John Clotaire Daguia Zambe, Zhang Bin, Wei Na, Gao Rongfang, Zhao Fei, Wang Yunfei, Lei Ming, Huang Weiwei
College of Life Sciences, Northwest A&F University, 3 Taicheng Road, Yangling, Shaanxi Province, 712100, China; Laboratoire des Sciences Agronomiques et Biologiques pour le Développement (LASBAD), Faculty of Science, University of Bangui, Central African Republic.
College of Life Sciences, Northwest A&F University, 3 Taicheng Road, Yangling, Shaanxi Province, 712100, China.
Biochem Biophys Res Commun. 2016 Mar 25;472(1):194-200. doi: 10.1016/j.bbrc.2016.02.093. Epub 2016 Feb 23.
Autophagy is a catabolic process widely conserved among eukaryotes that permits the rapid degradation of unwanted proteins and organelles through the lysosomal pathway. The Serine/threonine protein kinase ULK2 (unc-51 like kinase 2) plays an important regulatory role in autophagy thanks to its involvement in mTOR-regulated-initiation and downstream ATG protein-related progression of this catabolic process. An increasing number of miRNAs have been found to modulate autophagy by targeting some ATG genes. In this study, we focus on the role of mir-26b in autophagy in prostate cancer (PCa) cells. We found that miR-26b inhibited autophagy in PC-3 and C4-2 cells, through down-regulation of ULK2 expression. Dual luciferase reporter assays showed that miR-26b binds the 3'UTR of ULK2, suggesting that ULK2 is a direct target of miR-26b. Real-time PCR and Western blot analysis confirmed that over-expression of miR-26b reduced ULK2 mRNA and protein levels. Our results showed also that miR-26b was down-regulated in LNCaP, DU145, C4-2 and PC-3 cells compared to the two normal prostate cells RWPE-1 and WPMY-1 except DU145 cells. This inversely correlates with ULK2 level in the same cell lines. Expression level of ULK2 in tissues microarray (TMA) of prostate cancer derived from 96 patients positively correlated with the pathologic stage of the patients (∗P < 0.05). Over-expression of ULK2 significantly reversed miR-26b-mediated autophagy inhibition. Taken together, our findings indicate that mir-26b inhibits autophagy through targeting ULK2 which is up-regulated in PCa.
自噬是一种在真核生物中广泛保守的分解代谢过程，它允许通过溶酶体途径快速降解不需要的蛋白质和细胞器。丝氨酸/苏氨酸蛋白激酶ULK2（unc-51样激酶2）由于参与了mTOR调节的启动以及该分解代谢过程的下游自噬相关基因（ATG）蛋白的进展，在自噬中发挥着重要的调节作用。越来越多的微小RNA（miRNA）被发现通过靶向一些自噬相关基因来调节自噬。在本研究中，我们聚焦于mir-26b在前列腺癌细胞自噬中的作用。我们发现miR-26b通过下调ULK2的表达来抑制PC-3和C4-2细胞中的自噬。双荧光素酶报告基因检测表明miR-26b与ULK2的3'非翻译区（3'UTR）结合，这表明ULK2是miR-26b的直接靶点。实时定量聚合酶链反应（Real-time PCR）和蛋白质免疫印迹分析证实，miR-26b的过表达降低了ULK2的mRNA和蛋白质水平。我们的结果还表明，与两种正常前列腺细胞RWPE-1和WPMY-1相比，除DU145细胞外，miR-26b在LNCaP、DU145、C4-2和PC-3细胞中表达下调。这与同一细胞系中ULK2的水平呈负相关。来自96例患者的前列腺癌组织芯片（TMA）中ULK2的表达水平与患者的病理分期呈正相关（*P < 0.05）。ULK2的过表达显著逆转了miR-26b介导的自噬抑制。综上所述，我们的研究结果表明mir-26b通过靶向在前列腺癌中上调的ULK2来抑制自噬。
