辐射诱发胸腺淋巴瘤Kras基因的表达及其启动子甲基化

摘要

目的：研究辐射诱发小鼠胸腺淋巴瘤 Kras基因 mRNA和蛋白表达的变化，并检测其启动子 CpG岛甲基化状态，探讨辐射致癌的发生机制。方法：采用 X射线照射30只 BALB/c小鼠，建立胸腺淋巴瘤动物模型，取胸腺淋巴瘤和正常胸腺组织，分别提取总 mRNA、合成 cDNA和总蛋白，采用逆转录-聚合酶链式反应（RT-PCR）和 Western blotting法分别检测Kras基因mRNA和蛋白表达水平，采用硫化测序 PCR （BSP）法检测Kras基因启动子CpG岛甲基化状态。结果：RT-PCR法检测，胸腺淋巴瘤 Kras基因 mRNA相对表达水平明显高于正常胸腺组织（P＜0.01）；Western blotting法检测，胸腺淋巴瘤Kras基因蛋白表达水平高于正常胸腺组织近1.41倍；BSP法检测，正常胸腺组织Kras基因启动子有4个CpG位点发生甲基化，而辐射诱发胸腺瘤 Kras基因启动子有1个CpG位点发生甲基化，呈去甲基化状态。结论：电离辐射可能通过Kras基因启动子去甲基化而引起Kras基因mRNA和蛋白表达水平改变，进而导致肿瘤的发生，其可能是辐射致癌的发生机制之一。%Objective To study the changes of mRNA and protein expressions of Kras gene in thymic lymphomas induced by ionizing radiation,and to detect the methylation of CpG islands in promoter region of Kras gene,then to investigate the mechanisms for the occurrence of radiation carcinogenesis.Methods The thymic lymphoma models of BALB/c mice were made by X-ray irradiation,then the total RNA was extracted,cDNA was synthesized and the total protein was extracted from both thymic lymphoma tissue and normal thymus tissue;the mRNA and protein expressions of Kras gene in thymic lymphoma tissue and normal thymus tissue were detected by RT-PCR and Western blotting method, and the methylation of CpG islands in promoter region of Kras gene was detected by bisulfite sequencing PCR. Results The mRNA expression level of Kras gene in thymic lymphoma tissue was significantly higher than that in normal thymus tissue(P<0.01).The protein expression level in thymic lymphoma tissue was about 1.41 times higher than that in normal thymus tissue;4 CpG sites were methylated detected by bisulfite sequencing PCR in normal thymus tissue, however, 1 CpG site was methylated in thymic lymphoma tissue,the CpG islands in promoter region of Kras gene were demethylation state in thymic lymphoma. Conclusion Ionizing radiation can cause the changes of mRNA and protein expression levels of Kras gene in thymic lymphoma tissue by demethylation state of Kras gene,eventually lead to the occurrence of tumor;it might be one of the mechanisms for the occurrence of radiation carcinogenesis.