乳腺癌患者血浆循环核酸表观遗传变异调控的研究

Breast cancer is one of malignant diseases in female. Now many data showed that the cancer cells release their nucleic acid into the blood, normally there are few of nucleic acid in the plasma of healthy person, but more were found in the plasma of the patients with cancer. Moreover, the character of the plasma nucleic acid is same as that of the nucleic acid in cancer tissue. Abnormal expression of miRNA and DNA methylation had been found during breast cancer formation. the structure of miRNA is similar to that of mRNA, 7-methyl G cap structure and poly A tail were found in pri-miRNA, and more than 47% of coding gene of miRNA are associated with CpG island in human, which suggested that DNA methylation modulate the expression of miRNA. In this study, we will evaluate the modulation of DNA methylation on miRNA which abnormal expressed in plasma of the patients with breast cancer with QM-MSP、westen blot 、 miRNA chip and so on. The results will elucidate the relationship between miRNA and DNA methylation further and the effect of DNA methylation and miRNA on the expression of suppressive gene, which will apply theoretical support for the questions in epigenetic regulation in breast cancer formation and might found new methods for clinical diagnosis and treatment of breast cancer.

乳腺癌发生、发展过程中会持续释放肿瘤细胞核酸进入外周血,其miRNA的表达及DNA的甲基化状态均出现异常。与mRNA相似, pri-miRNA也具有7-甲基鸟嘌呤核苷帽结构和多聚腺苷酸尾部, 且人类miRNA编码基因中约有47%与CpG岛相关。这提示: miRNA可能像一般的蛋白编码基因一样, 可以被DNA甲基化等表观遗传修饰调控。因此本项目拟运用miRNA芯片、定量多重甲基化特异性PCR(QM-MSP)、和Western Blot等生物学技术探究乳腺癌DNA甲基化对在血浆循环中异常表达的microRNA转录调控情况进行研究。旨在进一步明确异常miRNA与DNA甲基化的关系，揭示DNA甲基化、microRNA在抑癌基因表达沉默与缺失中的作用，为乳腺癌的发生发展中表观遗传调控网络的相互作用等问题提供理论基础。为临床乳腺癌诊治提供理论依据及可能的治疗策略。

乳腺癌的发生发展是一个复杂的过程，涉及到甲基化水平变化及miRNA水平的改变。本项目主要通过荧光定量PCR、westen blotting,双荧光素酶报告基因等方法研究了影响乳腺癌DNA甲基转移酶DNMT3B与DNMT3A的miRNA及其在乳腺癌患者血清中的表达。并应用DNA甲基转移酶抑制剂及siRNA干涉改变DNA甲基转移酶的表达，以探究其对miRNA表达的影响。同时采用BSP及HRM检测进一步了乳腺癌患者血清循环DNA中抑癌基因BRCA1与FHIT启动子区甲基化水平，以探讨乳腺癌患者血清循环核酸异常变异调控的原因，为乳腺癌早期诊断及靶向治疗提供理论依据。实验结果显示乳腺浸润性导管癌组织中DNA甲基转移酶DNMT3B与DNMT3A均存在异常表达。将人工合成miRNA摸拟体(mimics)转染至乳腺癌细胞后，经Westen bolting检测可见miR-29c mimics可以使二都表达均下调，通过双荧光素酶报告基因进一步确认hsa-miR-29c可以通过与mRNA 3’端结合影响DNMT3B与DNMT3A蛋白的表达，并抑制细胞生长；将乳腺癌细胞株MCF-7进行DNA甲基转移酶抑制剂5-aza-cdr处理后，67个miRNA表显著改变(p<0.05)，用siRNA干涉DNMT3B的表达后，miRNA的表达也出现改变，其中has-miR-200a的表达出现显著上调(p<0.05),同时也影响相关抑癌基因BRCA1、BRCA2与Survivin的表达。血清BSP结果显示乳腺浸润性导管癌患者血清循环核酸BRCA1与FHIT启动子甲基化水平均明显高于乳腺纤维腺病患者与健康对照组，进一步进行HRM定量检测结果发现超过83.3%的乳腺恶性导管癌患者血清中FHIT启动子甲基化水平超过50% ，与另外两组存在显著性差异， FHIT启动子区甲基化程度与乳腺导管癌存在着显著相关性(p<0.05)。综上所述，乳腺癌表观遗传变异调控是一个复杂的网络，DNA甲基转移酶的异常表达，会影响到miRNA网络的改变及相关抑癌基因的表达；反过来miRNA会在翻译水平上通过与DNA甲基转移酶3’非编码区结合影响其表达，两者相互协调，影响乳腺癌的发生发展。而乳腺癌患者血清循环DNA中抑癌基因FHIT启动子区甲基化水平与乳腺导管癌的发生发展也存在着显著相关性，因此血清游离核酸的检测可以反应表观遗传变异的状