Hsa-mir-34a作為新型的自噬調節劑，可抑制STX-17介導的自噬體與溶酶體的融合

何肇晏1、張安辰2、仇光宇1、陳宏恩1、蔡德甫1、黃一勝1,3,4

1新光醫院 泌尿科，2中央研究室；3台北醫學大學 醫學院；4輔仁大學 醫學院

Hsa-mir-34a, as a novel autophagy regulator, inhibits STX-17-mediated fusion of autophagosomes with lysosomes

Chao-Yen Ho1, An-Chen Chang2, Kuang-Yu Chou1, Hung-En Chen 1, Te-Fu Tsai 1 and Thomas I-Sheng Hwang1,3,4

Department of Urology1 and Central Laboratory2, Shin Kong Wu Ho-Su Memorial Hospital; Department of Urology, Taipei Medical University3; Division of Urology, School of Medicine, Fu-Jen Catholic University4, Taipei, Taiwan.

Purpose:

High basal level of autophagy activity in BC rises up therapeutic issue by increasing cell survival rate and by inhibiting apoptotic cell death and eventual contribute to develop resistance to current treatment. Hsa-miR 34a is described to display anti-tumor activity in several types of cancer. However, the functional mechanism of hsa-miR 34a in regulating autophagy of BC remains largely unknown.

Materials and Methods:

Human BC cell lines 5637 and T24 were obtained from the Bioresource Collection and Research Center (BCRC; Hsinchu, Taiwan). Both cell lines were cultured in RPMI-1640 medium. The miRanda database was used to screen the potential targets of hsa-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:

Transfection of BC cells with hsa-miR-34a mimic exhibited LC3-II and p62 protein up-regulation, indicates LC3-II and p62 accumulation in BC cells. From miRanda database, we screen the potential targets of hsa-miR-34a and found four candidates (EPG5, SNAP29, STX17 and RAB7L1) which play important role in the fusion of autophagosomes with lysosomes. We proved that hsa-miR-34a reduces STX17 mRNA expression; however, no effect was seen on EPG5, SNAP29 and RAB7L1 mRNA expression. Moreover, further data revealed that miR-34a directly binds on STX17 3’UTR and suppress STX17 mRNA translation. Therefore, hsa-mir-34a is a negative autophagy regulator in BC.

Conclusions:

Our results provide insights into how hsa-miR-34a sabotage the fusion of autophagosomes with lysosomes via STX17 silencing in BC. Overall, miR-34a can benefit BC treatment efforts by blocking autophagic flux.