抑制中心体相关蛋白TACC3表达对膀胱尿路上皮癌T24细胞周期的影响

发布时间：2018-05-10 21:59

本文选题：膀胱癌尿路上皮癌 + TACC3　；参考：《天津医科大学》2017年硕士论文

【摘要】：目的:探讨中心体相关转录相关酸性卷曲蛋白3(TACC3)在膀胱癌T24细胞系中的表达情况,并探讨抑制中心体相关蛋白TACC3在膀胱尿路上皮癌T24细胞系中的表达水平对细胞周期的影响及相关周期蛋白的变化,初步探讨TACC3影响细胞周期变化的基本分子机制,为临床膀胱癌的诊疗与预后提供相关的参考信息。方法:Western blot半定量实验方法检测T24细胞系及膀胱上皮永生化细胞SV-HUC-1细胞系中TACC3蛋白的表达情况;转染特异性TACC3-sh RNA相关质粒下调TACC3蛋白表达水平并验证其转染效果;流式细胞技术检测抑制TACC3蛋白表达后,膀胱癌T24细胞系细胞周期的变化,同时检测被阻滞位点周期相蛋白蛋白量的表达水平,初步探讨TACC3影响膀胱癌T24细胞系细胞周期的基本分子机制。结果:实验结果发现TACC3蛋白水平在膀胱尿路上皮癌T24细胞中高表达,Western Blot结果灰度扫描表明:两组灰度值(0.30±0.03、0.85±0.07)。差异明显符合统计学要求(P0.05)。在Hela细胞中应用荧光定量PCR(q PCR)技术初步验证4个靶点的干扰效率,获得了对TACC3干扰效率达到92.6%的p Yr-LVX-TACC3-sh RNA质粒(即sh RNA4);下调TACC3蛋白表达水平后,流式细胞技术结果显示,sh RNA组细胞周期被阻滞在G1期,与Untreated组和Negative control组相比差异具有统计学意义(P0.05);Western Blot检测G1期相关周期蛋白表达水平结果显示,CDK4、CDK6及Cyclin D1的表达均受到抑制,与Untreated组和Negative control组相比差异具有统计学意义(P0.05);在探讨TACC3影响膀胱癌T24细胞系细胞周期的基本分子机制时。p53蛋白是细胞增殖和分化的重要调节因子,文献表明探索p53上游通路p38(细胞分裂素活化蛋白激酶)作为一个对G1期停滞的重要贡献者介导了相关应激反应。TACC3蛋白水平的抑制导致p53蛋白水平增加,同样的现象也发生在p21和p38蛋白上。我们发现TACC3蛋白表达的抑制激活了p38,从而使p53和p21的水平增加,与Untreated组和Negative control组相比差异具有统计学意义(P0.05)。进一步研究发现,特异性抑制p38表达后,由TACC3蛋白表达的抑制诱导的细胞G1期阻滞被释放且与sh RNA组比较差异具有统计学意义,同时我们发现,p53及p21蛋白的表达也受到抑制。结论:TACC3在膀胱尿路上皮癌T24细胞系中高表达,转染重组质粒(p Yr-LVX-TACC3-sh RNA4)下调TACC3蛋白的表达后,膀胱尿路上皮癌T24细胞系被阻滞在G1期且G1期相关周期蛋白CDK4、CDK6及Cyclin D1的表达均受到抑制,进一步研究结果提示TACC3蛋白表达的抑制激活了p38,从而使p53和p21的水平增加,我们猜测TACC3的抑制通过p38 p53 p21应激信号通路诱导G1期阻滞。
[Abstract]:Objective: to investigate the expression of centrosomal transcription-associated acid convoluted protein 3tact CC3 in bladder cancer cell line T24. The effects of inhibition of centrosomal associated protein (TACC3) expression in bladder urothelial carcinoma cell line T24 on cell cycle and the changes of related cyclin were investigated. The basic molecular mechanism of TACC3 affecting cell cycle was also discussed. To provide reference information for the diagnosis, treatment and prognosis of bladder cancer. Methods the expression of TACC3 protein in T24 cell line and SV-HUC-1 cell line of bladder epithelial immortalized cell line was detected by the semi-quantitative method of 1: Western blot, and the expression level of TACC3 protein was down-regulated by transfection specific TACC3-sh RNA related plasmids and its transfection effect was verified. Flow cytometry was used to detect the cell cycle changes of bladder cancer cell line T24 after inhibiting the expression of TACC3 protein, and the expression level of cyclin at the blocked site was also detected. To explore the basic molecular mechanism of TACC3 affecting the cell cycle of bladder cancer cell line T 24. Results: the results showed that the expression of TACC3 protein was high in T24 cells of bladder urothelial carcinoma. The results of gray-scale scanning showed that the gray values of the two groups were 0.30 卤0.03 and 0.85 卤0.07 respectively. The difference was obviously in line with the statistical requirements (P 0.05). The interference efficiency of the four target sites was preliminarily verified by fluorescence quantitative PCR(q PCR in Hela cells. The plasmid of p Yr-LVX-TACC3-sh RNA (sh RNA4), which had the interference efficiency of 92.6% to TACC3, was obtained, and the expression level of TACC3 protein was down-regulated. The results of flow cytometry showed that the cell cycle was blocked in G1 phase in RNA group, and there was a significant difference compared with Untreated group and Negative control group. The results showed that the expression of CDK4, CDK6 and Cyclin D1 were inhibited by Western Blot. Compared with Untreated group and Negative control group, the difference was statistically significant (P 0.05). In order to explore the basic molecular mechanism of TACC3 affecting the cell cycle of bladder cancer cell line T24, p53 protein is an important regulatory factor for cell proliferation and differentiation. It has been reported that exploring p53 upstream pathway p38 (cytokinin-activated protein kinase), as an important contributor to G1 phase arrest, mediates the inhibition of the related stress response. TACC3 protein level may lead to the increase of p53 protein level. The same is true of p21 and p38 proteins. We found that the inhibition of the expression of TACC3 protein activated p38, which increased the levels of p53 and p21. The difference was statistically significant compared with that of Untreated and Negative control groups. Further studies showed that after the specific inhibition of p38 expression, the G1 phase arrest induced by the inhibition of TACC3 protein expression was significantly different from that of sh RNA group, and the expression of p53 and p21 proteins was also inhibited. Conclusion the expression of T24 cell line in bladder urothelial carcinoma cell line T24 was highly expressed by T24, and the expression of TACC3 protein was down-regulated by transfection of recombinant plasmid p Yr-LVX-TACC3-sh RNA4. T24 cell line of bladder urothelial carcinoma was blocked in G1 phase and the expression of CDK4, CDK6 and Cyclin D1 was inhibited. The further results indicated that the inhibition of TACC3 protein activated p38, which increased the levels of p53 and p21. We speculate that the inhibition of TACC3 induces G 1 arrest through p38 p53 p21 stress signaling pathway.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.14

【参考文献】