| 摘要: | 背景：乳癌是女性發生率以及死亡率最高的癌症，及早監測到治療無效、復發或轉移徵兆，以利於及早調整治療方式，可提高乳癌病人的存活率。目前檢測乳癌病人接受治療效果反應的檢測方式以影像學檢查和抽血測腫瘤指數指標（CEA、CA-153）為主，但影像學檢查往往無法即時檢測病人的治療效果疾復發轉移狀態，而即使臨床目前合併使用血液腫瘤指數CEA與CA-153來檢測病患復發，敏感度僅約70%，過去文獻指出異常的DNA甲基化與癌症的發生及惡化有關，血漿中游離DNA中有特定基因的甲基化可用於監測病人的預後及追蹤治療效果，可及早監測乳癌復發，並提升乳癌存活率。
研究目的：使用The Cancer Genome Atlas（TCGA）數據庫和Illumina 450K甲基化微陣列篩選乳癌異常甲基化的生物指標，TMEM240 和 ITPRIPL1 基因，並分析此二基因與乳癌病人藥物治療的關聯性以及開發新的潛在預後生物指標。
實驗設計：使用 TCGA 數據庫來分析乳癌患者的存活率以及治療反應與甲基化的關係。透過使用乳癌細胞（T-47D 和 MDA-MB-231）分析了細胞增殖、爬行、侵襲和藥物敏感度、與TMEM240 和 ITPRIPL1 基因大量表現和基因沉默的影響。也使用液體檢體（來自乳癌患者的血漿）分析治療後的乳癌病人血漿中游離DNA中有TMEM240 和 ITPRIPL1 基因的甲基化程度。
結果：在 TCGA 資料庫中，我們發現 TMEM240 和 ITPRIPL1基因的高度甲基化與乳癌患者存活率較低以及接受化療和荷爾蒙治療反應不佳有顯著關係。在乳癌細胞 （T-47D 和 MDA-MB-231）中給予去甲基化藥物，發現低 TMEM240 和 ITPRIPL1 mRNA 表現量受異常啟動子高度甲基化調節。發現 TMEM240 的減少可誘導乳腺細胞（T-47D 和 MDA-MB-231）中的細胞增殖、侵襲和遷移。過度表現 TMEM240 會降低乳癌細胞的生長能力。 在T-47D乳癌細胞中TMEM240沉默會導致乳癌細胞對Tamoxifen藥物治療的反應不佳。預後不良的乳癌患者血漿游離DNA中TMEM240和ITPRIPL1基因的甲基化程度上升，在血漿游離DNA中沒有異常循環甲基化 TMEM240 的患者預後良好。
結論：TMEM240 和 ITPRIPL1 基因的高度甲基化造成兩基因表現量下降，增加乳癌細胞的存活率、侵襲與爬行能力以及降低藥物治療反應，此外血液中TMEM240 和 ITPRIPL1基因高度甲基化訊號是可用於檢測乳癌患者治療反應和預後不佳的潛在生物指標。
Introduction：Breast cancer is the most common woman cancer and the leading cause of cancer deaths worldwide. The most widely used prognosis monitoring tool of breast cancer are imaging examination and measurement of serum tumor markers （CEA, CA-153）in clinical. However, the imaging examination cannot immediately response the treatment effectiveness and measurement of CEA and CA-153 biomarkers are inadequate to predict poor prognosis of breast cancer. Previous studies indicate the altered DNA methylation is involved in the occurrence of cancer. The specific genes methylation of circulating cell-free DNA in plasma can be used to monitor patients' prognosis, response to treatment, predict the recurrence and improve the survival of breast cancer.
Purpose：To identify the methylation biomarkers of breast cancer was analyzed by the data from The Cancer Genome Atlas（TCGA）database and the Illumina 450K methylation array. To demonstrate the relevance of the candidate genes, TMEM240 gene, and ITPRIPL1 gene to the drug treatment of breast cancer was analyzed in cell model and breast cancer patients. Then, develop new prognostic biomarkers by clinical validation.
Method：We used the TCGA database to analyze the treatment response of breast cancer patients. Then, we performed SRB assay, trans-well assay, and wound healing analysis for breast cancer cells (T-47d and MDA-MB-231). The cell proliferation, migration, invasion, and drug sensitivity, gene expression and mechanisms for gene silencing were analyzed. And liquid biopsies (plasma from breast cancer patients) were also detected by specific gene methylation in progressive breast cancer patients.
Results：In TCGA database, we found that hypermethylation of TMEM240 and ITPRIPL1 genes was associated with poor overall survival and worse chemotherapy and hormone treatment response in breast cancer patients. Low TMEM240 and ITPRIPL1 mRNA expression was observed. Demethylating drug treatment found that low TMEM240 and ITPRIPL1 mRNA expression was regulated by aberrant promoter hypermethylation. Knockdown assay and overexpression transfection were performed in breast cancer cells T47D and MDA-MB-231. A decrease of TMEM240 induced cell proliferation, invasion and migration in breast cancer cells (T-47D and MDA-MB-231). Overexpression TMEM240 can decreased cell viability in breast cancer cells. Silencing of TMEM240 expression can induce the poor response for Tamoxifen drugs treatment. The methylation level of TMEM240 and ITPRIPL1 gene are increased in cell-free DNA of the plasma in breast cancer patients with poor prognosis. Patients without aberrant circulating methylated TMEM240 in blood revealed good prognosis.
Conclusion：Hypermethylation of TMEM240 and ITPRIPL1 genes induced low expression of TMEM240 and ITPRIPL1, increase of breast cancer cell viability, invasion, and poor drug response in breast cancer cells. In addition, circulation of Hypermethylated TMEM240 and ITPRIPL1 in plasma is a potential biomarker for treatment response and poor prognosis in breast cancer patients. |
