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OA北大核心CSTPCD
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-qPCR)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mRNA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-Iβ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 type Ⅰ,Ⅱ and Ⅲ ROP16 protein and empty carrier control group were constructed,and the expression of type Ⅰ/Ⅱ/Ⅲ ROP16 was verified by real-time fluorescent quantitative PCR(RT-qPCR)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-siRNA was designed and synthesized,and transfected into A549 cells overexpressing type Ⅰ,Ⅱ and Ⅲ ROP,respectively.And the proliferation of cells in each group was detected by CCK-8 method,and the mRNA and protein expression levels of apoptosis-relat-ed proteins and cell cycle-related proteins were detected by RT-qPCR and Western-blot.Ⅰ Ⅱ,and Ⅲ type 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 type Ⅰ and type Ⅲ ROP16,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 CyclinD1 were significantly down-regulated,suggesting that type Ⅰ and Ⅲ ROP16 proteins could cause apoptosis and cycle arrest.In the silent-NKRF group,the pro-apoptotic effect of A549 cells overexpressing type Ⅰ and Ⅲ ROP16 was weakened.There was no significant cell cycle stagnation and compared with that of type Ⅱ ROP16 protein,there no statistical difference.Toxoplasma Ⅰ and Ⅲ ROP16 proteins can induce apoptosis,cell cycle arrest and inhibit proliferation of A549 cells.
周毓宁;赵志军;李光琪;李佳铭;党甜甜;林永仙;于欣
宁夏医科大学临床医学院,宁夏银川 750004宁夏医科大学总医院医学实验中心,宁夏银川 750004||宁夏临床病原微生物重点实验室,宁夏银川 750004宁夏医科大学总医院医学实验中心,宁夏银川 750004宁夏医科大学检验学院,宁夏银川 750004
畜牧业
非小细胞肺癌ROP16蛋白刚地弓形虫NF-κB通路NF-κB抑制因子
Non-small cell lung cancerROP16 proteinToxoplasma gondiiNF-κB pathwayNF-κB in-hibitor
《中国兽医科学》 2024 (007)
982-992 / 11
国家自然科学基金青年基金项目(NSFC81560333);宁夏自然科学基金一般项目(2023AAC03576)
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