DNA甲基化抑制HOXB13基因在乳腺癌中的表达及其对β-catenin/TCF-4信号通路的调控研究

Breast cancer (BrCa) is a complex disease driven by multiple factors including both genetic and epigenetic alterations. Recent studies revealed that abnormal gene expression induced by epigenetic changes, including aberrant promoter methylation and histone modification, plays a critical role in human breast carcinogenesis. Silencing of tumor suppressor genes (TSGs) by promoter CpG methylation facilitates cells growth and survival advantages and further results in tumor initiation and progression, thus directly contributing to breast tumorigenesis. Homeobox protein Hox-B13 (HOXB13) is a homeodomain protein implicated to play a role in growth arrest and apoptosis in breast cancer cells, which was frequently downregulated or silenced in breast cancer cell lines and tumor tissues, but readily expressed in normal breast tissues. However，the mechanism underlying this silencing has not been fully understood. In this study, we try to analyze the DNA methylation level of HOXB13 in breast cancer cells and primary tumors with clinicopathological features to see whether HOXB13 can work as a target to detect the malignancy with its stage. How can HOXB13 take part in tumorigenesis is also a part of our project. The expression of HOXB13 can inhibits TCF4-mediated transcriptional activity in prostate cells, we want to investigate whether HOXB13 can regulate beta-catenin/TCF4 pathway in breast cancer. Altogether, data included in this project will contribute to elucidating the unique silencing mechanisms and function of HOXB13 in breast cancer, which can provide more details and evidence for the tumor diagnosis and treatment.

乳腺癌在女性中的高发率及高死亡率使乳腺癌的早期诊断和治疗成为目前亟待解决的重要课题。DNA甲基化属于表观遗传学调控机制，通过检测基因的甲基化状态可以监控乳腺癌的发生和发展。我们的前期工作发现，HOXB13在正常乳腺组织中表达正常，但在乳腺癌细胞株中表达存在下调或缺失的现象，去甲基化作用可以恢复其表达，表明DNA甲基化改变可致HOXB13基因表达沉默。HOXB13是一种非转录因子，能够抑制TCF4分子的转录激活能力，从而负调控Wnt信号通路抑制细胞生长，促进其凋亡。本研究通过对HOXB13在乳腺癌中的甲基化状态进行研究，结合临床病理资料分析HOXB13作为评价乳腺癌分级分期新靶点的可行性；对HOXB13参与的调控的β-catenin/Tcf-4信号通路及凋亡途径的分子机制进行探讨，以期筛选出潜在的乳腺癌治疗靶点，为乳腺癌的干预和治疗提供理论基础。

乳腺癌在女性中的高发率及高死亡率使乳腺癌的早期诊断和治疗成为目前亟待解决的重要课题。DNA 甲基化属于表观遗传学调控机制，通过检测基因的甲基化状态可以监控乳腺癌的发生和发展。HOXB13是一类转录因子，已有文献报道其在前列腺癌、肾癌、黑色素瘤等肿瘤中出现不同程度的甲基化和表达水平的明显降低，并可能具有显著性抑制肿瘤细胞生长的生物学功能。. 本研究通过逆转录聚合酶链式反应（Reverse transcription polymerase chain reaction，RT-PCR）、实时荧光定量聚合酶链式反应（Real-time fluorescence quantitative polymerase chain reaction，Real-time PCR）、甲基化特异性聚合酶链式反应（Methylation specific PCR，MSP）、蛋白免疫印迹（Western blot，WB）和在线数据库的方法分析了乳腺癌组织及其正常组织中HOXB13基因的表达差异及甲基化状态；采用5-氮杂-2-脱氧胞苷（5-Aza-dC）联合组蛋白去乙酰化酶抑制剂曲古菌素A（Trichostatin A，TSA）的方法处理 HOXB13表达沉默或下调的肿瘤细胞株。通过软琼脂或平板克隆形成实验、MTS细胞增殖实验和Annexin V-FITC/PI双标流式细胞术等体外实验检测了HOXB13对乳腺癌细胞增殖和凋亡的影响，并进一步采用定量PCR和WB的方法检测了HOXB13对TGFβ信号通路及p53分子信号通路的影响。结果发现，相对于正常组织或癌旁组织，乳腺癌组织或细胞株的HOXB13表达水平均有不同程度的下调或沉默。对有HOXB13甲基化的肿瘤细胞株去甲基化处理后出现了其mRNA表达水平的恢复。体外细胞功能实验结果示，HOXB13能够有效抑制肿瘤细胞的增殖并促进凋亡，从而发挥其抑制肿瘤细胞生长的生物学功能，并且进一步发现其可能是通过靶向转录激活p53通路的活化进而发挥其抑制肿瘤细胞生长的作用。