摘要目的 筛选可能受甲基化调控的,与髓母细胞瘤(medulloblastoma,MB)WNT分子亚型相关的微小RNA(miRNA)分子标志物.方法 采用NanoString技术对45例原发性MB石蜡样本进行了包含22个经典MB亚型特异性基因的mRNA表达水平的检测,从而对肿瘤进行分子亚型鉴定.同时通过miRNA表达芯片检测去甲基化药物处理前后MB细胞系(Daoy、D283、D341)的miRNA表达情况,筛选出可能受甲基化调控的miRNA分子标志物,并用甲基化特异性PCR(MSP)和即时荧光定量PCR方法对其甲基化状态和表达水平分别进行验证.最后,对照分子亚型找到与MB亚型相关的受甲基化调控的miRNA.结果 45例MB除1例无法准确划分亚型外,其余44例中4例为WNT组,8例为SHH组,16例为Group3,16例为Group4.miR-449a在去甲基化药物处理前后的MB细胞系中表达水平具有明显差异,生物信息学分析确认了其启动子区CpG岛的存在,MSP方法进一步证明了部分肿瘤受启动子高甲基化的调控,且WNT亚型MB肿瘤样本中发现miR-449a存在相对高表达.结论 基于金标准(NanoString技术)对MB肿瘤进行分子亚型鉴定,并发现miR-449a在MB细胞系中受到甲基化调控,其可能为WNT亚型MB的分子标志物.

abstractsObjective To identify the candidate epigenetic biomarkers of Wnt subtype of medulloblastoma ( MB ) . Methods MicroRNAs ( miRNAs ) expression array was used to detect the expression of miRNAs in MB cell lines with or without treatment by demethylation reagent. Nanostring gene expression array was used to detect the expression level of mRNA in 45 samples of primary MB. Molecular subtyping was performed based on the NanoString data. The status of methylation was confirmed by methylation specific PCR. The expression of candidate miRNA was confirmed by real-time PCR. Results All 45 MBs except one were classified into the four molecular groups:4 in WNT group,8 in SHH group,16 were in Group3 and 16 in Group4. Methylation specific PCR (MSP) assay confirmed miR-449a was silenced due to aberrant DNA methylation in MB cell lines. WNT subtype of MBs showed relatively higher expression of miR-449a comparing with other subgroups. Conclusion MiR-449a, a candidate tumor suppressor gene regulated by hypermethylation, is a novel potential epigenetic marker for WNT subtype of MBs.