Effection of different genotypes of Toxoplasma gondii ROP16 on apoptosis and cell cycle of human lung epithelial cells
To investigate the effects and molecular mechanisms of Toxoplasma gondii type Ⅰ,Ⅱ,andⅢROP16 proteins on apoptosis and cell cycle in human lung epithelial BEAS-2B cells.Lentiviral over-expression vectors were constructed for overexpression of type Ⅰ,Ⅱ and Ⅲ ROP16 and transfected into BEAS-2B cells,which were screened by puromycin to obtain stable overexpression of the three different genotypes of ROP16 cell lines,and the cells were cultured and the whole proteins and total RNA were extracted.The effects of type Ⅰ,type Ⅱand typeⅢ ROP16 on the relative expression of apoptosis-asso-ciated proteins BAX,BCL-2,Caspase9(CAS9),P53 and cell cycle-associated proteins CDK6,P21 and Cyclin Dl at the Mrna and protein levels were detected by Western-blot and RT-Qpcr,and the effects of the ROP16 on the phosphorylation level of STAT3 in cells were also further investigated.Western-blot and RT-Qpcr results showed that three different genotypes of lentiviral overexpression vectors transfected BEAS-2B cells with ROP16 Mrna and protein were obviously expressed,suggesting that the cell model was successfully constructed.The detection results of apoptosis and cell cycle associated proteins showed that compared with the blank control group and negative control group,all three genotypes of ROP16 could cause down-regulation of apoptosis-related proteins BAX and BCL-2,and the expression levels of CAS9 and P53 could be reduced by type Ⅰ and type Ⅲ ROP16,and there was a statistically significant difference;Western-blot results showed that compared with the blank control group and negative control group,the expression levels of cellcycle-associated proteins P21 and Cyclin D1 were down-regulated by the three genotypes of ROP16,and CDK6 expression was down-regulated by the three genotypes.CDK6 expression was down-regulated in type Ⅰ and type Ⅲ ROP16 compared with the blank control and negative control groups,and the expression of STAT3 was down-regulated in BEAS-2B cells of type Ⅰ and type Ⅲ ROP16 com-pared with the blank control and negative control groups,while the expression of P-STAT3 was down-reg-ulated in BEAS-2B cells of type Ⅱ ROP16,and both of them were statistically significant.In conclu-sion,Type Ⅰ and Ⅲ Toxoplasma gondii ROP16 more significantly regulate the expression of apoptosis and cell cycle-related factors and cellular STAT3 phosphorylation in BEAS-2B cells compared to type Ⅱ.
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