限制性标记基因组扫描分析在前列腺腺癌基因组CpG岛的异常甲基化

Restriction landmark genomic scanning for screening aberrant Cp G methylations in prostate cancer

目的用限制性标记基因组扫描(RLGS)分析前列腺腺癌基因组Cp G岛的异常甲基化,为前列腺腺癌相关基因的甲基化研究奠定基础。方法采用激光捕获显微切割技术从20例前列腺腺癌和18例良性前列腺增生组织中捕获均一同质的腺体细胞,提取DNA,利用RLGS技术对这些样本基因组Cp G岛扫描,比较前列腺腺癌和良性前列腺增生的Cp G岛甲基化差异,筛选出参与前列腺腺癌发生的Cp G岛甲基化基因。将其上传至DAVID数据库进行GO分析,并对筛选出甲基化最显著的基因DPYS进行焦磷酸测序。结果前列腺腺癌和良性前列腺增生分别有10245个和8658个Cp G岛发生甲基化,甲基化发生率分别为37.2%和30.3%,其中超过60%为启动子区Cp G岛。前列腺腺癌与良性前列腺增生存在差异甲基化的Cp G岛有735个,其中高甲基化458个,去甲基化256个。筛选出DPYS、P16、APC、GSTP1、TMEM122、RARB、ARHGAP20等7个前列腺腺癌甲基化最显著的基因。生物信息学分析提示这些基因涉及多个分子功能和生物过程,其中DPYS参与了13个GO注释的生物功能,50种疾病的发生发展和47个蛋白间的相互作用。对Cp G岛7个位点进行焦磷酸测序,平均甲基化频率为32.7%。可能在前列腺腺癌发生发展中存在重要的意义。结论 RLGS发现前列腺腺癌和良性前列腺增生之间存在大量Cp G岛高甲基化和去甲基化改变。筛选出甲基化显著的基因DPYS在前列腺腺癌发生和发展中起重要作用,机制有待进一步研究。

Objective To screen methylations of Cp G islands in prostate cancer using restriction landmark genomic scanning（RLGS）. Methods The DNA was extracted from homogeneous cells captured by laser capture microdissection in 20 prostate cancer and 18 benign prostatic hyperplasia（BPH） tissues for scanning the Cp G islands using RLGS. The methylation status of each Cp G island was compared between the cancer and BPH samples to screen the genes involved in prostate cancer development. The screened genes were uploaded to DAVID database for GO analysis, and the genes with the most significant methylation were analyzed by pyrosequencing. Results and Conclusion Among all the tested Cp G islands, 10245（37.2%） in prostate cancer and 8658（30.3%） in BPH samples were found to be abnormally methylated, and 60% of the methylated Cp G islands were in the promoter region. Compared with BPH samples, the prostate cancer samples showed differential methyation in 735 Cp G islands, including 458 hepermethyated and 256 hypomethelated ones. Seven genes（DPYS, P16, APC,GSTP1, TMEM122, RARB, and ARHGAP20） in prostate cancer were identified to have distinct methylations. Bioinformatics analysis suggested that these genes were associated with several biomolecular and biological processes, and among them DPYS gene was involved in 13 GO anotated biologic functions, development of 50 diseases and 47 protein interactions.Pyrosequencing of 7 sites of the CPG island in DPYS gene showed a methylation frequency of 32.7%, suggesting the importance of DPYS gene in the carcinogenesis and progression of prostate cancer.