弓形虫ROP16蛋白与NKRF互作对人非小细胞肺癌A549细胞凋亡和周期的影响

Effect of Toxoplasma gondii ROP16 protein interaction with NKRF on apoptosis and cycle of human non-small cell lung cancer A549 cells

为探讨刚地弓形虫Ⅰ/Ⅱ/Ⅲ型ROP16蛋白与NKRF互作对A549细胞凋亡、周期及增殖的影响。本研究通过构建过表达Ⅰ、Ⅱ、Ⅲ型ROP16蛋白和空载体对照组的慢病毒感染A549细胞,并通过实时荧光定量PCR(RT-q PCR)和Western-blot对Ⅰ/Ⅱ/Ⅲ型ROP16的表达进行验证。利用免疫共沉淀与液相色谱-质谱联用技术获取同ROP16可能发生互作的蛋白,并利用Venn图、Score分值、Intensity分值筛选出评分较高的互作蛋白NKRF进行验证。设计并合成NKRF-siRNA,分别转染至过表达Ⅰ/Ⅱ/Ⅲ型ROP16细胞,并利用CCK-8法检测各组细胞增殖情况,利用RT-qPCR和Western-blot分别对凋亡相关蛋白及细胞周期相关蛋白的m RNA和蛋白表达水平进行检测。此次试验成功构建了Ⅰ/Ⅱ/Ⅲ型ROP16过表达A549稳转细胞株。利用IP-MS及CO-IP技术筛选并验证ROP16互作蛋白NKRF。筛选并构建NKRF沉默的过表达各型ROP16蛋白A549细胞。在过表达Ⅰ型、Ⅲ型ROP16的A549细胞中,促凋亡蛋白Bax表达水平上调,抑凋亡蛋白BCL-2、Caspase-9、p53表达水平明显下调;细胞周期抑制蛋白p21表达明显上调,而G1/S期相关蛋白CDK6和CyclinD1蛋白表达明显下调,提示Ⅰ、Ⅲ型ROP16蛋白会引起细胞凋亡和周期停滞,而在沉默NKRF组中,过表达Ⅰ型、Ⅲ型ROP16蛋白的A549细胞中Ⅰ型、Ⅲ型ROP16蛋白的促凋亡作用减弱,细胞周期无明显停滞,与Ⅱ型ROP16蛋白之间没有统计学差异。综上所述得出弓形虫Ⅰ、Ⅲ型ROP16蛋白通过与NKRF互作,诱导A549细胞凋亡、细胞周期阻滞并抑制A549细胞增殖。

To investigate the effects of interaction between ROP16 protein and NKRF on apoptosis,cell cycle and proliferation in A549 cells.In this study,lentivirus-infected A549 cells overexpressing typeⅠ,ⅡandⅢROP16 protein and empty carrier control group were constructed,and the expression of typeⅠ/Ⅱ/ⅢROP16 was verified by real-time fluorescent quantitative PCR(RT-q PCR)and Western-blot analysis.The proteins that may interact with ROP16 were obtained by co-immunoprecipitation and liquid chromatography-mass spectrometry,and the interacting protein NKRF with high score was selected by Venn,score and intensity for verification.NKRF-si RNA was designed and synthesized,and transfected into A549 cells overexpressing typeⅠ,ⅡandⅢROP,respectively.And the proliferation of cells in each group was detected by CCK-8 method,and the m RNA and protein expression levels of apoptosis-related proteins and cell cycle-related proteins were detected by RT-q PCR and Western-blot.ⅠⅡ,andⅢtype ROP16 overexpressing A549 stable cell line was constructed successfully.The ROP16 interacting protein NKRF was screened and verified by IP-MS and CO-IP techniques.Nkrf-silenced A549 cells overexpressing ROP16 protein were screened and constructed.In A549 cells overexpressing typeⅠand typeⅢROP16,the expression level of pro-apoptotic protein Bax was up-regulated,the expression level of anti-apoptotic protein BCL-2,Caspase-9 and p53 was down-regulated.The expression of cell cycle suppressor protein p21 was significantly up-regulated,while the expressions of G1/S phase related proteins CDK6 and Cyclin D1 were significantly down-regulated,suggesting that typeⅠandⅢROP16 proteins could cause apoptosis and cycle arrest.In the silent-NKRF group,the pro-apoptotic effect of A549 cells overexpressing typeⅠandⅢROP16 was weakened.There was no significant cell cycle stagnation and compared with that of typeⅡROP16 protein,there no statistical difference.ToxoplasmaⅠandⅢROP16 proteins can induce apoptosis,cell cycle arrest and inhibit proliferation of A549 cells.