ABSTRACT
Objective:To investigate the effects of centromere protein-A (CENP-A) on the invasion and migration of ovarian cancer (OC) cells and explore the related mechanism.Methods:OC cell line A2780 was cultured in vitro, and they were divided into Ng Group (Blank Control Group) , pcDNA group (negative transfection group:PCDNA vector plasmid) , pcDNA-CENP-A group (over-expression Group: pcDNA-CENP-A Vector Plasmid) and pathway inhibitor group (TRANSFECTION-CENP-A+ PI3K pathway inhibitor LY294002) . The cell proliferation was detected by CCK-8 method; the cell migration and invasion was detected by Scratch test and Transwell test; the expression of CENP-A, E-cadherin, N-cadherin and phosphatidylinositol 3-kinase/protein kinase B/nuclear factor-kappa B (PI3K/AKT/NF-κB) pathway related proteins was detected by Western blot.Results:A2780 cells were successfully transfected. After 24 hours, with the extension of culture time, compared with that in NG group [ (0.50±0.07) , (0.72±0.11) , (0.99±0.14) ] and pcDNA group [ (0.55±0.08) , (0.78±0.12) , (1.02±0.15) ], the viability of A2780 cells in pcDNA-CENP-A group [ (0.78±0.12) , (1.03±0.15) , (1.67±0.25) ] and pathway inhibitor group [ (0.63±0.09) , (0.87±0.13) , (1.39±0.20) ] increased significantly ( P<0.05) , compared with that in the pcDNA-CENP-A group, the viability of A2780 cells in the pathway inhibitor group was significantly decreased ( P<0.05) , in a time-dependent manner. Compared with those in NG group [ (15.83±1.46) %, (105.32±15.78) individual] and pcDNA group [ (16.79±1.46) %, (108.98±16.35) individual], the migration rate [ (37.96±5.80) %, (25.15± 2.19) %] and invasion number [ (327.87±49.18) individual, 206.53±30.97) individual] of A2780 cells, protein expression of CENP-A, N-cadherin, Vimentin, p-PI3K/PI3K, p-AKT/AKT, NF-κB, interleukin (IL-1β) , tumor necrosis factor-α (TNF-α) in pcDNA-CENP-A group and pathway inhibitor group were significantly higher ( P<0.05) , the expression of E-cadherin was significantly lower ( P<0.05) ; compared with those in the pcDNA-CENP-A group, the migration rate and invasion number of A2780 cells, protein expression of CENP-A, N-cadherin, Vimentin, p-PI3K/PI3K, p-AKT/AKT, NF-κB, interleukin (IL-1β) , tumor necrosis factor-α (TNF-α) in pathway inhibitor group were significantly lower ( P<0.05) , and the expression of E-cadherin was significantly higher ( P<0.05) . Conclusion:Overexpression of CENP-A can promote the proliferation, invasion and migration of ovarian cancer cells, which may be achieved by activating PI3K/AKT/NF-κB signaling pathway.
ABSTRACT
Objective To investigate the effects of trefoil factor 3 (TFF3) on the proliferation and apoptosis of human thyroid papillary carcinoma cell line (TPC-1) and its molecular mechanism. Methods The lentiviral expression vector of overexpression and knockdown of TFF3 gene were constructed, 293T cell was packaged to produce lentiviral particles, virus solution was collected and transfected into TPC-1 cells, enhanced cell TFF3-TPC-1 and enhanced control group ConTFF3-TPC-1; silencing cell shRNA-TFF3-TPC-1 and silencing control cells shCon -TPC-1. Western blotting, and Real-time PCR were used to detect the expression of TFF3 protein and mRNA of four groups. Growth curve and colony formation assay were used to detect the proliferation. Flow cytometry was used to analyze the apoptosis level of the four groups; Western blotting and immunocytochemistry were used to detect apoptosis-related protein and pathway protein phosphatidylinositol 3-kinase/ protein kinase B (PI3K/ Akt), nuclear factor-κB (NF-κB) expression. Results 1. Overexpression and inhibition of expression of TFF3 stable cell TFF3-TPC-1 and shRNA-TFF3-TPC-1 were constructed suscessfully. 2. The proliferation and cloning ability of TFF3-TPC-1 cells were significantly higher than those of ConTFF3-TPC-1 cells(P<0. 05 or P< 0. 01), the proliferation and cloning ability of shRNA-TFF3-TPC-1 cells were significantly lower than those of shCon-TPC-1 cells(P<0. 01); 3. The apoptosis rate of TFF3-TPC-1 cells was lower than that of ConTFF3-TPC-1 (0. 75%±0. 08% vs 5. 62%±0. 3%, P<0. 01),and the apoptosis rate of shRNA-TFF3-TPC-1 was higher than that of shConTPC-1 (22. 2% ± 1. 2% vs 5. 34% ± 0. 4%, P<0. 01); 4. After silencing TFF3 gene, the expressions of Bax, cytochrome C (Cyt-C), cleaved-Caspase-9, cleaved-Caspase-3 were up-regulated, and the expressions of Bcl-2, Akt, p-Akt and NF-κB-P65 were down-regulated (P<0. 05 or P<0. 01). Conclusion TFF3 may regulate the proliferation and apoptosis of TPC-1 cells by affecting the PI3K/ Akt/ NF-κB signaling pathway.