Identification of miRNAs as promising biomarkers for diagnosis with combined analysis of mRNA transcriptome using next-generation sequencing in glioblastoma patients. Identification of miRNAs as promising biomarkers for diagnosis with combined analysis of mRNA transcriptome using next-generation sequencing in glioblastoma patients

Applying Next Generation Sequencing technique we compared the mRNA expression pattern of tumor tissue sample of 5 GPs and peritumoral region of 5 lower grade (I-II) Glioma patients, serving as control group. To determine the difference on mRNA expresion level between GBM and control cases, we performed cluster analysis on the NGS dataset of 6 replicates for each of the two goups of samples with iDEP 96 software. In order to characterize the extent of up- or downregulation, log2FC values were calculated using the iDEP.96 web tool applying the DESeq2 algorithm. On the base of that 6125 known mRNAs were identified to be differentially expressed using a threshold of false discovery rate (FDR) 2 during the analysis. Among them, 2253 mRNAs were upregulated (log2FC > 2) and 3872 mRNAs were downregulated (log2FC < -2) with biological revelance in tissue samples comparing with the control samples. To validate our results obtained by NGS, seven upregulated mRNAs: E2F2, HOXD13, VEGFA, CDC45, AURKB, HOXC10, MYBL2 and seven downregulated mRNAs:FABP6, PRLHR, NEUROD6, CBLN1, HRH3, HCN1, RELN were chosen for RT-qPCR analysis. As the result of that E2F2, HOXD13, VEGFA, CDC45, AURKB, HOXC10, MYBL2 was significantly upregulated while FABP6, PRLHR, NEUROD6, CBLN1, HRH3, HCN1, RELN was significantly downregulated, compared with those in the control samples. Overall design: Tissue samples were gathered from peritumoral region of 5 individuals diagnosed with lower grade (Grade I-II.) glioma, serving as control group, and from 5 patients with GBM. All samples in both group were confirmed histopathologically. These intraoperative quick-frozen tissue samples were stored at -80⁰C until further processing. Total RNA was isolated from the tissue samples of GBM and control group as well. Global transcriptomic profile was studied by RNA-sequencing applying the Illumina NextSeq500 platform including mRNA sequencing. In the case of all samples 3 replicates were included. In order to identify up-, or down-regulated miRNAs the GBM samples were compared to the control samples.

本研究采用下一代测序（Next Generation Sequencing，NGS）技术，对比了5例胶质母细胞瘤（GBM）患者的肿瘤组织样本与5例低级别（I-II级）胶质瘤患者的癌旁组织（作为对照组）的mRNA表达谱。为明确胶质母细胞瘤与对照组样本间的mRNA表达水平差异，本研究借助iDEP 96软件，对两组样本各6个生物学重复的NGS数据集开展聚类分析。为表征基因的上调/下调幅度，本研究通过iDEP.96网页工具，采用DESeq2算法计算了log2FC值。基于此分析流程，本研究以错误发现率（false discovery rate, FDR）阈值为2作为筛选标准，共鉴定出6125个差异表达的已知mRNA。其中，相较于对照组样本，本研究的组织样本中2253个mRNA呈上调表达（log2FC > 2），3872个mRNA呈下调表达（log2FC < -2），且具备生物学相关性。为验证NGS测序所得结果，本研究选取7个上调mRNA（E2F2、HOXD13、VEGFA、CDC45、AURKB、HOXC10、MYBL2）与7个下调mRNA（FABP6、PRLHR、NEUROD6、CBLN1、HRH3、HCN1、RELN）进行实时定量聚合酶链反应（RT-qPCR）验证。结果显示，与对照组样本相比，E2F2、HOXD13、VEGFA、CDC45、AURKB、HOXC10、MYBL2均显著上调，而FABP6、PRLHR、NEUROD6、CBLN1、HRH3、HCN1、RELN均显著下调。整体实验设计：组织样本采集自5例确诊为低级别（I-II级）胶质瘤患者的癌旁组织（作为对照组），以及5例胶质母细胞瘤患者的肿瘤组织。两组所有样本均经组织病理学确认。这些术中快速冷冻的组织样本保存于-80℃环境中，以待后续处理。本研究从胶质母细胞瘤组与对照组的组织样本中均提取了总RNA，采用Illumina NextSeq500平台进行RNA测序（含mRNA测序），以分析全局转录组谱。所有样本均设置3个生物学重复。为鉴定差异表达的miRNA，本研究将胶质母细胞瘤样本与对照组样本进行了对比。