The Suppression Of Centrosome Prote TACC3 And Its Influence To Cell Cycle In Bladder Urothelial Carcinoma T24 Cells

Objective:To detect the expression of centrosome protein TACC3 protein in bladder cancer T24 cells.we investigate the effect of the expression of centrosome protein TACC3 on the cell cycle in the bladder carcinoma T24 cell line.Then we examined the levels of cell cycle-associated proteins expression.Final we investigated the role of the p38–p53–p21 stress signaling pathway in the TACC3 depletion-induced effects in bladder cancer T24 cells,providing a new way for the diagnosis and prognosis of bladder cancer.Methods:Western blot was used to detect the expression of TACC3 protein in T24 cell line and bladder epithelium immortalized cell SV-HUC-1 cell line.A group of sh RNA was screened as the highest transfection efficiency.Then we use the most effective TACC3-sh RNA transfects bladder cancer T24 cell to suppress TACC3 level.Then we use the flow cytometry to observe the cell cycle and cycle-associated proteins expression.Finally,Western Blot technique was used to investigate the basic molecular mechanism of TACC3 on cell cycle of bladder cancer T24 cell line..Results:1.The result of western blot showed that the expression level of TACC3 protein in T24 cells was obviously higher than that in bladder epithelium immortalized cell SV-HUC-1 cells,the difference was statistically significant(P < 0.05).Western Blot results showed that the gray scale values(0.30 ± 0.03,0.85 ± 0.07).2.The p Yr-LVX-TACC3-sh RNA(sh RNA4)with 92.4% interference was obtained by fluorescence quantitative PCR(q PCR)technique in Hela cells.3.We first determined the effect of TACC3 depletion on cell cycle progression in bladder T24 cells.After transfection with TACC3 sh RNA for 48 h,the cells were stained with PI,and the DNA profiles were analyzed by flow cytometry.Suppression of TACC3 significantly increased the percentage of G1 cells.This result was statistically significant compared to the control.So we confirm that TACC3-depletion blocked the cells to proceed from G1 to S phase.4.Using western blot to detect the expression levels of cell cycle-associated proteins: TACC3 depletion decreased the levels of G1-associated proteins(CDK4CDK6 and Cyclin D1).Together,these results demonstrate that the suppression of TACC3 arrests cells at G1.5.p53 is a key regulator of cell cycle progression and apoptosis.Therefore,we next investigated whether TACC3 depletion-induced G1 arrest are dependent on p53.For this,we analyzed the effects of TACC3 depletion on cell cycle progression in bladder T24 cells.TACC3 depletion increased the levels of p53.As the p38-mediated signal transduction pathway is known to activate p53 under cellular stress.Next,we determined the role of p21 in this pathway.It should be noted that the cell cycle profiles of the p21 or p38 were similar to that of the p53 in bladder T24 cells.(not shown).We found that pharmacological inhibition of p38 by a small-molecule inhibitor,SB202190,followed by TACC3 depletion released cells from G1 arrest.Our findings suggest that TACC3 depletion-induced G1 arrest are mediated via a stress response involving p38 and activation of the p38–p53–p21 signaling pathway.Conclusion:TACC3 was highly expressed in bladder urothelial carcinoma T24 cell line.Then we use the most effective TACC3-sh RNA transfects bladder cancer T24 cell to suppress TACC3 level(The interference effect was as high as 92.6%).TACC3-depletion blocked the cells to proceed from G1 to S phase and decreased the levels of G1-associated proteins(CDK4 CDK6 and Cyclin D1).Our findings suggest that TACC3 depletion-induced G1 arrest are mediated via a stress response involving p38 and activation of the p38–p53–p21 signaling pathway.