Activation of STAT3 phosphorylation by Toxoplasma gondii ROP16 Ⅰ/Ⅲ protein regulates the cell cycle,proliferation,and apoptosis of THP-1 cells
10.3969/j.issn.1002-2694.2024.00.171
- VernacularTitle:弓形虫ROP16 Ⅰ/Ⅲ蛋白激活STAT3磷酸化调节THP-1细胞周期、增殖及凋亡
- Author:
He YIN
1
;
Lei MA
;
Tian-tian DANG
;
Jia-ming LI
;
Ting-ting TIAN
;
Shan-ni MA
;
Zhi-jun ZHAO
Author Information
1. 宁夏医科大学总医院,银川 750004;宁夏临床病原微生物重点实验室,银川 750004
- Publication Type:Journal Article
- Keywords:
ROP16 protein;
THP-1 cells;
cell cycle;
cell proliferation;
cell apoptosis
- From:
Chinese Journal of Zoonoses
2024;40(12):1115-1121,1127
- CountryChina
- Language:Chinese
-
Abstract:
This study was aimed at exploring the effects of Toxoplasma gondii ROP16 Ⅰ/Ⅲ protein on the cell cycle,pro-liferation,and apoptosis of THP-1 cells via activation of STAT3 phosphorylation.ROP16 empty vector and recombinant lenti-viral overexpression vector were transfected into THP-1 cells,the fluorescence intensity was observed,and overexpression was verified.STAT3 and P-STAT3 protein expression in THP-1 cells overexpressing ROP16 Ⅰ/Ⅲ was detected by Western blot-ting.The cells were treated with the STAT3 phosphorylation inhibitor Stattic,and divided into a THP-1 group,THP-1+Stattic group,THP-1-ROP16 Ⅰ/Ⅲ group,and THP-1-ROP16Ⅰ/Ⅲ+Stattic group.Changes in the cell cycle,proliferation curve,and apoptosis rate were detected with flow cytometry and CCK-8 assays,and the expression levels of the apoptosis-related proteins Bax,Cleaved Caspase3,Caspase9,and Bcl-2 were detected by Western blotting.A strong fluorescence signal was ob-served after transfection of ROP16 recombinant lentivirus overexpression vector into THP-1 cells,and ROP16 mRNA and pro-tein expression significantly increased(FmRNA=314.1,Fprotein=758.7,P＜0.001),thus indicating successful construction of cell strains with stable overexpression.In addition,P-STAT3 protein showed higher expression in THP-1-ROP16 Ⅰ/Ⅲ group than the THP-1 group(F=606.1,P＜0.001).Flow cytometry and CCK-8 results indicated that,compared with the THP-1 group,the THP-1-ROP16 Ⅰ/Ⅲ group showed a cell cycle blocked in G1 phase,lower cell proliferation ability(t Ⅰgroup=19.2,tⅢgroup=24.0,P＜0.01),and a higher apoptosis rate(t Ⅰgroup=175.1,tⅢgroup=205.2,P＜0.001).Western blotting results also demonstrated significantly elevated expression of the intracellular pro-apoptotic proteins Bax,Cleaved Caspase3,and Caspase9(t Bax Ⅰgroup=15.8,tBaxⅢgroup=11.1,t Caspase9 Ⅰ group=9.1,t Caspaseg Ⅲ group=10.2,t Cleaved Caspase3 Ⅰ group=16.5,t Cleaved Caspase3 Ⅲ group=12.9,all P＜0.001),and significantly diminished expression of the anti-apoptotic protein Bcl-2(t Ⅰgroup=18.4,tⅢgroup=26.2,P＜0.001).However,after Stattic treatment,the cell cycle,proliferation ability,apoptosis rate,and expression of apoptosis-re-lated proteins in the THP-1-ROP16 Ⅰ/Ⅲ+Stattic group were restored to near baseline levels.Thus,Toxoplasma gondii ROP16 Ⅰ/Ⅲ proteins were found to block the THP-1 cell cycle at G1 phase,inhibit cell proliferation,and promote apoptosis by activating the phosphorylation of STAT3.
