Wang Yunfeng, Zhang Songyan, Dang Shuwei, Fang Xuan, Liu Ming
Department of General Surgery, The Fourth Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang, People's Republic of China.
Onco Targets Ther. 2019 Jun 12;12:4621-4629. doi: 10.2147/OTT.S196992. eCollection 2019.
Autophagy executes the rapid degradation of unneeded proteins and organelles through the lysosomal pathway, and is a crucial catabolic process widely conserved among eukaryotes. miRNAs can modulate autophagy by targeting genes encoding proteins involved in the process. A great deal of researchhas indicated that miR-216a was a functional miRNA related to tumorigenesis. However, the contribution of miR-216a to autophagy in colorectal cancer (CRC) remains unclear. The purpose of this study was to investigate the role of miR-216a in autophagy in CRC cells. The expression levels of miR-216a in 67 paired CRC patients were evaluated by qRT-PCR. Direct gene targeting predicted by TargetScan and miRanda was confirmed by luciferase activity. Western blot and flow cytometry were used to identify the regulatory mechanism of miR-216a on autophagy in CRC cells. We determined that miR-216a is downregulated in CRC by screening its expression in 67 CRC tissue samples. Dual luciferase reporter assays showed that miR-216a binds the 3'-UTR of MAP1S, suggesting that MAP1S is a direct target of miR-216a. miR-216a could inhibit autophagy in HCT-116 and HT-29 CRC cells through downregulating MAP1S expression. Flow cytometry and Western blot analysis demonstrated that overexpression of miR-216a reduced MAP1S mRNA and protein levels. Moreover, we determined that miR-216a-regulated inhibition of autophagy via MAP1S regulation involves the TGF-β pathway. Taken together, our findings indicate that miR-216a was a tumor-suppressor miRNA in human CRC, which can inhibit autophagy via the TGF-β/MAP1S pathway.
自噬通过溶酶体途径对不需要的蛋白质和细胞器进行快速降解，是真核生物中广泛保守的关键分解代谢过程。微小RNA（miRNA）可通过靶向编码参与该过程的蛋白质的基因来调节自噬。大量研究表明，miR-216a是一种与肿瘤发生相关的功能性miRNA。然而，miR-216a在结直肠癌（CRC）自噬中的作用仍不清楚。本研究的目的是探讨miR-216a在CRC细胞自噬中的作用。通过qRT-PCR评估67对CRC患者中miR-216a的表达水平。通过荧光素酶活性证实了TargetScan和miRanda预测的直接基因靶向。采用蛋白质免疫印迹法和流式细胞术鉴定miR-216a对CRC细胞自噬的调控机制。通过筛选67个CRC组织样本中的miR-216a表达，我们确定其在CRC中表达下调。双荧光素酶报告基因检测显示，miR-216a与MAP1S的3'-UTR结合，表明MAP1S是miR-216a的直接靶点。miR-216a可通过下调MAP1S表达抑制HCT-116和HT-29 CRC细胞的自噬。流式细胞术和蛋白质免疫印迹分析表明，miR-216a的过表达降低了MAP1S mRNA和蛋白质水平。此外，我们确定miR-216a通过MAP1S调控对自噬的抑制作用涉及TGF-β途径。综上所述，我们的研究结果表明，miR-216a是人类CRC中的一种肿瘤抑制性miRNA，可通过TGF-β/MAP1S途径抑制自噬。
