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目的观察RNA干扰细胞周期检测点激酶CHK1和CHK2表达对食管癌细胞照射后G2期阻滞的影响。方法CHK1和CHK2基因各选择4段序列分别设计合成短发卡状RNA (shRNA),分别与pENTRTM／U6质粒连接后转染Eca109食管癌细胞。采用Western blotting检测CHK1和CHK2蛋白表达,RT-PCR检测其mRNA表达,流式细胞仪检测5 Gy照射后细胞周期变化,克隆法检测5 Gy照射后细胞存活率。结果CHK1和CHK2基因各成功建立4个序列的shRNA连接质粒,转染Eca109细胞后均使其蛋白表达降低。用抑制效果最好的CHK1和CHK2 shRNA转染Eca109细胞后,其mRNA表达下降;在转染后72 h,子一代细胞CHK1和CHK2蛋白仍明显降低,并使5 Gy照射后1 h的CHK2-T68磷酸化水平降低,而对CHK1-S345磷酸化水平无明显影响。CHK1 shRNA转染的Eca109细胞在5 Gy照射后12 h时,明显减轻G2期阻滞程度;转染后72 h和5 Gy照射后12 h,子一代Eca109细胞G2期阻滞仍明显低于单纯5 Gy照射组;而CHK2 shRNA转染不影响照射后G2期阻滞。CHK1和CHK2 shRNA转染可降低5 Gy照射后Eca109细胞存活率。结论shRNA转染对CHK1和CHK2蛋白表达的抑制效应至少可以持续3 d,且可传递给子代细胞;CHK1 shRNA转染可以减轻Eca109细胞照射后G2期阻滞,增加放射敏感性。
Objective To observe the effects of RNA interference on cell cycle checkpoint kinase CHK1 and CHK2 on G2 arrest in esophageal cancer cells. Methods CHK1 and CHK2 genes were selected and sequenced to generate short hairpin RNA (shRNA), respectively, and then transfected into Eca109 esophageal cancer cells after being ligated with pENTRTM / U6 plasmid respectively. The expression of CHK1 and CHK2 protein was detected by Western blotting, the mRNA expression of CHK1 and CHK2 was detected by RT-PCR, the cell cycle was detected by flow cytometry after 5 Gy irradiation, and the cell survival rate was detected by cloning method. Results Four shRNA sequences of CHK1 and CHK2 genes were successfully constructed and transfected into Eca109 cells. The expression of CHK1 and CHK2 decreased significantly in Eca109 cells transfected with CHK1 and CHK2 shRNAs with the best inhibitory effect. After 72 h of transfection, CHK1 and CHK2 proteins in the progeny cells were still significantly decreased, and CHK2- T68 phosphorylation decreased, while CHK1-S345 phosphorylation level had no significant effect. Eca109 cells transfected with CHK1 shRNA significantly reduced G2 arrest at 12 h after 5 Gy irradiation. G2 arrest in Eca109 cells was still significantly lower at 72 h after transfection and 12 h after 5 Gy irradiation 5 Gy irradiation group; however, CHK2 shRNA transfection did not affect G2 arrest after irradiation. Transfection of CHK1 and CHK2 shRNAs reduced the survival rate of Eca109 cells after 5 Gy irradiation. Conclusion The inhibitory effect of shRNA transfection on the expression of CHK1 and CHK2 protein can persist for at least 3 days and can be transmitted to progeny cells. CHK1 shRNA transfection can reduce G2 block after Eca109 cell irradiation and increase radiosensitivity.