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

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。

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

Objective To evaluate effects of downregulation of glucose?6?phosphate dehydrogenase(G6PD) expression on proliferation and cell cycle distribution of cutaneous squamous cell carcinoma(CSCC)cells. Methods Western blot analysis was performed to measure the protein expression 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 expressions in A431 cells, and flow cytometry to analyze cell cycle distribution in A431 cells after 48 hours of additional culture. Results The protein expression 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 expressions 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.