performed to analysis p62 and LC3-II expression density in BC cells.
Results:
First, transfected BC cells with miR-34a mimic exhibited LC3-II and p62 accumulation. We demonstrated that syntaxin 17 (STX17), which is required for autophagosome–lysosome fusion, was downregulated upon miR-34a mimic treatment. Mechanistically, miR-34a reduced the expression of STX17 proteins that directly bind on STX17 3' untranslated regions (UTRs) and thus suppresses STX17 mRNA translation to eventually inhibit protective autophagy in BC. The overexpression of miR-34a in BC cells enhances the chemosensitivity of cisplatin, doxorubicin, epirubicin, and mitomycin C. Furthermore, miR-34a inhibited cell proliferation and triggered G0/G1 cell cycle arrest by inhibiting cyclin D1 and cyclin E2 protein expression. Moreover, miR-34a suppressed cell motility through the downregulation of epithelial–mesenchymal transition.
Conclusions:
Our findings reveal new clinical potential for miR-34a as an antitumor agent in the treatment of human BC through blocking the cell cycle, inhibiting motility, and impairing protective autophagy. miR-34a can be administered with anti-BC chemotherapeutic drugs to improve their efficacy.