MARK2基因协同调控HeLa细胞极性与增殖的机制研究

MARK2 regulates cell polarity and cell proliferation in HeLa cells

目的:观察MARK2基因对He La细胞极性与增殖的影响,探讨其可能的机制。方法:脂质体介导MARK2质粒转染He La细胞,G418抗性筛选获得稳定转染的细胞株。TRITC-Phalloidin染色观察细胞极性,平板克隆形成试验研究细胞贴壁生长能力,流式细胞术分析细胞周期各时相细胞的比例,Western blot检测总Rb蛋白及磷酸化Rb蛋白的水平。结果:成功构建稳定转染MARK2的He La细胞系,与空质粒对照组细胞相比,MARK2蛋白表达增强后He La细胞形态改变、上皮细胞极性恢复;MARK2表达增强后He La细胞形成平板克隆数量明显减少[(303.67±7.77)vs.(111.67±7.64),P=0.000],细胞周期G1期比例明显增加[(48.82±0.84)%vs.(72.01±2.13)%,P=0.000]、S期比例减少[(36.60±0.58)%vs.(16.03±3.85)%,P=0.010],Rb蛋白的磷酸化水平明显降低[(7.66±0.74)vs.(1.10±0.13),t=15.053,P=0.003]。结论:在He La细胞中,MARK2通过抑制Rb蛋白的磷酸化,使细胞周期停滞在G1期,抑制细胞增殖。MARK2能够重建He La细胞上皮极性,协同调控细胞极性与细胞增殖两条通路。

Objective：To observe the effect of MARK2 expression on cell polarity and proliferation in HeLa cells,and to investigate the underlying mechanisms. Methods：A stable HeLa cell line highly expressing MARK2 protein was established by using G418 se- lection. Cell polarity was observed with TRITC-Phalloidin staining. Cell proliferation was examined with plate colony formation assay. Proportions of cells in different phases of cell cycle were analyzed by flow cytometry. Levels of total Rb protein and phosphorylated Rb protein were analyzed by Western blot. Results ： High expression of MARK2 in Hela stable cells led to phenotypic changes con- sistent with reversed epithelial polarity. Decreased cell proliferation, increased Gl-phase cell proportion,reduced S-phase cell pro- portion and inhibited Rb phosphorylation were observed in HeLa stable cells. Conclusion：Enforced expression of MARK2 protein in HeLa cells leads to decreased Rb phosphorylation,arrests cell cycle at Gl-stage thus inhibits cell proliferation. MARK2 can reconstruct epithelial polarity of HeLa ceils, and modulate both signaling pathways.regulating cell polarity and cell proliferation.