血清microRNA 在多形性胶质母细胞瘤术预后评估中的研究

目的:筛选多形性胶质母细胞瘤（glioblastoma multiform，GBM）患者术前、术后血清中差异表达的microRNAs（miRNAs），并探讨差异表达的miRNAs与患者术后预后的相关性。方法:收集2006年1 月至2009年6 月48例北京天坛医院经临床病理诊断为GBM患者的术前术后血清样本。采用Solexa 测序的方法初步筛选出术前术后表达量有差异的 miRNA ，用实时荧光定量PCR（quantitative real-time PCR ，RT-qPCR）的方法对每个样本进行逐一验证，应用t 检验的方法筛选出满足条件的miRNA（两组之间的平均值差异在2 倍以上，且P < 0.05），对48例患者进行随访，统计生存时间，根据48例患者中位生存时间494 d，将所有标本分为长生存期组和短生存期组，应用Kaplan-Meier 法和Log-rank 检验，研究患者术后血清miRNAs的表达量与患者生存时间之间是否存在统计学意义的相关性。结果:Solexa 结果显示，有63个miRNA 表达量存在差异，基于本研究先前的研究成果和其他文献的报道，从中选出4 个miRNA（miR-26b，miR-30e ，miR-129- 3p，miR-206）进行逐一验证并进行统计学分析，结果只有1 个miRNAs（miR-30e）在术后患者血清中的表达水平有明显上调现象（术前与术后表达水平平均值差异≥ 2 倍且P < 0.05），随访结果显示，生存时间> 494 d，患者术后血清miR-30e 的表达水平有降低的趋势（P < 0.05），但生存分析显示，患者术后血清中miR-30e 的表达量与患者总生存时间之间差异无统计学意义（P = 0.101）。 结论:GBM患者术前术后血清中差异表达的miRNA 只有miR-30e ，且术后患者血清中的miR-30e 水平与肿瘤负荷成负相关关系。生存分析结果显示，术后患者血清miR-30e 的表达水平与患者的预后没有明显的相关性。 

Serum microRNA profiles as novel biomarkers for the post-operative evaluation and survival of patients with glioblastoma multiform

Objective:To investigate the differentially expressed miRNAs in serum collected post operation and compared these miR-NAs with those collected pre-surgery among patients suffering from glioblastoma multiform (GBM) and undergoing regular clinical fol-low-up. These miRNAs may be potential biomarkers for the post-operative evaluation of patients with GBM. Methods:Forty-eight pa-tients with GBM and clinical pathological diagnosis were enrolled in this study. In the initial biomarker screening stage, total RNAs were extracted and subjected to Solexa sequencing to select miRNAs with significantly altered expression pre-and post-operation. Some of these differentially expressed miRNAs were chosen and verified through TaqMan probe-based qRT-PCR assay. A t-test was performed to determine the miRNAs that satisfied the two criteria, namely, fold change>2 and P<0.05. All of the patients were fol-lowed-up, and survival data were collected. The patients were then classified into two groups, namely, long-and short-survival groups, on the basis of the median of the miR-30e expression levels in the sera collected post-operation. Kaplan-Meier method and Log-rank test (SPSS version 19.0, IBM) were employed to determine the possible relationships between miR-30e expression levels in the sera collected post-operation and patients' overall survival. Results: Solexa revealed 63 differentially expressed miRNAs. Four miRNAs, namely, miR-26b, miR-30e, miR-129-3p, and miR-206, were selected on the basis of previous and present findings. These miRNAs were then verified in the RT-qPCR phase. Among these miRNAs, only miR-30e was significantly upregulated post-operation. The serum miR-30e expression level post-operation was not significantly associated with the overall survival of the patients. A low miR-30e expression level corresponded to prolonged survival. Conclusion:miR-30e was upregulated in the sera collected post-operation from patients with GBM. This miRNA may be negatively related to the tumor load of these patients. The miR-30e expression level in the serum col-lected post-surgery serum was not significantly associated with overall survival. Therefore, miR-30e may serve as a novel potential non-invasive biomarker for the post-operative evaluation of patients with GBM.