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