Chk1基因对人卵巢癌细胞HO8910增殖及细胞周期调控的影响及可能机制

Effects of checkpoint kinase 1 gene on the proliferation and cycle of human ovarian carcinoma HO8910 cells and its potential mechanisms

摘要:

目的：探讨siRNA沉默检测点激酶1（Chk1）基因对人卵巢癌细胞HO8910增殖和周期调控的影响及其作用机制。方法：设计并合成3条Chk1 siRNA干扰片段，转染进入卵巢癌细胞HO8910，Western blot检测转染前后HO8910细胞中Chk1蛋白的表达情况，筛选出有效片段。将Chk1 siRNA有效片段转染进入HO8910细胞，采用MTT法和流式细胞仪分析Chk1 siRNA对HO8910细胞增殖和周期的影响；Western blot方法分别检测细胞周期相关基因CyclinA、CDK4、Cdc25A蛋白表达。 结果：3个siRNA中，1条是有效siRNA。该有效Chk1 siRNA片段转染后，HO8910细胞中Chk1蛋白水平下降约53.6%，明显低于未转染组和脂质体转染组（P=0.000）。转染48 h后，Chk1 siRNA转染组HO8910细胞增殖活性下降，抑制率为（52.1±5.1）%，明显高于脂质体转染组的（7.6±3.8）%（P=0.000）。流式细胞仪检测显示Chk1 siRNA转染组HO8910细胞G2/M期比例为（5.03±2.62）%，明显低于未转染组（19.35±2.19）%和脂质体转染组（19.76±2.67）%（P=0.000）。Western blot结果显示转染Chk1 siRNA后，细胞内与细胞周期相关的CyclinA、CDK4、Cdc25A蛋白水平表达明显增强，与未转染组和脂质体组比较，差异有统计学意义（P=0.000）。结论：siRNA沉默Chk1基因后，可明显抑制卵巢癌细胞HO8910细胞的增殖，其抑制增殖作用与减弱G2/M期阻滞有关。

Abstract:

Objective：To investigate the effects of checkpoint kinase 1 siRNA transfection on the proliferation and cell cycle of human ovarian carcinoma HO8910 cells and its mechanisms. Methods：Three Chk1 siRNA targeting fragments were designed and synthe－sized. After the transfection，Western blot was conducted to determine the expression of Chk1 at protein level. The effective Chk1 siR－NA fragment was transfected into HO8910 cells，and MTT method and flow cytometry analysis were used for determining the influence of Chk1 siRNA on the proliferation and cell cycle，respectively. Then，Western blot was used to detect cell cycle of related genes Cy－clinA，CDK4 and Cdc25A at protein level. Results：One fragment among 3 Chk1 siRNAs was valid. In HO8910 cells，after transfec－tion with the effective Chk1 siRNA，the protein level of Chk1 was decreased about 75.3%，significantly lower than that in the non-transfected group and liposome transfection group（P=0.000）. At 48 h after transfection，the activity of cell proliferation was inhibited with an inhibition rate of （52.1±5.1）%，significantly higher than that in the liposome transfection group（7.6±3.8）%（P=0.000）. Flow cytometry showed that the proportion of HO8910 cells in G2/M phase was （5.03±2.62）%，significantly lower than that in the non-transfected group（19.35±2.19）% and liposome transfection group（19.76±2.67）%（P=0.000）. Western blot results showed that in the HO8910 cells transfected with Chk1 siRNA，the protein levels of CyclinA，CDK4 and Cdc25A were enhanced，which were statis－tically different compared with those of non-transfection group and liposome group（P=0.000）. Conclusion：After Chk1 gene silencing by siRNA，the proliferation of the human ovarian carcinoma HO8910 cells was inhibited，which was related with the block－ade of G2/M phase arrest.