RNA干扰Dub3表达对鼻咽癌细胞增殖和凋亡的影响

Effect of siRNA targeting Dub3 gene on the proliferation and apoptosis of human nasopharyngeal carcinoma CNE-2 cells

目的探讨小干扰RNA(siRNA)靶向抑制去泛素化酶3(Dub3)基因表达对人鼻咽癌CNE-2细胞增殖、凋亡及细胞周期的影响。方法优化siRNA转染条件,将2条靶向抑制Dub3基因的siRNA载体片段(siRNA-1、siRNA-2)分别高效转染人鼻咽癌CNE-2细胞,同时设转染无义序列的空转染组及无任何处理的对照组。分别于转染24、48、72、96 h后采用实时定量PCR(q PCR)检测Dub3表达水平以筛选抑制率高的感染载体用于后续试验。噻唑蓝法检测各组转染24、48、72、96 h后的增殖抑制率,流式细胞术检测各组转染48、96 h后的细胞凋亡和细胞周期情况,采用Western blotting检测各组转染96 h后细胞分裂周期蛋白25A(Cdc25A)的表达情况。结果转染组的Dub3 mRNA水平均低于空转染组和对照组(P<0.05),且选择干扰效率高的siRNA-1载体进行功能学研究;与其余两组相比,抑制组转染后的增殖抑制率、凋亡率及caspase-3活化率均升高,且G_0/G_1期细胞比例升高,但S期和G_2/M期细胞比例均降低,以上差异均有统计学意义(P<0.05);抑制组转染后的Cdc25A水平降低,与空载体组和对照组的差异有统计学意义(P<0.05)。空载体组和对照组以上指标的差异均无统计学意义(P>0.05)。结论通过siRNA抑制Dub3基因表达可抑制鼻咽癌细胞的增殖并诱导凋亡和细胞周期阻滞,对鼻咽癌的防治有一定价值。

Objective To explore the effect of siRNA targeting deubiquitinating enzyme 3（ Dub 3） gene on the proliferation and apoptosis of human nasopharyngeal carcinoma CNE-2 cells. Methods After the optimization of the transfection conditions,2 siRNA vector fragments（ siRNA-1 and siRNA-2） targeting Dub3 gene were effectively transfected into CNE-2 cells,respectively. There were empty transfection group whose cells were transfected with the antisense sequence and control group without any treatment. The Dub3 expression levels were detected by real-time quantitative PCR（ q PCR） at 24,48,72 and 96 h after transfection,and the siRNA vector with the higher inhibition rate was used for the following experiments. Thiazolyl blue was used to detect the proliferation inhibition rates at 24,48,72,96 h after transfection. The apoptosis and cell cycle of the cells were detected by flow cytometry after 48 and96 h. The expression of cell division cycle 25A（ Cdc25A） after 96 h was detected by Western blotting. Results The levels of mRNA Dub3 in the transfection group were lower than those in the empty transfection group and the control group（ P〈0. 05）. Since the interference efficiency of the siRNA-1 fragment was higher than that of the siRNA-2,so the subsequent experiments chose the siRNA-1 fragment. Compared with other two groups,the proliferation inhibitory rates,apoptosis rates and caspase-3 activation rates together with the proportion of cells in G0/ G1 phase increased,but the proportion of cells in S and G2/ M phase decreased in inhibition group with statistically significant difference（ P〈0. 05）; After transfection,the levels of Cdc25 A were decreased in inhibition group versus other groups（ P〈0. 05）. Conclusion The inhibition of Dub3 gene expression by siRNA can inhibit the proliferation and induce apoptosis and cell cycle arrest,and it can be valuable for the prevention and treatment of nasopharyngeal carcinoma.