shRNA沉默CCAT2通过抑制自噬对非小细胞肺癌细胞A549化疗敏感性的影响

Effects of shRNA silencing CCAT2 on chemosensitivity of non-small cell lung cancer cell line A549 by inhibiting autophagy

目的:探究shRNA沉默CCAT2抑制自噬对非小细胞肺癌细胞A549化疗敏感性的影响及机制。方法:细胞分为A549/DDP组、Scramble组、CCAT2 shRNA组,转染相应的shRNA处理细胞,RT-PCR检测CCAT2基因表达水平,CCK8检测A549/DDP的细胞活力,Hoeschst和流式细胞法检测细胞凋亡,Western blot检测B细胞淋巴瘤/白血病(Bcl-2)、Bcl-2相关X蛋白(Bax)、活性半胱天冬酶3(cl-caspase-3)、微管相关蛋白1轻链3Ⅱ(LC3Ⅱ)、LC3Ⅰ、Beclin1、p62蛋白表达水平。结果:与A549细胞株相比,A549/DDP细胞株中CCAT2表达水平显著升高。与A549/DDP组相比,CCAT2 shRNA组CCAT2表达水平显著降低,细胞活力显著降低,细胞凋亡率显著升高,凋亡相关蛋白Bax、cl-caspase-3蛋白表达水平显著升高,Bcl-2蛋白表达水平显著降低,自噬相关蛋白LC3Ⅱ/LC3Ⅰ蛋白水平比率显著降低,Beclin1蛋白表达水平降低,p62蛋白表达水平升高。结论:沉默CCAT2可提高非小细胞肺癌细胞A549株化疗敏感性,作用机制可能与抑制自噬作用有关。

Objective:To explore the effects of shRNA silencing CCAT2 on chemosensitivity of non-small cell lung cancer cell line A549 by inhibiting autophagy and its mechanism.Methods:Cells were divided into A549/DDP,Scramble,CCAT2 shRNA group,each group was transfected with corresponding shRNA,RT-PCR was performed for measuring the level of CCAT2,cell viability of A549/DDP was measured by CCK8,cell apoptosis was determined by Hoeschst and flow cytometry.Western blot was used to determine the protein levels of B-cell lymphoma/leukemia 2(Bcl-2),Bcl-2 associated X protein(Bax),Cleave Caspase-3(cl-caspase-3),microtubulesas sociated protein light chain 3Ⅱ(LC3Ⅱ),LC3Ⅰ,Beclin1,p62.Results:Compared with A549 cell lines,the gene level of CCAT2 was increased notably in A549/DDP cell lines.Compared with A549/DDP group,the gene level of CCAT2 was decreased significantly,the cell viability was decreased markedly,the cell apoptosis was increased notably,and the apoptosis related protein levels of Bax,cl-caspase-3 were increased markedly,the protein level of Bcl-2 was decreased significantly,the autophagy related protein ratio of LC3Ⅱ/LC3Ⅰ,protein level of Beclin1 were decreased significantly,the protein level of p62 was increased notably.Conclusion:Silence of CCAT2 may induced the chemosensitivity of non-small cell lung cancer cell line A549,and the mechanism may related to inhibit autophagy.