Abstract:

Objective To investigate the effect and mechanisms of Toxoplasma gondii type Ⅰ and Ⅲ rhoptry protein 16 (ROP16) on the proliferation and apoptosis of human monocytic leukemia THP-1 cells via TATA-binding protein-associated factor 15 (TAF15). Methods THP-1 cells were transfected with entiviruses overexpressing T. gondii type Ⅰ and Ⅲ ROP16 to generate cell lines that stably expressed ROP16 (THP-1-ROP16 Ⅰ/Ⅲ), and cell transfected with lentiviruses containing empty vectors (THP-1-Venus) served as an empty vector control, while non-transfected cells (THP-1) served as controls. The efficiency of overexpression was checked using quantitative real-time PCR (RT-qPCR) assay and Western blotting. The proteins interacting with ROP16 were identified using immunoprecipitation-mass spectrometry (IP-MS) in THP-1-ROP16 Ⅰ/Ⅲ cell lines, and the expression of the ROP16-interacting protein TAF15 was quantified using RT-qPCR and Western blotting assays. Three siRNA targeting different sites of TAF15 gene (siRNA 1215, siRNA 825, siRNA 288) were used to interfere with THP-1-ROP16 Ⅰ/Ⅲ cell lines and divided into THP-1-ROP16 Ⅰ/Ⅲ + siRNA 1215/825/288 groups, while an undisturbed control group (THP-1-ROP16 Ⅰ/Ⅲ + siRNA NC) was set up and the silencing efficiency of TAF15 was checked using Western blotting. In addition, the cell proliferation and apoptosis was measured using cell counting kit-8 (CCK-8) assay and flow cytometry, and the expression of cyclin-dependent kinase inhibitor 1A (p21), cyclin-dependent kinase 6 (CDK6), G1/S-specific cyclin (CyclinD1), B-cell lymphoma/leukemia-2 protein (Bcl-2), Bcl-2-associated X protein (Bax), cleaved caspase-3, caspase-9 and phosphorylated signal transducer and activator of transcription 3 (P-STAT3) was determined using Western blotting. Results The relative ROP16 mRNA expression was 2 679.427 ± 250.600 in the THP-1-ROP16 Ⅰ group and 2 395.410 ± 325.700 in the THP-1-ROP16 Ⅲ group, which was both higher than in the THP-1-Venus group (1.036 ± 0.102) (F = 153.3, P < 0.01), and the relative ROP16 protein expression was higher in the THP-1-ROP16 Ⅰ group (4.526 ± 0.020) and THP-1-ROP16 Ⅲ group (5.457 ± 0.250) than in the THP-1 Venus group (1.688 ± 0.653) (F = 76.4, P < 0.01). TAF15 was identified as a protein interacting with type Ⅰ and Ⅲ ROP16, and the relative TAF15 mRNA and protein expression was both higher in the THP-1 ROP16 Ⅰ group (6.027 ± 0.313 and 1.789 ± 0.145) and THP-1 ROP16 Ⅲ group (5.567 ± 0.088 and 1.593 ± 0.029) than in the THP-1 Venus group (0.985 ± 0.027 and 1.010 ± 0.365) (F = 869.4 and 50.6, P < 0.01). The relative TAF15 protein expression was 0.384 ± 0.047, 0.246 ± 0.072, and 0.125 ± 0.026 in the THP-1 ROP16 Ⅰ + siRNA 1215 group, the THP-1 ROP16 Ⅰ + siRNA 825 group, and the THP-1 ROP16 Ⅰ + siRNA 288 group 48 hours post-transfection with TAF15 siRNA, which was all lower than in the THP-1-Venus group (1.007 ± 0.019) (F = 