微型核糖核酸基因let-7a-3在急性骨髓性白血病病人之甲基化情形

Abstract

微型核糖核酸 let-7家族成員可藉由調控多種參與細胞週期的基因，包括致癌基因RAS、HMGA2等來調控細胞增生、分化。目前已知道成員之一的let-7a-3可藉由表觀遺傳來調控基因表現，而且在上皮性卵巢癌病人中發現其let-7a-3基因有高度甲基化，且基因甲基化情形和病人預後具統計上的意義。故本研究的目的即是想了解急性骨髓性白血病病人其let-7a-3基因甲基化的情形，並探討其在急性骨髓性白血病的重要性。 本研究以兩種偵測DNA甲基化方法對病人檢體作分析：以組合重亞硫酸鹽限制酶分析法對90個病人檢體作分析，發現73個病人分析的結果是甲基化（81.1％）、11個病人是部份甲基化（12.2％）和6個是未甲基化的病人（6.7％）；並以重亞硫酸鹽定序法對其中之16個檢體的let-7a-3基因啟動子區域內的33個CpG位點做分析，同時與組合重亞硫酸鹽限制酶分析法結果進行確認。結果發現兩種方法實驗結果一致，組合重亞硫酸鹽限制酶分析法結果是甲基化的檢體其重亞硫酸鹽定序法得到的甲基化情形在56~86％；部分甲基化的在45~58％；未甲基化的在12~15％。將組合重亞硫酸鹽限制酶分析法結果和病人臨床資料作卡方測定分析，發現let-7a-3基因啟動子區域甲基化情形和染色體核型分型及CEBPA轉錄起點上游－1423位置上的CpG是否甲基化則具有統計上的意義，p值分別是0.006和0.002。顯示在unfavorable和intermediate的病人中，let-7a-3多為甲基化的情形，而在favorable的病人中，甲基化和部分甲基化比例約為一比一；CEBPA未甲基化的病人其let-7a-3多為甲基化。 在5年的存活分析結果中，發現核型分型是intermediate的病人中，let-7a-3 基因啟動子區域甲基化的病人預後比較好，p值為0.046；在CEBPA未甲基化的病人中，let-7a-3 基因啟動子區域甲基化的病人預後也比較好，p值為0.02。另外，根據細胞株和僅有的6支病人檢體RNA結果顯示，let-7a-3 基因啟動子區域甲基化情形和RNA表現是有負相關的，r值是－0.82。

Let-7 family miRNAs repress several genes involved in cell cycle, including RAS and HMGA2 . By this way, they play specific roles in cell proliferation, differentiation and the other biological processes. Let-7a-3 belongs to the archetypal let-7 miRNA gene family and was found to be regulated by epigenetic regulation. Recent study showed that let-7a-3 was methylated in epithelial ovarian cancer and methylation status was associated with survival of patients. In this study, we speculate the methylation status of let-7a-3 and its significance in acute myeloid leukemia. Combined bisulfite restriction analysis（COBRA）were used to evaluate the let-7a-3 methylation status in 90 AML patients. We found that let-7a-3 was methylated in 73 patients（81.1％）, partial methylated in 11 patients（12.2％）and unmethylated in 6 patients（6.7％）. Of them, 16 patients were simultaneously analyzed for methylation status of 33 CpG sites in let-7a-3 promoter region by using bisulfite sequencing PCR method （BSSP）. BSSP demonstrated similar results to the COBRA method, showing the methylation levels ranged from 56％ to 86％ in patients with let-7a-3 methylated, 45％ to 58％ in patients with let-7a-3 partial methylated and 12％ to 15％ in patients with let-7a-3 unmethylated. Chi-square test was performed to assess the association of let-7a-3 methylation status with patients’ age, gender and FAB subtype . We found that let-7a-3 methylation status was negatively correlated with karyotype and CEBPA methylation significantly（0.006 and 0.002, respectively）. Kaplan-Meier survival analysis showed that let-7a-3 methylation in AML patients with intermediate karyotype or CEBPA unmethylation had better prognosis.