JQ1下调SKP2和上调p27诱导HeLa细胞凋亡的研究

Study on JQ1 Ιnducing Αpoptosis by Down-regulation of SKP2 and Up-regulation of p27 in HeLa Cells

[目的]观察JQ1对宫颈癌HeLa细胞增殖的影响及可能的作用机制。[方法]采用CCK-8比色法检测JQ1对HeLa细胞增殖的影响,分为二甲亚砜(DMSO)对照组和JQ1处理组(终浓度分别为0.01、0.1、1和10μmol/L),分别处理24、48、72和120h。将细胞分为DMSO对照组和10μmol/L JQ1处理组进行后续实验,细胞培养12d后计算细胞克隆形成数量;细胞培养72h后采用流式细胞仪检测细胞凋亡的变化,采用实时荧光定量PCR(RT-q PCR)和蛋白印迹法检测S期激酶相关蛋白2(SKP2)和p27的表达变化。[结果]JQ1抑制HeLa细胞增殖,且作用呈剂量与时间依赖性,10μmol/L JQ1处理细胞72h后细胞存活数量减半;与DMSO对照组相比,10μmol/L JQ1显著抑制HeLa细胞克隆形成(细胞克隆形成数量:3±2 vs240±10,P<0.001);且能诱导HeLa细胞凋亡(细胞凋亡百分比:12.80±0.88 vs 2.90±0.27,P<0.01);与DMSO对照组相比,JQ1能抑制细胞SKP2 mRNA和蛋白表达(其中SKP2 mRNA表达倍数:0.43±0.02 vs 1.00±0.03,P<0.01),并上调p27 mRNA和蛋白表达(p27 mRNA表达倍数:2.60±0.13 vs 1.00±0.11,P<0.01)。[结论]JQ1通过诱导细胞凋亡来抑制HeLa细胞的增殖,其可能的机制是抑制SKP2表达和上调p27表达。

[Objective] To investigate the effect of JQ1 on proliferatoin of HeLa cells and its potential mechanism. [Methods] HeLa cells were exposed to dimethyl sulfoxide（DMSO,control group） or different concentrations of JQ1（0.01,0.1,1 and 10μmol/L,JQ1 group） in the culture medium for 24,48,72 and 120 h respectively,and cell proliferation analysis was performed using CCK-8 method. HeLa cells were exposed to DMSO or 10μmol/L JQ1 in the culture medium for 12 d,and the number of colony formation was assayed by counting. HeLa cells were exposed to DMSO or 10μmol/L JQ1 in the culture medium for 72 h,and the apoptosis was assayed by flow cytometry,and the expression of S-phase kinase-associated protein 2（SKP2） and p27 were detected by real-time quantitative PCR（RT-q PCR） and Western blot methods. [Results] The proliferation of HeLa cells was inhibited by JQ1 in a time- and dose-dependent manner,a fifty percent inhibition of growth was seen after treated with 10μmol/L concentration of JQ1 for 72 h. Compared with DMSO control group,the colony formation was inhibited significantly in 10μmol/L JQ1-treated group（the number of colony formation：3±2 vs 240±10,P0.001）;and cell apoptosis was induced signiticantly by 10μmol/L JQ1（the percentage of apoptosis：12.80±0.88 vs 2.90±0.27,P0.01）. Compared with DMSO control group,the expression of SKP2 was significantly decreased and the expression of p27 was significantly increased in JQ1-treated group（among them SKP2 mRNA expression folds：0.43 ±0.02 vs 1.00 ±0.03;p27 m RNA expression folds：2.60 ±0.13 vs1.00±0.11,both with P0.01）. [Conclusion] JQ1 inhibits cell proliferation of HeLa cells via inducing apoptosis,which might depend on down-regulation of SKP2 and up-regulation of p27.