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目的探讨沉默整合素连接激酶(integrin-linked kinase,ILK)基因表达对人舌鳞癌Tb细胞生长、迁移和侵袭能力的影响。方法将ILK siRNA表达质粒和阴性对照质粒在脂质体介导下转染人舌鳞癌Tb细胞,稳定表达细胞株分别命名为Tb siILK和Tb vector组,并设正常Tb细胞对照组(Tb组)。Western blot法检测细胞中ILK蛋白的表达;细胞免疫荧光法检测细胞中ILK、p-Akt和p-GSK3β的表达;光镜和HE染色观察细胞的形态学变化;划痕试验检测细胞的迁移能力;Transwell法检测细胞的侵袭能力;流式细胞术检测细胞的细胞周期和凋亡情况;MTT法检测细胞的增殖活力;Western blot法检测沉默ILK基因后细胞中p-Akt、Akt、p-GSK3β、GSK3α/β、Snail的表达。结果与Tb和Tb vector组相比,Tb siILK组细胞中ILK蛋白的表达水平显著降低(P<0.01);ILK、p-Akt、p-GSK3β在胞质中的荧光信号明显减弱;大部分细胞的上皮形态特征更为明显;细胞的迁移距离和侵袭细胞数显著减少(P<0.05);G2-M期和G0-G1期细胞比例均显著增加(P<0.01);细胞的增殖活力显著减低;ILK基因沉默后,细胞中p-Akt、p-GSK3β和Snail蛋白的表达水平显著降低(P<0.05)。3组细胞的凋亡率差异无统计学意义(P>0.05)。结论抑制ILK基因的表达可通过Akt/GSK3β/Snail途径显著抑制人舌鳞癌Tb细胞的生长、迁移和侵袭能力,ILK有望作为治疗人舌鳞癌的靶基因。
Objective To investigate the effect of silencing integrin-linked kinase (ILK) gene on the growth, migration and invasion of human tongue squamous cell carcinoma Tb cells. Methods ILK siRNA expression plasmid and negative control plasmid were transfected into human tongue squamous cell carcinoma Tb cells by lipofectamine. Stably expressing cell lines were named as Tb siILK and Tb vector, and normal Tb cells control group (Tb group ). The expression of ILK protein in cells was detected by Western blot. The expression of ILK, p-Akt and p-GSK3β in cells was detected by immunofluorescence method. The morphological changes of cells were observed by light microscopy and HE staining. Transwell method was used to detect cell invasion ability. Cell cycle and apoptosis were detected by flow cytometry. Cell viability was detected by MTT assay. Expression of p-Akt, Akt, p-GSK3β , GSK3α / β, Snail expression. Results The expression of ILK protein in Tb siILK group was significantly lower than that in Tb and Tb vector group (P <0.01). The fluorescence signal of ILK, p-Akt and p-GSK3β in the cytoplasm was significantly decreased. Most of the cells (P <0.05). The proportion of cells in G2-M phase and G0-G1 phase were significantly increased (P <0.01), and the proliferation activity of cells was significantly decreased After ILK gene silencing, the expression of p-Akt, p-GSK3β and Snail protein were significantly decreased (P <0.05). There was no significant difference in apoptosis rate between the three groups (P> 0.05). Conclusion Inhibition of ILK gene expression significantly inhibits the growth, migration and invasion of human tongue squamous cell carcinoma cell line Tb-Akt through the Akt / GSK3β / Snail pathway. ILK is expected to be used as a target gene in the treatment of human tongue squamous cell carcinoma.