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目的:研究eIF3I蛋白在细胞中的泛素化修饰,阐明其对人宫颈癌细胞系Hela增殖的影响。方法:通过点突变技术获得突变体K282R,与野生型eIF3I比较泛素化的水平,研究细胞内的泛素化修饰调控。经流式细胞仪分析细胞周期,研究野生型蛋白eIF3I和突变体K282R对Hela细胞的细胞周期影响。再从周期蛋白水平研究eIF3I对细胞增殖的调控作用。结果:突变体K282R比野生型eIF3I蛋白的外源表达量大。在Hela细胞中K282R突变体的泛素化水平低,抑制了该蛋白的泛素-蛋白酶体途径降解。过表达eIF3I能上调周期蛋白Cyclin D1的表达量,促进细胞进入由G1期进入S期。同时,泛素化程度低的突变体K282R具有较强的促进细胞增殖的作用。结论:抑制eIF3I的泛素-蛋白酶体途径降解能上调周期蛋白CyclinD1的表达,促进肿瘤细胞增殖,提示eIF3I在细胞增殖和肿瘤发生发展中发挥作用。
OBJECTIVE: To study the ubiquitination of eIF3I in cells and elucidate its effect on the proliferation of human cervical cancer cell line Hela. Methods: Mutant K282R was obtained by point mutation. The level of ubiquitination was compared with wild-type eIF3I to study the regulation of ubiquitination in cells. The cell cycle was analyzed by flow cytometry to study the cell cycle effects of the wild-type protein eIF3I and the mutant K282R on Hela cells. Then we study the regulatory effect of eIF3I on cell proliferation from the cyclin level. Results: The mutant K282R had more exogenous expression than the wild-type eIF3I protein. The ubiquitination level of the K282R mutant in Hela cells is low, inhibiting the ubiquitin-proteasomal pathway degradation of this protein. Overexpression of eIF3I can up-regulate the expression of cyclin D1 and promote cell entry into the S phase from G1 phase. Meanwhile, K282R, a mutant with low ubiquitination, has a strong effect of promoting cell proliferation. CONCLUSION: Inhibition of eIF3I degradation by ubiquitin-proteasome pathway can up-regulate cyclinD1 expression and promote tumor cell proliferation, suggesting that eIF3I may play a role in cell proliferation and tumorigenesis.