RNA干扰抑制pyk2表达对大肠癌细胞凋亡和增殖的影响

Suppression of pyk2 expression in colorectal cancer cells by RNA interference and its contribution to cell proliferation and apoptosis

目的探讨富含脯氨酸的酪氨酸蛋白激酶2(proline-rich tyrosin kinase-2,pyk2)对Lovo大肠癌细胞凋亡和增殖的影响。方法利用两组构建有pyk2 shRNA(短发夹RNA,会在细胞内顺序反应而产生siRNAs)的质粒载体瞬时转染Lovo细胞系,设立空白对照组和阴性对照组,应用PCR和western blot方法检测转染组和对照组细胞中pyk2 mRNA与蛋白表达水平的变化,通过细胞计数法来绘制细胞生长曲线,利用流式细胞技术(FCM)检测转染组与对照组细胞在凋亡和细胞周期S期分布的差异。结果转染pyk2shRNA质粒的Lovo细胞凋亡水平明显低于对照组,S期细胞数显著增多,细胞绝对数增加,生长更加活跃。结论 pyk2能够升高Lovo大肠癌细胞的凋亡水平,降低其增殖水平。

Objective To clarify the role of Pyk2 in cell proliferation and apoptosis in Lovo rectal cancer cells in vitro. Methods Two sets of shRNA （ short hairpin RNA which will be subsequently processed to siRNAs） expression plasmid vectors were used to transfeet lovo cells separetely. The lovo cells and the cells transfected with a non - targeting, negative shRNA expression plasmid were used as controls. The downregulation of the protein level and mRNA level was further confirmed by Western blotting and RT - PCR. The growth curve of lovo cells was studied by cell counting. The changes in apoptosis and cell cycle were deremlined by flow cytometry. Results Cells transfected with one of the shRNA expression vectors obtained knockdown of pyk2, with a significantly lower apoptosis level. The number of cells in the S stage of cell cycle increased as cell count did. Conclusion Pyk2 elevates the apoptosis level of lovo cells and lowers their proliferation.