小分子干扰RNA沉默组蛋白去乙酰化酶1基因对皮肤鳞状细胞癌细胞增殖和凋亡的影响

Effects of histone deacetylase 1 silence by RNA interference on proliferation and apoptosis of cutaneous squamous cell carcinoma

目的观察沉默组蛋白去乙酰化酶1（HDAC1）基因对人皮肤鳞状细胞癌细胞增殖、凋亡和细胞周期的影响。方法人皮肤鳞状细胞癌细胞株（A431）分为3组：空白对照组（不予任何处理）、阴性对照小干扰RNA（siRNA）组（予以30nmol／L阴性siRNA）、siRNA组（予以30nmol／LHDAC1siRNA）。siRNA转染48h后，用反转录-聚合酶链反应（RT—PCR）法和Western blot分别检测3组细胞HDAC1、p21和细胞周期蛋白（Cyclin）E的表达；噻唑蓝（MTF）法检测阴性对照组和siRNA组细胞增殖；流式细胞术检测3组细胞周期和凋亡变化。结果转染HDAC1siRNA48h后A451中HDAC1mRNA表达量为0．22±0．04，显著低于空白对照组（1．01±0．05）和阴性对照siRNA组（1．03±0．05），差异有统计学意义（P〈0．05）；Westernb lot显示HDAC1和CyclinE表达减少（P〈0．05），而p21表达增加（P〈0．05）；MTr法检测显示细胞增长抑制率增加呈时间依赖性，差异有统计学意义（P〈0．05）；流式细胞术检测结果显示细胞阻滞于G2／M期（P〈0．05），细胞凋亡增加（P〈0．05）。结论HDAC1siRNA能抑制A4S1HDAC1的表达、抑制细胞增殖、诱导细胞凋亡。

Objective To investigate the effects of histone deacetylase 1 （ HDAC1 ） gene silencing on cell proliferation, apoptosis and cell cycle of squamous cell carcinoma. Methods A431 cells were cultured and divided into three groups： control group （untreated）, negative small interfering RNA （siRNA） group （treated with 30 nmol/L negative control siRNA）, siRNA group （treated with 30 nmol/L HDAC1 siRNA）. Reverse transcriptase -polymerase chain reaction （RT -PCR） and Western blotting were used to detect the gene expression of HDAC1 in 3 groups 48 h after HDAC1 siRNA transfection. The expression of p21 and Cyclin E was detected by Western blotting. Methyl thiazol tetrazolium （MTF） assay was employed to evaluate the inhibitory effect of HDAC1 RNA interference （RNAi） on the growth of human squamous cell carcinoma cell line A431 in vitro. The cell cycle distribution and apoptotic ratio in 3 groups were determined by flow cytometry. Results After HDAC1 siRNA transfection, the expression of HDAC1 mRNA in A431 cells was 0. 22 ± 0. 04 in siRNA group, which was significantly lower than that in control group （ 1. 01 ± 0. 05, P 〈 0.05 ） and negative siRNA group （ 1.03 ± 0.05, P 〈0. 05 ）. Western blotting showed that the expression of HDAC1 protein was decreased （P 〈 0.05 ）, p21 protein increased and Cyclin E protein decreased （P 〈0. 05）. HDAC1 RNAi inhibited the growth of A431 cells in a time -dependent manner by MTY assay （ P 〈0.05 ）. Flow eytometry showed that the ceils were blocked at the G2/M phase and cell apoptosis was increased as compared with control group （ P 〈 0. 05 ）. Conclusion HDAC1 siRNA can inhibit the expression of HDAC1 and the proliferation of A431 cells, and meanwhile induce cell apoptosis.