论文部分内容阅读
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,P<0.001)和HT-29(F=167.3,P<0.001)细胞中的TEAD4蛋白水平显著降低,同时观察到Caco2(F=45.78,P<0.001)和HT-29(F=40.71,P<0.001)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,P<0.001)和HT-29(F=141.0,P<0.001)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,P<0.001)和HT-29(F=305.1,P<0.001)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(P<0.001)、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。
Objective The high incidence of colorectal cancer at home and abroad, is one of the major causes of cancer death. TEAD4 is a key factor in the YAP / TAZ signaling pathway. This study aimed to investigate the effect and mechanism of knockdown of TEAD4 expression on colorectal cancer cell proliferation. Methods Transfection of TEAD4 siRNA and transfection of lentivirus in colorectal cancer cell lines Caco2 and HT-29 in order to achieve transient or stable knockdown of TEAD4. The transfection efficiency and related protein expression were detected by Western blot. The survival rates of Caco2 and HT-29 cells after knocking down TEAD4 were detected by SRB method, and the DNA synthesis efficiency was determined by EdU incorporation method. Results TEAD4 siRNA was used to knock down the expression of TEAD4 in intestinal cancer cells for 48h. Western blotting showed that Caco2 (F = 99.22, P <0.001) and HT-29 (F = 167.3, P <0.001), and the expression of p27 protein in Caco2 (F = 45.78, P <0.001) and HT-29 (F = 40.71, P <0.001) Of the rise, the protein expression difference was statistically significant. The results of SRB showed that TEAD4 gene silencing TEAC4 gene Caco2 (F = 142.9, P <0.001) and HT-29 (F = 141.0 , P <0.001), the cell growth rate became slower. After 3d, the inhibitory effect was more obvious. The proliferation rate of Caco2 (F = 394.5, P <0.001) and HT-29 SLOW. The effects of knockdown of TEAD4 on DNA synthesis in Caco2 and HT-29 cells were detected by EdU incorporation assay. The results showed that Caco2 and HT-29 cells, Caco2 / Consi, Caco2 / TEAD4si # 2, Caco2 / TEAD4si # DNA synthesis rates of (29.20 ± 2.18)%, (27.19 ± 2.34)% and (30.14 ± 1.02)% and (28.23 ± 3.11 ), (11.89 ± 1.20)% and (17.91 ± 2.23)%, respectively. The levels of Caco2 / TEAD4si # 2 and Caco2 / TEAD4si # = 0.001) and HT-29 / TEAD4si # 3 (P = 0.011) cells were significantly lower than that of the control cells, indicating that DNA synthesis was significantly inhibited after knockdown of TEAD4 in Caco2 and HT-29 cells. CONCLUSION: Knockdown of TEAD4 can inhibit the proliferation and DNA synthesis of colorectal cancer cells in vitro, which may be partly through the up-regulation of p27 expression. TEAD4 may be a new potential target for clinical diagnosis or treatment of colorectal cancer.