TEAD4对结直肠癌细胞增殖影响及机制探讨

发布时间：2018-05-22 07:51

  本文选题：结直肠癌 + TEAD　； 参考：《中华肿瘤防治杂志》2017年15期

【摘要】：目的结直肠癌国内外高发,是肿瘤死亡的主要病因之一。TEAD4是YAP/TAZ信号通路中的关键因子。本研究旨在探讨敲低TEAD4的表达后对结直肠癌细胞增殖的影响和机制。方法通过在结直肠癌细胞系Caco2和HT-29中通过转染TEAD4siRNA和感染慢病毒,以达到瞬时或稳定敲低TEAD4的目的,利用蛋白质印迹方法检测转染效率及相关蛋白的表达。用SRB法分别检测敲低TEAD4后Caco2和HT-29细胞的存活率,以及使用EdU掺入法测定细胞DNA合成效率。结果在肠癌细胞中使用TEAD4的siRNA,瞬时敲低TEAD4的表达48h后,蛋白质印迹法检测结果提示,与对照组相比,Caco2(F=99.22,P0.001)和HT-29(F=167.3,P0.001)细胞中的TEAD4蛋白水平显著降低,同时观察到Caco2(F=45.78,P0.001)和HT-29(F=40.71,P0.001)细胞中p27蛋白的表达量有明显的上升,蛋白表达差异有统计学意义。SRB结果显示,使用shRNA慢病毒载体稳定敲低肠癌细胞中TEAD4的表达,与对照组细胞相比,2d沉默TEAD4基因的Caco2(F=142.9,P0.001)和HT-29(F=141.0,P0.001)细胞的生长速度变慢;于3d后这种抑制作用更加明显,Caco2(F=394.5,P0.001)和HT-29(F=305.1,P0.001)细胞增殖速度明显变慢。使用EdU掺入法检测敲低TEAD4后对Caco2和HT-29细胞DNA合成的影响,结果显示,Caco2和HT-29细胞,Caco2/Consi、Caco2/TEAD4si#2、Caco2/TEAD4si#3、HT-29/Consi、HT-29/TEAD4si#2、HT-29/TEAD4si#3的DNA合成率分别为(43.20±2.18)%、(27.19±2.34)%和(30.14±1.02)%及(28.23±3.11)%、(11.89±1.20)%和(17.91±2.23)%,干扰组Caco2/TEAD4si#2(P=0.001)、Caco2/TEAD4si#3(P0.001)、HT-29/TEAD4si#2(P=0.001)及HT-29/TEAD4si#3(P=0.011)细胞的DNA合成率显著低于对照组细胞,说明在Caco2和HT-29细胞中敲低TEAD4后,DNA的合成明显受到了抑制。结论敲低TEAD4对结直肠癌细胞体外增殖和DNA的合成有抑制作用,可能部分通过上调p27的表达,TEAD4可能是结直肠癌发生、发展中潜在的临床诊断或治疗新靶点。
[Abstract]:Objective Colorectal cancer is a major cause of death at home and abroad. TEAD4 is a key factor in YAP/TAZ signaling pathway. The aim of this study was to investigate the effect and mechanism of knockout TEAD4 expression on the proliferation of colorectal cancer cells. Methods transfection of TEAD4siRNA and lentivirus infection in colorectal cancer cell lines Caco2 and HT-29 were carried out to achieve transient or stable knockdown of TEAD4. Western blotting was used to detect transfection efficiency and expression of related proteins. The survival rate of Caco2 and HT-29 cells after TEAD4 knockout was detected by SRB assay, and the DNA synthesis efficiency was measured by EdU incorporation method. Results TEAD4 siRNAs were used in intestinal cancer cells, and the expression of TEAD4 was instantly knocked down for 48 hours. The results of Western blot analysis showed that the TEAD4 protein levels in the cells were significantly lower than those in the control group (P 0.001) and HT-29FN 167.3P 0.001) cells. It was also observed that the expression of p27 protein increased significantly in Caco2FN 45.78 (P0.001) and HT-29 FN 40.71 P0.001) cells, and the difference in protein expression was statistically significant. The results showed that the expression of p27 protein was stabilized by shRNA lentivirus vector, and the expression of p27 protein in low intestinal cancer cells was stabilized by shRNA lentivirus vector. Compared with the control cells, the growth rate of the cells silencing TEAD4 gene for 2 days was slower than that of the control cells, and the growth rate of the cells was much slower than that of the control cells at 3 days after silencing the TEAD4 gene, and the proliferation rate of the cells was significantly slower than that of the control cells, and the proliferation rate of the cells was significantly slower than that of the control cells. The proliferation rate of the cells was significantly slower than that of the control cells. 浣跨敤EdU鎺哄叆娉曟��娴嬫暡浣嶵EAD4鍚庡�笴aco2鍜孒T-29缁嗚優DNA鍚堟垚鐨勫奖鍝，

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