313.1, P < 0.01), and the relative TAF15 protein expression was 0.186 ± 0.020, 0.180 ± 0.015, and 0.112 ± 0.019 in the THP-1 ROP16 Ⅲ + siRNA 1215 group, the THP-1 ROP16 Ⅲ + siRNA 825 group, and the THP-1 ROP16 Ⅲ + siRNA 288 group 48 hours post-transfection with TAF15 siRNA, which was all lower than in the THP-1 Venus group (0.995 ± 0.052) (F = 3 046.0, P < 0.01). CCK-8 assay measured the A₄₅₀ values of 0.803 ± 0.015 and 0.813 ± 0.011 in the THP-1-ROP16 Ⅰ + siRNA NC group and the THP-1-ROP16 Ⅲ + siRNA NC group, which were both lower than in the THP-1 Venus group (0.997 ± 0.010 and 0.995 ± 0.016) (t = 19.2 and 24.0, both P < 0.01). A₄₅₀ values of THP-1-ROP16 Ⅰ/Ⅲ + siRNA 825/288 groups were 0.986 ± 0.010, 0.983 ± 0.004; 0.980 ± 0.006, 0.984 ± 0.010 (F = 3.5, 2.9; both P > 0.05), respectively. The apoptotic rates of THP-1 cells were (38.19 ± 0.45)% in the THP-1-ROP16 Ⅰ + siRNA NC group and (38.06 ± 0.84)% in the THP-1-ROP16 Ⅲ + siRNA NC group, which were both higher than in the THP-1-Venus group [(28.41 ± 0.69)% ] (t = 20.5 and 17.7; both P < 0.01). The apoptotic rates of THP-1-ROP16 Ⅰ/Ⅲ + siRNA 825/288 group were (30.03 ± 1.83)%, (28.78 ± 0.72)%; (29.33 ± 0.80)%, (28.94 ± 0.58)% (F = 1.5, 0.4，both P > 0.05). The relative expression of p21, Bax, Caspase-9, Cleaved Caspase-3, and P-STAT3 proteins was 1.322 ± 0.027, 1.493 ± 0.030, 1.349 ± 0.021, 1.324 ± 0.020, and 10.500 ± 1.005 in the THP-1-ROP16 Ⅰ + siRNA NC group, which was all higher than in the THP-1-Venus group (1.000 ± 0.026, 0.996 ± 0.016, 0.989 ± 0.019, 0.994 ± 0.010 and 1.000 ± 0.001) (t = 14.8, 25.4, 22.3, 25.0 and 15.6; all P < 0.01), and the relative CDK6, CyclinD1, and Bcl-2 protein expression was 0.387 ± 0.040, 0.424 ± 0.030, and 0.438 ± 0.035 in the THP-1-ROP16 Ⅰ + siRNA NC group, which was all lower than in the THP-1-Venus group (0.989 ± 0.018, 1.000 ± 0.074 and 0.991 ± 0.016) (t = 23.6, 12.4 and 25.0; all P < 0.01). The relative expression of p21, Bax, caspase-9, cleaved caspase-3, and P-STAT3 proteins was 1.409 ± 0.020, 1.493 ± 0.030, 1.349 ± 0.021, 1.324 ± 0.020, and 16.210 ± 0.664 in the THP-1-ROP16 Ⅲ + siRNA NC group, which was all higher than in the THP-1-Venus group (1.004 ± 0.032, 0.996 ± 0.015, 0.989 ± 0.019, 0.994 ± 0.010 and 1.000 ± 0.001) (t = 18.7, 25.4, 22.3, 25.0 and 39.7; all P < 0.01), and the relative expression of CDK6, CyclinD1, and Bcl-2 proteins was 0.418 ± 0.021, 0.357 ± 0.040, and 0.411 ± 0.019 in the THP-1-ROP16 Ⅲ + siRNA NC group, which was all lower than in the THP-1-Venus group (1.000 ± 0.001, 1.001 ± 0.042 and 0.991 ± 0.016) (t = 47.7, 19.1 and 40.7; all P < 0.01). Conclusion T. gondii type Ⅰ and Ⅲ ROP16 proteins inhibit THP-1 cell proliferation and promote cell apoptosis through promoting TAF15 expression, which may be associated with inhibition of activation of the STAT3 signaling pathway in THP-1 cells.

Key words: Toxoplasma gondii, ROP16 protein, TAF15, THP-1 cell