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FAT10双甘酸突变体对宫颈癌细胞增殖和凋亡的影响
作者:李翠 刘亚伟 肖霄 刘爱华 罗海华 姜勇 
单位:510515 广州 南方医科大学基础医学院病理生理学教研室 广东省蛋白质组学重点实验室(李翠、刘亚伟、肖霄、罗海华、姜勇) 510515 广州 南方医科大学南方医院呼吸内科(刘爱华) 
关键词:宫颈肿瘤 泛素类 细胞增殖 细胞凋亡 
分类号:R737.33
出版年,卷(期):页码:2014,39(12):931-935
摘要:

目的 探讨FAT10的碳末端双甘酸缺失突变体对宫颈癌HeLa细胞增殖和凋亡的影响。方法 收集宫颈原位癌组织标本和正常宫颈组织标本各5例,Western blotting法检测组织中FAT10蛋白的表达。采用定点突变技术构建pcDNA3.0-flag-FAT10?GG质粒,分别将野生型FAT10FAT10?GG及空载体(阴性对照)瞬时转染至HeLa细胞中,以野生型Hela细胞作为空白对照组,采用Western blotting法检测转染效率,CCK-8法检测细胞增殖情况。转染24h后,各组细胞经顺铂诱导凋亡,采用流式细胞仪检测细胞凋亡率。结果 FAT10蛋白在宫颈癌组织中的表达明显高于正常组织。过表达野生型FAT10对宫颈癌细胞的增殖有明显促进作用,但FAT10发生双甘酸突变之后,该作用受到明显抑制。流式细胞仪检测结果显示:与空白对照组(22.7%±4.2%)和阴性对照组(24.1%±3.8%)相比,过表达野生型FAT10组的细胞凋亡率(10.9%±2.0%)明显降低(P<0.05),而FAT10?GG组的细胞凋亡率(25.7%±5.1%)无明显变化(P>0.05)。结论 FAT10可通过其碳末端双甘酸结构促进肿瘤细胞增殖,减少凋亡,从而促进肿瘤的发生发展。

Objective To investigate the effects of FAT10?GG, a carboxyl-terminal diglycine deficient mutant, on the proliferation and apoptosis of cervical cancer cell line HeLa. Methods Specimens of cervical carcinoma in situ and normal cervix tissue, 5 each, were collected. The expressive levels of FAT10 protein in these specimens were detected by Western blotting. Site-directed mutagenesis was applied to construct the mutant pcDNA3.0-flag-FAT10?GG plasmid. The HeLa cells were then transiently transfected with wild-type FAT10, FAT10?GG and empty vector (used as negative control), and the wild-type HeLa cells served as blank control. The transfection efficiency of FAT10 or FAT10?GG was detected by Western blotting, and cell proliferation was determined by CCK-8 assay. Cisplatin was used to induce cell apoptosis after cells were transfected for 24h, and the cell apoptotic rates of all groups were determined by flow cytometry. Results Western blotting showed a significantly increased expression of FAT10 protein in cervical carcinoma tissues compared with that in normal cervical tissue. Over-expression of wild FAT10 in HeLa cells obviously promoted cell proliferation, but this promotion was significantly inhibited in cells transfected with its diglycine mutant. Compared with blank control group (22.7%±4.2%) and negative control group (24.1%±3.8%), the apoptotic rate was significantly reduced in wild FAT10 group (10.9%±2.0%, P<0.05), while no obvious changes were found in cells transfected with its diglycine deficient mutant (25.7%±5.1%, P>0.05). Conclusion FAT10 can promote cell proliferation and inhibit cell apoptosis through its carboxyl-terminal diglycine motif, and it may play an essential role in carcinogenesis and development of cancer.

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