GPP34激活结肠癌细胞Wnt信号通路促进癌细胞增殖的研究

GPP34 promotes colon cancer cell proliferation through activation of Wnt signaling pathway

摘要:

探讨人结肠癌细胞中高尔基相关蛋白34（ 34）基因高表达，激活经典Wnt信号通路，促进癌细胞增殖的机制。方法将SW620 结肠癌细胞株分为4 组：对照组、siRNA 转染组、Tricinbine 组及TWS119组，分别培养；逆转录聚合酶链反应（RT-PCR）检测结肠癌细胞转染后的沉默效果；分别用Topflash报告基因活性法、噻唑蓝（MTT）、流式细胞技术检测各组癌细胞Wnt 信号通路活性、生长抑制率、细胞凋亡率；Western blot检测各组结肠癌细胞中GPP34、β-catenin、pS9- 糖原合酶激酶-3β（pS9-GSK-3β）蛋白的表达。结果转染siRNA 后，沉默结肠癌细胞GPP34 表达效果佳；与对照组比较，各实验组癌细胞的生长抑制率和凋亡率升高，且Wnt信号通路活性抑制。Western blot检测结果显示，siRNA-GPP34 组GPP34 蛋白表达下降，其余两组GPP34蛋白表达比较，差异无统计学意义；siRNA转染组、Tricinbine组和TWS119组的β-catenin和pS9-GSK3β蛋白表达降低。结论SW620 结肠癌细胞中34 基因的高表达可通过激活经典Wnt 细胞信号通路，促进癌细胞增殖并抑制其凋亡。

Abstract:

To investigate the mechanism of high-expression of Golgi-associated protein of 34(GPP34 ) gene in activation of Wnt signaling pathway to promote colon cancer cell proliferation. Methods Colon cancer cell lines were divided into four groups: control group, siRNA transfection group, the group of added inhibitor of Akt/Protein Kinase B (Tricinbine group), and the group of added inhibitor of GSK-3β (TWS119 group). The cells were cultured respectively. The silencing effect of gene was detected by RT-PCR. The proliferation and apoptosis of the cancer cells in all groups were detected by MTT and flow cytometry respectively. Topflash reporter gene activity assay was used to detect the activity of Wnt signaling pathway in the cancer cells of each group. The expressions of GPP34, β-catenin and pS9-GSK-3βproteins in the cancer cells of all groups were detected by Western blot. Results The expression level of mRNA in the transfection group was significantly lower than that in the control group ( p< 0.05). Compared to the control group, the growth inhibition rate and the apoptosis rate were significantly increased ( p< 0.05), and the activity of Wnt signaling pathway was significantly inhibited in each experimental group ( p< 0.05). However, the growth inhibition rate, the apoptosis rate and the activity of Wnt signaling pathway showed no significant differences among the experimental groups ( p> 0.05). Compared to the control group, GPP34 protein expressionsignificantly decreased in the siRNA transfection group ( p< 0.05), while that in the other two experimental groups had no significant difference (p > 0.05); β-catenin and pS9-GSK-3βprotein expressions were significantly decreased in the three experimental groups ( p< 0.05). Conclusions High-expression of GPP34 can activate the classical Wnt signaling pathway to promote proliferation and inhibit apoptosis of SW620 colon cancer cells.