葡萄糖-6-磷酸脱氢酶的表达抑制对A431细胞增殖和细胞周期的影响

中华皮肤科杂志 ›› 2016, Vol. 49 ›› Issue (11): 766-770.

葡萄糖-6-磷酸脱氢酶的表达抑制对A431细胞增殖和细胞周期的影响

李敏¹,夏永华²,刘冬²,张孟杰¹,田中伟³,李占国⁴

1. 新乡医学院第一附属医院
2. 新乡医学院第一附属医院皮肤性病科
3. 河南省新乡医学院第一附属医院皮肤科
4.

收稿日期:2016-02-03 修回日期:2016-07-17 发布日期:2016-10-28
通讯作者: 田中伟 E-mail:zhonwt@163.com
基金资助:
河南省教育厅科学技术研究重点项目;河南省中青年卫生科技创新型人才工程专项基金;河南省医学科技攻关项目

Effects of inhibition of glucose-6-phosphate dehydrogenase on proliferation and cell cycle distribution of A431 cells

Received:2016-02-03 Revised:2016-07-17 Published:2016-10-28

摘要/Abstract

摘要：

目的研究葡萄糖?6?磷酸脱氢酶（G6PD）表达下调对皮肤鳞状细胞癌（鳞癌）细胞增殖和细胞周期的影响。方法正常培养人永生化上皮细胞HaCaT、皮肤鳞癌SCL?1和A431细胞，采用Western印迹法检测细胞中G6PD蛋白的表达。当A431细胞生长至85% ~ 90%融合时，将siRNA对照（siRNA对照组）和G6PD siRNA（G6PD siRNA组）分别转染A431细胞，未转染的A431细胞则为未转染组。Western印迹法检测3组不同处理的A431细胞中G6PD蛋白及细胞周期蛋白D1、CDK4的表达，CCK?8法检测3组A431细胞的增殖情况，流式细胞仪分析3组A431细胞周期的变化。结果正常培养的2株皮肤鳞癌SCL?1和A431细胞中G6PD蛋白的表达水平（分别为0.308 ± 0.023和0.643 ± 0.046）均显著高于HaCaT细胞（0.100 ± 0.019），且A431细胞显著高于SCL?1细胞（均P < 0.05）。A431细胞G6PD siRNA组G6PD、细胞周期蛋白D1和CDK4蛋白的表达（0.134 ± 0.027、0.154 ± 0.017、0.166 ± 0.017）显著低于未转染组（0.425 ± 0.029、0.344 ± 0.024、0.330 ± 0.020）和siRNA对照组（0.444 ± 0.033、0.350 ± 0.027、0.348 ± 0.018），差异均有统计学意义（P < 0.05）。G6PD siRNA组在24 ~ 96 h各时间点的细胞增殖活性均明显低于siRNA对照组和未转染组（P < 0.001），而siRNA对照组与未转染组间细胞增殖差异无统计学意义（均P > 0.05）。G6PD siRNA组G0/G1期A431细胞比例显著高于siRNA对照组及未转染组（P < 0.001），而G6PD siRNA组S期A431细胞比例又显著低于siRNA对照组及未转染组（P < 0.001）。结论 G6PD可能在调控皮肤鳞癌细胞增殖和细胞周期中发挥重要作用。

Abstract:

Li Min, Xia Yonghua, Liu Dong, Zhang Mengjie, Tian Zhongwei, Li Zhanguo Department of Dermatology, First Affiliated Hospital of Xinxiang Medical University, Xinxiang 453100, Henan, China Corresponding author: Tian Zhongwei, Email: zhonwt@xxmu.edu.cn 【Abstract】 Objective To evaluate effects of downregulation of glucose-6-phosphate dehydrogenase （G6PD） on proliferation and cell cycle distribution of cutaneous squamous cell carcinoma （CSCC） cells. Methods Western blot analysis was performed to measure the protein of G6PD in normally cultured human HaCaT keratinocytes, SCL-1 and A431 CSCC cells. When A431 cells grew to 85% - 90% confluence, a small interfering RNA （siRNA） targeting G6PD （G6PD-siRNA group） and a negative control siRNA （siRNA control group） were transfected into them separately, and untransfected A431 cells served as the untransfected group. CCK-8 assay was performed to evaluate proliferative activity of the A431 cells on days 0, 1, 2, 3 and 4 after transfection, Western blot analysis to measure G6PD, cyclin D1 and CDK4 protein s in A431 cells, and flow cytometry to analyze cell cycle distribution in A431 cells after 48 hours of additional culture. Results The protein of G6PD was significantly higher in normally cultured SCL-1 cells （0.308 ± 0.023） and A431 cells （0.643 ± 0.046） than in HaCaT cells （0.100 ± 0.019, both P < 0.05）, and significantly higher in A431 cells than in SCL-1 cells （P < 0.05）. The G6PD-siRNA group showed significantly decreased protein s of G6PD, cyclin D1 and CDK4 （0.134 ± 0.027, 0.154 ± 0.017 and 0.166 ± 0.017, respectively） compared with the untransfected group （0.425 ± 0.029, 0.344 ± 0.024 and 0.330 ± 0.020 respectively） and siRNA control group （0.444 ± 0.033, 0.350 ± 0.027 and 0.348 ± 0.018 respectively）（all P < 0.05）. Besides, the G6PD-siRNA group showed significantly decreased cellular proliferative activity on days 1 - 4 compared with the siRNA control group and untransfected group （all P < 0.001）, while there were no significant differences between the untransfected group and siRNA control group at any of the time points （all P > 0.05）. Compared with the untransfected group and siRNA control group, the G6PD-siRNA group showed significantly higher proportions of A431 cells in G0/G1 phase （both P < 0.001）, but significantly lower proportions of A431 cells in S phase （both P < 0.001）. Conclusion G6PD may play important roles in the regulation of proliferation and cell cycle distribution of CSCC cells.

李敏夏永华刘冬张孟杰田中伟李占国. 葡萄糖-6-磷酸脱氢酶的表达抑制对A431细胞增殖和细胞周期的影响[J]. 中华皮肤科杂志, 2016,49(11):766-770. doi:

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