ECT2-siRNA转染对肺腺癌A549细胞周期分布和凋亡的影响
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摘要
目的:探讨ECT2-siRNA转染对肺腺癌A549细胞周期分布和凋亡的影响及其机制。方法:将体外培养的A549细胞分为空白组(未转染)、阴性对照组(转染阴性对照NC-siRNA)和干扰组(转染ECT2-siRNA),采用PI染色检测细胞周期分布,采用Annexin V-FITC和PI双染法检测凋亡,用Western blot检测细胞中ECT2、Cyclin D1、P53和Cleaved Caspase-3蛋白的表达。结果:干扰组细胞中ECT2蛋白的表达水平低于空白组和阴性对照组(P <0. 05)。与其他2组相比,干扰组S期和G2/M期细胞百分比降低,而G0/G1期细胞百分比和细胞凋亡率均升高(P <0. 05),Cyclin D1蛋白的表达水平降低,而P53和Cleaved Caspase-3蛋白的表达水平升高(P <0. 05)。结论:靶向沉默ECT2的表达可诱导A549细胞周期阻滞和凋亡,其作用机制可能与下调Cyclin D1,上调P53、Cleaved Caspase-3蛋白的表达有关。
Aim: To investigate the effects of ECT2-siRNA transfection on cell cycle distribution and apoptosis of lung adenocarcinoma A549 cells. Methods: : A549 cells cultured in vitro were divided into blank group(untransfected),negative control group(transfected with negative control NC-siRNA) and interference group(transfected with ECT2-siRNA).The cell cycle distribution and apoptosis were detected by PI staining and Annexin V-FITC and PI staining,respectively.The expressions of ECT2,Cyclin D1,P53 and Cleaved Caspase-3 protein were detected by Western blot. Results: The expression of ECT2 protein in the interference group was significantly lower than those in the blank group and negative control group(P < 0. 05). Compared with the other 2 groups,the percentage of cells at S phase and G2/M phase decreased significantly in the interference group,while the percentage of cells at G0/G1 phase and apoptotic rate increased significantly(P < 0. 05); at the same time,the expression level of Cyclin D1 protein in the interference group was significantly lower,while the expression levels of P53 and Cleaved Caspase-3 protein were higher(P < 0. 05). Conclusion: ECT2-siRNA transfection could induce cell cycle arrest and apoptosis in A549 cells,and its mechanism may be related to down-regulation of Cyclin D1 and up-regulation of P53 and Cleaved Caspase-3 protein expression.
Aim: To investigate the effects of ECT2-siRNA transfection on cell cycle distribution and apoptosis of lung adenocarcinoma A549 cells. Methods: : A549 cells cultured in vitro were divided into blank group(untransfected),negative control group(transfected with negative control NC-siRNA) and interference group(transfected with ECT2-siRNA).The cell cycle distribution and apoptosis were detected by PI staining and Annexin V-FITC and PI staining,respectively.The expressions of ECT2,Cyclin D1,P53 and Cleaved Caspase-3 protein were detected by Western blot. Results: The expression of ECT2 protein in the interference group was significantly lower than those in the blank group and negative control group(P < 0. 05). Compared with the other 2 groups,the percentage of cells at S phase and G2/M phase decreased significantly in the interference group,while the percentage of cells at G0/G1 phase and apoptotic rate increased significantly(P < 0. 05); at the same time,the expression level of Cyclin D1 protein in the interference group was significantly lower,while the expression levels of P53 and Cleaved Caspase-3 protein were higher(P < 0. 05). Conclusion: ECT2-siRNA transfection could induce cell cycle arrest and apoptosis in A549 cells,and its mechanism may be related to down-regulation of Cyclin D1 and up-regulation of P53 and Cleaved Caspase-3 protein expression.
引文
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[2]GE N,CHU XM,XUAN YP,et al.Associations between abnormal vitamin D metabolism pathway function and nonsmall cell lung cancer[J].Oncol Lett,2017,14(6):7538
[3]王莉洁,梁志欣,喻航,等.肺癌的驱动基因与靶向治疗研究进展[J].国际呼吸杂志,2017,37(4):297
[4]袁媛,周闯,翟文龙.上皮细胞转化序列2诱导肝癌细胞上皮-间充质转化促进侵袭转移[J].中华实验外科杂志,2016,33(9):2144
[5]CHEN CJ,SUNG WW,CHEN HC,et al.Early assessment of colorectal cancer by quantifying circulating tumor cells in peripheral blood:ECT2 in diagnosis of colorectal cancer[J].Int J Mol Sci,2017,18(4):743
[6]CHENG YS,LIN C,CHENG YP,et al.Epithelial cell transformation sequence 2 is a potential biomarker of unfavorable survival in human gliomas[J].Neurol India,2014,62(4):406
[7]WANG HB,YAN HC,LIU Y.Clinical significance of ECT2expression in tissue and serum of gastric cancer patients[J].Clin Transl Oncol,2016,18(7):735
[8]TAN HY,WANG XS,YANG XG,et al.Oncogenic role of epithelial cell transforming sequence 2 in lung adenocarcinoma cells[J].Exp Ther Med,2016,12(4):2088
[9]ZHOU SJ,WANG P,SU XL,et al.High ECT2 expression is an independent prognostic factor for poor overall survival and recurrence-free survival in non-small cell lung adenocarcinoma[J].PLo S One,2017,12(10):e0187356
[10]WANG HK,LIANG JF,ZHENG HX,et al.Expression and prognostic significance of ECT2 in invasive breast cancer[J].J Clin Pathol,2018,71(5):442
[11]GUO ZH,CHEN XJ,DU T,et al.Elevated levels of epithelial cell transforming sequence 2 predicts poor prognosis for prostate cancer[J].Med Oncol,2017,34(1):13
[12]IYODA M,KASAMATSU A,ISHIGAMI T,et al.Epithelial cell transforming sequence 2 in human oral cancer[J].PLo S One,2010,5(11):e14082
[13]XU J,YAO Q,HOU Y,et al.MiR-223/Ect2/p21 signaling regulates osteosarcoma cell cycle progression and proliferation[J].Biomed Pharmacother,2013,67(5):381
[14]XIE J,LEI PF,HU YH.Small interfering RNA-induced inhibition of epithelial cell transforming sequence 2 suppresses the proliferation,migration and invasion of osteosarcoma cells[J].Exp Ther Med,2015,9(5):1881
[15]肖安,彭蓉蓉.ECT2基因沉默调控人乳腺癌细胞增殖和凋亡及其机制探讨[J].肿瘤,2017,37(6):594
[16]詹启敏,陈杰.细胞周期与肿瘤转化医学[J].中国肿瘤临床,2014,41(1):1
[17]郭燕子,符健.肿瘤细胞凋亡及常用检测方法[J].中国热带医学,2017,17(4):413
[18]JOHN RR,MALATHI N,RAVINDRAN C,et al.Mini review:Multifaceted role played by cyclin D1 in tumor behavior[J].Indian J Dent Res,2017,28(2):187
[19]TUDZAROVA S,MULHOLLAND P,DEY A,et al.p53controls CDC7 levels to reinforce G1 cell cycle arrest upon genotoxic stress[J].Cell Cycle,2016,15(21):2958
[20]SUN YY,XIAO L,WANG D,et al.Triptolide inhibits viability and induces apoptosis in liver cancer cells through activation of the tumor suppressor gene p53[J].Int J Oncol,2017,50(3):847
[21]焦佩娟,应小平.Caspase-3与肿瘤细胞凋亡关系的中医药研究进展[J].山东中医杂志,2017,36(8):721