Experimental study on the effect of miR-21 targeting Atg5 on the proliferation, migration and invasion of non-small cell lung cancer A549 cells by regulating autophagy

GAO Xiaohui; ZHANG Yali; ZHANG Zhiye; GUO Yanzhen; CHEN Xiaobing

doi:10.3969/j.issn.1009-0460.2019.02.001

Abstract：Objective To investigate the mechanism of miR-21 regulating autophagy of non-small cell lung cancer (NSCLC) by targeting autophagy-related target gene 5 (Atg5) and its role in proliferation, migration and invasion of A549 cells. Methods Nonsense nucleic acid sequence NC (NC group), miR-21 mimics (miR-21 mimics group) and miR-21 inhibitor (miR-21 inhibitor group) were transfected into A549 cells, respectively. CCK-8 was used to detect the proliferation of A549 cells, scratch test was used to detect the migration ability of A549 cells, and Transwell invasion test was used to detect the invasion ability of A549 cells. Dual luciferase reporter gene assay verified the targeting relationship between miR-21 and Atg5. Western blotting was used to detect the expression of LC3B-II, p62 and Atg5 proteins. Results Compared with NC group, the proliferation, migration and invasiveness of the cells in the miR-21 mimics group were up-regulated, while the proliferation, migration and invasiveness of the cells in the miR-21 inhibitor group were down-regulated (P＜0. 05). The results of double luciferase report experiment showed that miR-21 significantly inhibited the luciferase activity of wild-type Atg5 3'-UTR plasmid transfected cells (P＜0. 05), but had no effect on the luciferase activity after co-transfection of mutant Atg5 3'-UTR gene report plasmid with miR-21 mimics. The expression of LC3B-II protein in NC group was lower than that in miR-21 mimics group, higher than that in miR-21 inhibitor group (P＜ 0. 05);the expression of p62 and Atg5 protein in NC group was higher than that in miR-21 mimics group, lower than that in miR-21 inhibitor group (P＜0. 05). Compared with NC group, 3-MA treatment decreased the proliferation of A549 cells induced by miR-21 mimics transfection (P＜0. 05). Scratch and Transwell experiments showed that 3-MA treatment inhibited the migration and invasion of A549 cells induced by miR-21 mimics transfection (P＜0. 05). Conclusion MiR-21 targeting Atg5 regulates autophagy in non-small cell lung cancer cells and promotes proliferation, migration and invasion of lung cancer cells.