miR-21靶向Atg5对非小细胞肺癌A549细胞自噬调控促进细胞增殖、迁移和侵袭的实验研究
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摘要
目的探讨miR-21通过靶向作用自噬相关靶基因5(Atg5)调控非小细胞肺癌(NSCLC)自噬的作用机制及其在A549细胞增殖、迁移及侵袭中的作用。方法无义核酸序列NC(NC组)、miR-21模拟物(miR-21 mimics组)、miR-21抑制物(miR-21抑制组)分别转染A549细胞,CCK-8检测细胞增殖情况;划痕实验检测细胞迁移能力; Transwell侵袭实验检测细胞侵袭能力。双荧光素酶报告实验验证miR-21和Atg5之间的靶向关系。Western blotting检测LC3B-II、p62和Atg5蛋白的表达。结果与NC组比较,miR-21 mimics组细胞增殖、迁移、侵袭能力均上调,miR-21抑制组细胞增殖、迁移、侵袭能力均下调(P<0. 05)。双荧光素酶报告实验结果显示,miR-21显著抑制野生型Atg5 3’-UTR质粒转染细胞的荧光素酶活性(P<0. 05),但对突变型Atg5 3’-UTR的基因报告质粒与miR-21 mimics共转染之后,并未对荧光素酶活性产生影响。NC组LC3BII蛋白表达量为1. 24±0. 059,低于miR-21 mimics组的1. 98±0. 077,高于miR-21抑制组的0. 52±0. 021(P<0. 05); NC组p62蛋白表达量为0. 62±0. 021,高于miR-21 mimics组的0. 45±0. 020,低于miR-21抑制组的0. 79±0. 031(P<0. 05); NC组Atg5蛋白表达量为1. 17±0. 025,高于miR-21 mimics组的0. 38±0. 014,低于miR-21抑制组的1. 40±0. 039(P<0. 05)。与NC组比较,3-MA处理降低miR-21 mimics转染诱导的A549细胞增殖能力(P<0. 05);划痕实验和Transwell实验表明,3-MA处理抑制了miR-21mimics转染诱导的A549细胞的迁移和侵袭,差异有统计学意义(P<0. 05)。结论 miR-21靶向Atg5调控NSCLC自噬促进细胞增殖、迁移和侵袭。
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.
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.
引文
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[3] Yu H,Guan Z,Cuk K,et al. Circulating microRNA biomarkers for lung cancer detection in Western populations[J/OL]. Cancer Med, 2018[2018-08-26]. https://www. onacademic. com/detail/journal_1000040860576310_2e87.html.
[4] Aiso T,Ohtsuka K,Ueda M,et al. Serum levels of candidate microRNA diagnostic markers differ among the stages of nonsmall-cell lung cancer[J]. Oncol Lett,2018,16(5):6643-6651.
[5] Guo H,Sadoul R,Gibbings D. Autophagy-independent effects of autophagy-related-5(Atg5)on exosome production and metastasis[J]. Mol Cell Oncol,2018,5(3):1445941.
[6] Iachettini S,Trisciuoglio D,Rotili D,et al. Pharmacological activation of SIRT6 triggers lethal autophagy in human cancer cells[J]. Cell Death Dis,2018,9(10):996.
[7] Gill RR,Murphy DJ,Kravets S,et al. Success of genomic profiling of non-small cell lung cancer biopsies obtained by trans-thoracic percutaneous needle biopsy[J]. J Surg Oncol,2018,118(7):1170-1177.
[8] Qiu F,Gu WG,Li C,et al. Analysis on expression level and diagnostic value of miR-19 and miR-21 in peripheral blood of patients with undifferentiated lung cancer[J]. Eur Rev Med Pharmacol Sci,2018,22(23):8367-8373.
[9] Zheng W,Zhao J,Tao Y,et al. MicroRNA-21:A promising biomarker for the prognosis and diagnosis of non-small cell lung cancer[J]. Oncol Lett,2018,16(3):2777-2782.
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[12] Dong Z,Cui H. The Autophagy-lysosomal pathways and their emerging roles in modulating proteostasis in tumors[J]. Cells,2018,8(1):4.
[13] Sharma S. Autophagy-based diagnosis of pregnancy hypertension and pre-eclampsia[J]. Am J Pathol,2018,188(11):2457-2460.
[14] Ma R,Zhang Y,Wang W,et al. Inhibition of autophagy enhances the antitumour activity of tigecycline in multiple myeloma[J]. J Cell Mol Med,2018,22(12):5955-5963.
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