小鼠骨髓细胞中p16和Ralb基因启动区CpG岛的甲基化位点分析

抑癌基因p16和白血病致癌因子Ralb与白血病的发生密切相关,其启动子区CpG岛的甲基化对基因表达具有重要作用.本文旨在分析p16、Ralb基因启动子区CpG岛甲基化位点信息,并比较这两个基因在小鼠骨髓细胞和原代培养的骨髓细胞中甲基化状态的差异.运用"MethPrimer"软件预测p16、Ralb基因启动子区的CpG岛,设计甲基化特异性引物.利用重亚硫酸盐测序法(BSP)检测甲基化位点信息.结果显示,p16有1个CpG岛,岛上21个CpG位点全部未发生甲基化;Ralb有2个CpG岛,CpG岛1上的5个CpG位点全部呈甲基化状态,而CpG岛2上的17个CpG位点全部呈非甲基化状态,且小鼠骨髓细胞和体外原代培养的骨髓细胞中两基因的甲基化状态一致.表明p16、Ralb基因甲基化状态未受外界培养条件的影响而改变,提示在与两基因甲基化相关的研究中体外试验可替代体内试验.     

DNA methylation analysis of bone marrow cells at diagnosis of acute lymphoblastic leukemia and at remission

To detect genes with CpG sites that display methylation patterns that are characteristic of acute lymphoblastic leukemia (ALL) cells, we compared the methylation patterns of cells taken at diagnosis from 20 patients with pediatric ALL to the methylation patterns in mononuclear cells from bone marrow of the same patients during remission and in non-leukemic control cells from bone marrow or blood. Using a custom-designed assay, we measured the methylation levels of 1,320 CpG sites in regulatory regions of 413 genes that were analyzed because they display allele-specific gene expression (ASE) in ALL cells. The rationale for our selection of CpG sites was that ASE could be the result of allele-specific methylation in the promoter regions of the genes. We found that the ALL cells had methylation profiles that allowed distinction between ALL cells and control cells. Using stringent criteria for calling differential methylation, we identified 28 CpG sites in 24 genes with recurrent differences in their methylation levels between ALL cells and control cells. Twenty of the differentially methylated genes were hypermethylated in the ALL cells, and as many as nine of them (AMICA1, CPNE7, CR1, DBC1, EYA4, LGALS8, RYR3, UQCRFS1, WDR35) have functions in cell signaling and/or apoptosis. The methylation levels of a subset of the genes were consistent with an inverse relationship with the mRNA expression levels in a large number of ALL cells from published data sets, supporting a potential biological effect of the methylation signatures and their application for diagnostic purposes.     

Low Expression of miR-196b Enhances the Expression of BCR-ABL1 and HOXA9 Oncogenes in Chronic Myeloid Leukemogenesis

MicroRNAs (miRNAs) can function as tumor suppressors or oncogene promoters during tumor development. In this study, low levels of expression of miR-196b were detected in patients with chronic myeloid leukemia. Bisulfite genomic sequencing PCR and methylation-specific PCR were used to examine the methylation status of the CpG islands in the miR-196b promoter in K562 cells, patients with leukemia and healthy individuals. The CpG islands showed more methylation in patients with chronic myeloid leukemia compared with healthy individuals (P<0.05), which indicated that low expression of miR-196b may be associated with an increase in the methylation of CpG islands. The dual-luciferase reporter assay system demonstrated that BCR-ABL1 and HOXA9 are the target genes of miR-196b, which was consistent with predictions from bioinformatics software analyses. Further examination of cell function indicated that miR-196b acts to reduce BCR-ABL1 and HOXA9 protein levels, decrease cell proliferation rate and retard the cell cycle. A low level of expression of miR-196b can cause up-regulation of BCR-ABL1 and HOXA9 expression, which leads to the development of chronic myeloid leukemia. MiR-196b may represent an effective target for chronic myeloid leukemia therapy.     

EGFR基因启动子区甲基化状态分析

表皮生长因子受体(epidermal growth factor receptor,EGFR)是HER/ERB-B跨膜受体激酶家族成员之一.EGFR的过表达促进细胞的增殖、存活和迁移,与许多实体瘤病人的低存活率相关.EGFR的表达受其启动子DNA甲基化调控.EGFR的转录沉默与CpG岛高甲基化相关.EGFR基因5′调控区包括1个富含GC的启动子,缺保守序列TATA盒和CAAT盒,有多个位点可以起始转录.本实验运用Bisulfite Sequencing PCR(BSP)方法检测了2种肿瘤细胞HeLa(EGFR+)和K562(EGFR)EGFR基因-1300～+600的甲基化状态.所检测目的片段共包含178个CpG位点.发现EGFR阳性与EGFR阴性两种细胞系的甲基化状态不同:宫颈癌细胞系HeLa转录起始点附近包括第一外显子区(-244～+91)处于非甲基化状态,白血病细胞系K562转录起始点附近包括第一外显子区呈嵌合性的高甲基化状态.因此,第一外显子比启动子区的甲基化状态更能反映基因的活化状况.     

食管癌LAPTM4B mRNA表达及其启动子区甲基化

研究溶酶体相关4次跨膜蛋白B（lysosome associated protein transmembrane 4 beta,LAPTM4B）基因在食管癌中的表达,及其启动子区甲基化状态,为进一步揭示LAPTM4B在不同肿瘤中表达高低机理提供参考.采用半定量RT-PCR法,确定42对食管癌中LAPTM4B mRNA表达.采用5对肝癌中LAPTM4B mRNA表达做内对照（利用灰度值比较）,分析该基因在食管癌中的表达强度.选取其中3对食管癌组织样品（癌组织和癌旁正常组织）,提取基因组DNA,采用亚硫酸氢钠修饰法,联合基因测序法分析LAPTM4B启动子区是否有甲基化修饰位点存在.结果发现,在42对食管癌组织中,癌组织和癌旁正常组织LAPTM4B mRNA表达存在差异：癌组织中LAPTM4B mRNA表达阳性为37/42（88.1%）,癌旁正常组织中LAPTM4B mRNA表达阳性为26/42（61.9%）.经基因测序法分析3对食管癌组织经通用引物PCR扩增的片段,发现1例癌旁正常组织样品中有3个CpG位点.以上结果表明,LAPTM4B基因与肝癌比较在食管癌中低表达,其启动子区1例癌旁正常组织在靠近转录起始点上游-418、-416和-398位置,存在3个CpG位点,而其他2例癌旁正常组织和3例癌组织中,没有发现CpG位点.这提示,LAPTM4B基因启动子区甲基化是其表达调节的重要方式.