Lin28对胃腺癌细胞株增殖周期的影响及机制

Influence and mechanism of Lin28 on cell cycle regulation in gastric carcinoma cells

目的:通过检测可以调控微小RNA(microRNA)生成的RNA结合蛋白Lin28在不同分化程度胃腺癌细胞中的表达水平,探讨了Lin28对胃癌细胞周期的影响及其可能的机制。方法:用RT-PCR法在RNA水平检测Lin28的表达,免疫荧光染色分析Lin28蛋白和人细胞角蛋白18(CK-18)在细胞中的共表达情况,分别应用RNA干扰技术敲减Lin28,及细胞转染技术过表达Lin28,比较Lin28表达水平变化前后细胞周期调控相关蛋白的变化情况,碘化丙啶(propidiumiodide,PI)染色检测细胞周期变化。结果:Lin28在高分化胃腺癌细胞株MKN28中的RNA和蛋白水平均低表达,而在低分化胃腺癌细胞株MKN45中均高表达;Lin28与肿瘤干细胞的常见标志物CK-18共表达于极少数胃癌细胞中;敲减了Lin28的MKN45细胞株S期则明显增加,而G1期比对照组相应地减少。相反,Lin28过表达导致S期明显减少,而G1期比对照组相应增加。Westernblot检测发现,敲减了Lin28的MKN45细胞株细胞周期蛋白依赖性激酶2(CDK2)蛋白发生明显的上调,抑制其活性的蛋白p21和p27水平降低;而MKN28获得Lin28后,CDK2水平有所下调,p21和p27表达水平上调。结论:Lin28参与胃癌细胞分化和细胞周期相关的调控,且对细胞周期抑制蛋白p21/CDK2,p27/CDK2也发挥着一定的调节作用。

Objective Lin28 is a highly conserved RNA-binding protein and involved in stem cell self-renewal and maintenance of pluripotency of embryonic stem （ES） cells. In this study, we examined the expression and influence of Lin28 on cell cycle in gastric carcinoma cell lines including MKN45 and MKN28. Methods The expression of Lin28 at RNA and protein levels was analyzed by RT-PCR and Western blotting. Dual-immunofluorescence was used with antibody against Lin28 and cytokeratin 18 （CK-18）. Cell cycle was analyzed by flow cytometry. Results Lin28 expression was different between MKN45 and MKN28 at both RNA and protein levels, its expression was higher in MKN45 than in MKN28. Lin28 and CK-18 were co-expressed in a small group of gastric carcinoma cells indicating that Lin28 might be an additional marker for gastric stem cells, siRNA suppression of Lin28 led to a decrease in G1 phase [siRNA （51.13± 1.12）%, control （65.12±0.78）%], and accordingly an increase in S phase[siRNA （36.62±0.27）% ,control （23.78±0.23）%]. Conversely, overexpression of Lin28 resulted in a significant reduce in cell population in S phase [overexpression （29.44± 2.11） %,control （57.79±5.6） %], with an increased G1 phase [overexpression （52.38±0.36） %, control （38.75±0.69） %]. At the same time, it was found that one of positively functional proteins in cell cycle cyclin-dependent kinase 2（CDK2） would be promoted a lot in Western blotting, and p21, p27, correspondingly,acting on cycle in negative way would play down. An opposite outcome was explored after require of Lin28 in MKN28 interestingly. Conclusions Lin28 is highly expressed in poorly differentiated gastric cancer cell line MKN45. Experiments for loss and gain-function study of Lin28 indicated that Lin28 might be involved in cell cycle regulation in gastric carcinoma cells through affecting p21/CDK2 and p27/CDK2.