端粒相关基因启动子甲基化通过调节端粒长度促进乳腺癌发生的机制及其早期诊断价值研究

Methylation status variation and telomere length change are the early events in breast carcinogenesis. However, the regulatory mechanism of DNA methylation on telomere length and its role in breast carcinogenesis is still unclear. In our previous research, we had found that the promoters of helicase RTEL1,WRN and telomerase reverse transcriptase (TERT) genes were significantly hyper-methylated and telomere length was shortened in breast cancer. The gene expression analysis showed that RTEL1 and WRN expression was reduced and the TERT expression was elevated in breast cancer. We speculate that helicases lead to telomere shortening, chromosome instability and promote breast carcinogenesis through inhibiting their expression by promoter methylation. Firstly, we will research the role of RTEL1 and WRN genes promoter methylation in breast cancer carcinogenesis through intervention with de-methylation reagent AZA and siRNA technology in this study. Furthermore, we will use of telomerase inhibitor to treat the cells with or without siRNA inference to find the mechanism which regulating to telomere length. Finally, we will establish the breast cancer early diagnosis model through RTEL1, WRN and TERT genes promoter methylation detection and confirm its performance in clinical. The purpose of this study was to elucidate the regulatory mechanism of DNA methylation on telomere length and its role in promoting breast carcinogenesis. Besides, this study will lay foundation for breast cancer early diagnosis with methylation detection in cfDNA.

DNA甲基化和端粒长度改变均为乳腺癌发生的早期事件。但DNA甲基化调节端粒长度的机制及其在乳腺癌发生中的作用仍不清楚。我们前期研究发现乳腺癌中负责解开端粒高级结构的解旋酶RTEL1、WRN及负责端粒延长的TERT基因启动子显著高甲基化，端粒长度显著缩短。对应的基因表达分析发现这两个解旋酶表达降低，而TERT表达升高；我们推测这两个解旋酶通过启动子甲基化降低基因表达，导致端粒末端解旋受阻、端粒缩短，染色体不稳定，促进癌症发生。本课题通过去甲基化药物干预、siRNA技术研究RTEL1、WRN甲基化在乳腺癌发生中的作用，进一步利用端粒酶抑制剂处理siRNA干扰前后的细胞，研究RTEL1、WRN调节端粒长度的机制。最后在游离DNA中检测RTEL1、WRN、TERT甲基化水平，建立乳腺癌早期诊断模型并验证诊断效果。旨在阐明端粒相关基因甲基化调节端粒长度、促进乳腺癌发生的机制，并为早期诊断奠定基础。

DNA甲基化和端粒长度改变均为乳腺癌发生的早期事件。但DNA甲基化调节端粒长度的机制及其在乳腺癌发生中的作用仍不清楚。我们前期研究发现乳腺癌中负责解开端粒高级结构的解旋酶RTEL1、WRN及负责端粒延长的TERT基因启动子显著高甲基化，端粒长度显著缩短。对应的基因表达分析发现这两个解旋酶表达降低，而TERT表达升高；我们推测这两个解旋酶通过启动子甲基化降低基因表达，导致端粒末端解旋受阻、端粒缩短，染色体不稳定，促进癌症发生。本课题通过去甲基化药物AZA干预处理四种亚型的乳腺癌细胞株，发现，在三阴性乳腺癌细胞MDA-MB-231细胞，AZA处理后、RTEL1、WRN、TERT基因启动子区甲基化水平显著降低，RTEL1和WRN基因表达水平升高，TERT基因表达水平无显著变化，端粒延长，细胞增殖、侵袭能力减弱。在三阴性乳腺癌细胞中通过shRNA技术干扰RTEL1，发现RTEL1表达降低，端粒延长，细胞增殖、侵袭能力增强，凋亡减弱。最后在游离DNA中检测RTEL1、WRN、TERT甲基化水平，通过逻辑回归建立乳腺癌早期诊断模型AUC值为0.7296。本研究基本阐明了解旋酶端粒相关基因启动子区甲基化促进乳腺癌发生的机制，并建立了基于端粒相关基因甲基化的乳腺癌无创诊断模型。