卷曲螺旋结构域蛋白74B对纤毛生成和细胞周期调控作用研究

Regulatory Effect of CCDC74B on Cilia Formation and Cell Cycle

背景卷曲螺旋结构域蛋白(CCDC)参与基因转录、细胞凋亡及细胞周期过程,并调控恶性肿瘤细胞的侵袭和转移等多种生物学行为,然而多数CCDC的生物学功能目前尚未明确。目的探讨CCDC74B的细胞定位及对纤毛生成和细胞周期的影响。方法 2013年3—9月通过构建在人视网膜色素上皮(RPE1)中的Tet-On表达系统,实现CCDC74B在细胞内的蛋白表达,然后通过细胞免疫荧光染色观察CCDC74B的细胞定位,通过小干扰RNA(siRNA)转染敲除CCDC74B在RPE1细胞中的表达,分为对照组、siRNA#1组、siRNA#2组,采用反转录聚合酶链式反应(RT-PCR)法检测CCDC74B mRNA表达水平;采用细胞免疫荧光染色及流式细胞术观察下调CCDC74B表达后纤毛生成情况和对细胞周期分布的影响。结果在正常上皮细胞中,CCDC74B位于中心体,细胞免疫荧光染色示,下调CCDC74B表达后细胞初级纤毛生成。siRNA#1组及siRNA#2组转染后36 h及48 h纤毛生成率分别高于对照组(P<0.05)。RT-PCR法结果显示,siRNA#1组及siRNA#2组均有效抑制了CCDC74B mRNA表达。细胞免疫荧光染色示,下调CCDC74B表达后cyclin A表达减少,提示存在细胞周期停滞;流式细胞术检测示,下调CCDC74B表达后G_1期细胞比例增高,S期及G_2/M期细胞比例减少。结论中心体蛋白CCDC74B在细胞增殖过程中参与对纤毛生成和细胞周期的调控。

Background CCDC protein is involved in various biological behaviors including gene transcription, apoptosis, cell cycle and regulating invasion and metastasis of cancer ceils. However, most CCDC protein functions remain unknown. Objective To investigate the cellular localization and its influence on cilia formation and cell cycle. Methods From March to September in 2013, Tet - On expression system of RPE1 was built, and protein exPression of CCDC74B was realized The localization of exogenous CCDC74B was observed by immunofluorescence. After siRNA transfection was performed to decrease endogenous CCDC74B protein expression in RPE1 cells, the ceils were divided into control group, siRNA#1 group and siRNA#2 group. RT-PCR method was used to detect CCDC74B mRNA expression. Cilia formation and cell cycle distribution after the decrease of CCDC74B expression were further observed by immunofiuorescent staining and flow cytometry. Results In normal epithelial ceils, CCDC74B was in centrosome, and immunofluorescent staining showed that the decrease of CCDC74B leaded to the elementary cilia formation. 36 h and 48 h after transfection, siRNA#1 group and siRNA#2 group were higher than control group in the proportion of ciliated cells （P 〈 0. 05） . RT -PCR method showed that siRNA#1 group and siRNA#2 group effectively inhibited the expression of CCDC74B mRNA. The immunofluorescent staining showed that after the decrease of CCDC74B expression, the expression of cyclin A decreased, which suggested that cell cycle arrest may exist. FCM suggested that the decrease of CCDC74B leaded to the increase of the proportion of G1 ceils and the decrease of the proportion of cells at S and GE/M phrases. Conclusion Centrosomal protein CCDC74B is involved in the regulation of cilia formation in the process of cell proliferation.