title: Sequencing and bioinformatics analysis of the differentially expressed genes in herniated discs with or without calcification.
abstract: 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.
authors.affiliation: Department of Orthopedics, Peking University Third Hospital, Beijing 100191, P.R. China.
authors.forename: Jia
authors.initials: J
authors.lastname: Shao
authors.affiliation: Department of Orthopedics, Peking University Third Hospital, Beijing 100191, P.R. China.
authors.forename: Miao
authors.initials: M
authors.lastname: Yu
authors.affiliation: Department of Orthopedics, Peking University Third Hospital, Beijing 100191, P.R. China.
authors.forename: Liang
authors.initials: L
authors.lastname: Jiang
authors.affiliation: Department of Orthopedics, Peking University Third Hospital, Beijing 100191, P.R. China.
authors.forename: Fengliang
authors.initials: F
authors.lastname: Wu
authors.affiliation: Department of Orthopedics, Peking University Third Hospital, Beijing 100191, P.R. China.
authors.forename: Xiaoguang
authors.initials: X
authors.lastname: Liu
mesh_terms: D002114:Calcinosis / Q000150:complications* / Q000235:genetics*; D019295:Computational Biology / Q000379:methods*; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D063990:Gene Ontology; D006801:Humans; D007403:Intervertebral Disc / Q000378:metabolism / Q000473:pathology; D007405:Intervertebral Disc Displacement / Q000150:complications* / Q000000981:diagnostic imaging* / Q000235:genetics; D060066:Protein Interaction Maps; D015203:Reproducibility of Results; D017423:Sequence Analysis, RNA; D055114:X-Ray Microtomography