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Sequencing and bioinformatics analysis of the differentially expressed genes in herniated discs with or without calcification

机译:伴或不伴钙化的椎间盘突出表达差异基因的测序和生物信息学分析

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摘要

The purpose of this study was to detect the differentially expressed genes between ossified herniated discs and herniated discs without ossification. In addition, we sought to identify a few candidate genes and pathways by using bioinformatics analysis. We analyzed 6 samples each of ossified herniated discs (experimental group) and herniated discs without ossification (control group). Purified mRNA and cDNA extracted from the samples were subjected to sequencing. The NOISeq method was used to statistically identify the differentially expressed genes (DEGs) between the 2 groups. An in-depth analysis using bioinformatics tools based on the DEGs was performed using Gene Ontology (GO) enrichment, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment, and protein-protein interaction network analysis. The top 6 DEGs were verified using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). A total of 132 DEGs was detected. A total of 129 genes in the ossified group were upregulated and 3 genes were found to be downregulated as compared to the control group. The top 3 cellular components in GO ontologies analysis were extracellular matrix components. GO functions were mainly related to the glycoprotein in the cell membrane and extracellular matrix. The GO process was related to completing response to stimulus, immune reflex and defense. The top 5 KEGG enrichment pathways were associated with infection and inflammation. Three of the top 20 DEGs [sclerostin (SOST), WNT inhibitory factor 1 (WIF1) and secreted frizzled related protein 4 (SFRP4)] were related to the inhibition of the Wnt pathway. The ossified discs exhibited a higher expression of the top 6 DEGs [SOST, joining chain of multimeric IgA and IgM (IGJ; also known as JCHAIN), defensin alpha 4 (DEFA4), SFRP4, proteinase 3 (PRTN3) and cathepsin G (CTSG)], with the associated P-values of 0.045, 0.000, 0.008, 0.010, 0.015 and 0.002, respectively, as calculated by the independent sample t-test. The gene expression profiling of the 2 groups revealed differential gene expression. Thus, our data suggest that Wnt pathway abnormality and local inflammation may be related to disc ossification.
机译:这项研究的目的是检测骨化的椎间盘突出症和无骨化的椎间盘突出症之间差异表达的基因。另外,我们试图通过使用生物信息学分析来鉴定一些候选基因和途径。我们分析了6个样品,分别是骨化的椎间盘突出(实验组)和无骨化的椎间盘突出(对照组)。从样品中提取的纯化的mRNA和cDNA进行测序。使用NOISeq方法来统计地识别两组之间的差异表达基因(DEG)。使用基因本体论(GO)富集,京都市基因与基因组百科全书(KEGG)途径富集和蛋白质-蛋白质相互作用网络分析,使用基于DEG的生物信息学工具进行了深入分析。使用逆转录定量聚合酶链反应(RT-qPCR)验证了前6个DEG。总共检测到132个DEG。与对照组相比,骨化组中总共有129个基因被上调,并且发现3个基因被下调。 GO本体分析中排名前三的细胞成分是细胞外基质成分。 GO功能主要与细胞膜和细胞外基质中的糖蛋白有关。 GO过程与完成对刺激,免疫反射和防御的反应有关。排名前5位的KEGG富集途径与感染和炎症相关。前20个DEG中的三个[硬化蛋白(SOST),WNT抑制因子1(WIF1)和分泌的卷曲相关蛋白4(SFRP4)]与Wnt途径的抑制有关。骨化的椎间盘在前6个DEGs [SOST,多聚IgA和IgM(IGJ;也称为JCHAIN)的连接链,防御素α4(DEFA4),SFRP4,蛋白酶3(PRTN3)和组织蛋白酶G(CTSG)的连接中表现出更高的表达。 )],相关的P值分别为0.045、0.000、0.008、0.010、0.015和0.002,由独立样本t检验计算得出。两组的基因表达谱显示差异的基因表达。因此,我们的数据表明Wnt通路异常和局部炎症可能与椎间盘骨化有关。

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