镉转化细胞DNA异常甲基化对肿瘤相关基因表达的影响

Effect of aberrant DNA methylation on the expression of cancer-related genes in Cd-transformed cells

目的 对镉转化细胞DNA异常甲基化及其对肿瘤相关基因表达的影响进行研究 ,探讨镉的外遗传致癌机制。方法 从CdCl2 转化BALB/c 3T3细胞中提取基因组DNA ,经甲基化非敏感性酶 (Mse1 )单独消化或Mse1和甲基化敏感性酶 (BstU1 )联合消化 ,消化产物用甲基化敏感性内切酶指纹法 (MSRF)进行分析 ,差异显示出异常甲基化基因片段 ,进一步以异常甲基化DNA为探针进行Southern分子杂交加以证实 ,并进行DNA序列测定 ,与基因文库中的基因进行类比分析。结果 发现镉转化细胞存在异常甲基化DNA ,其中一个甲基化DNA片段为p1 6抑癌基因。结论 DNA高甲基化会导致基因表达抑制 ,因此 ,p1 6基因高甲基化会导致其抑癌功能减弱或丧失 。

Objective To study aberrant DNA methylation potentially resulting in changes in the expression of cancer related genes as a possible epigenetic mechanism for cadmium carcinogenesis. Methods Genomic DNA isolated from CdCl 2 transformed BALB/c 3T3 cells was digested with Mse1(methylation non sensitive) alone or with Mse1 and BstU1(methylation sensitive).The resulting DNA was analyzed for aberrant methylation using PCR based technique——Methylation Sensitive Restriction Fingerprinting(MSRF).Several DNA fragments differentially methylated in the transformed cells identified by MSRF were confirmed by Southern hybridization analysis using the aberrantly methylated DNA fragments as the probes. Results Aberrant DNA methyation was identified in the transformed cells.DNA sequencing and sequence similarity analysis identified one of the aberrantly methylated DNA fragments as the p16 tumor suppressor gene. Conclusion DNA hypermethylation is known to result in gene silencing,it appears that hypermethylation of p16 gene may represent a possible epigenetic mechanism for Cd induced cell transformation and carcinogenesis.