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目的构建整合素连接激酶(Integrin linked kinase,ILK)基因siRNA干扰质粒,探讨ILK基因沉默对膀胱癌BIU-87细胞增殖能力及糖原合成酶激酶3(Glycogen synthase kinase 3,GSK3)、β-环连蛋白(β-catenin)表达的影响。方法构建针对人ILK基因的2个特异性siRNA表达质粒和1个无同源性的阴性对照质粒,在脂质体介导下转染膀胱癌BIU-87细胞,通过RT-PCR及Western blot检测ILK基因mRNA和蛋白的表达水平,筛选出1个干扰效果较强的质粒用于后续试验,Western blot检测细胞GSK3和β-catenin蛋白的表达,Am-blue法检测细胞的增殖活力,流式细胞术检测细胞周期的分布。结果经酶切和测序证明重组干扰质粒构建正确;稳定转染pGenesil-1 siRNA1和pGenesil-1 siRNA2质粒的BIU-87细胞ILK基因mRNA和蛋白表达水平较BIU-87细胞组(未转染组)分别下降了69%、52%和70%、55%,选择pGenesil-1 siRNA1为最有效干扰序列;BIU-87siRNA1组GSK3的表达水平较阴性对照组和BIU-87细胞组分别升高了41.87%和30.12%,β-catenin的表达水平较阴性对照组和BIU-87细胞组分别降低了38.67%和48.05%;BIU-87 siRNA1组细胞增殖活力明显下降;BIU-87 siRNA1组G0-G1期细胞比例明显增加,S期减少。结论已成功构建ILK基因siRNA表达质粒,其可明显抑制膀胱癌BIU-87细胞的增殖,机制可能为ILK基因通过介导GSK3、β-catenin信号分子促进膀胱癌BIU-87细胞的增殖。
OBJECTIVE: To construct a siRNA plasmid targeting integrin linked kinase (ILK) gene to investigate the effect of ILK gene silencing on the proliferation of human bladder cancer cell line BIU-87 and the effects of glycogen synthase kinase 3 (GSK3) Effect of β-catenin expression. Methods Two specific siRNA expression plasmids targeting human ILK gene and one negative control plasmid without homology were constructed and transfected into bladder cancer BIU-87 cells by lipofectamine. ILK gene mRNA and protein expression level, screening a strong interference plasmids for follow-up test, Western blot detection of cell GSK3 and β-catenin protein expression, Am-blue method to detect cell proliferation activity, flow cytometry Surgery to detect the distribution of cell cycle. Results The recombinant plasmids were constructed correctly by restriction enzyme digestion and sequencing. The mRNA and protein expression of ILK gene in BIU-87 cells stably transfected with pGenesil-1 siRNA1 and pGenesil-1 siRNA2 were significantly lower than those in BIU-87 cells (untransfected group) The expression of GSK3 in BIU-87siRNA1 group was increased by 41.87% compared with the negative control group and the BIU-87 cell group respectively by 69%, 52% and 70%, 55% And 30.12%, the expression ofβ-catenin was decreased by 38.67% and 48.05% respectively compared with the negative control group and the BIU-87 cell group; the proliferation activity of BIU-87 siRNA1 group was significantly decreased; The proportion increased significantly, S reduced. Conclusion ILK gene siRNA expression plasmid has been successfully constructed, which can significantly inhibit the proliferation of bladder cancer BIU-87 cells. The possible mechanism is that ILK gene can promote the proliferation of bladder cancer BIU-87 cells by mediating GSK3 and β-catenin signaling molecules.