Aberrantly Expressed Long Noncoding RNAs In Human Degenerated Intervertebral Disc Nucleus Pulposus

As a major factor affecting the quality of life of the population, low back pain has asignificant social and economic impact worldwide. The main cause of low back pain isintervertebral disc degeneration (IDD). Numerous studies indicate that a variety of cellularevents are disordered in the progression of IDD. Underlying these alterations there is thedysregulated gene expression of particular molecules. Multiple lines of evidences suggestthat alterations of several regulatory factors at different levels result in the aforementionedeventual gene dysregulation. Amongst these, aberrantly expressed regulatory noncodingRNAs are gained more and more attention in the recent years. Noncoding RNA can begrouped into several subtypes. In addition to the well-known short noncoding RNAs asmiRNAs, increasing evidence suggests that long noncoding RNAs (LncRNA) act as keyregulators in a wide aspect of biologic processes. Dysregulated expression of LncRNAshas been demonstrated being implicated in a variety of human diseases. However, there isrelative paucity of information regarding the role of LncRNAs in intervertebral discdegeneration hitherto. In the present study we aimed to determine whether LncRNAs aredifferentially expressed in IDD and to predict and verify the roles of aberrantly expressed LncRNAs functioned in IDD.The current study is comprised of three parts, the main findings are showed in thefollowing.The first part is the study of the expression profile of the LncRNA in human nucleuspulposus (NP). In this part a LncRNA-mRNA microarray was applied for ten human NPsamples, five of them were normal samples, the others were derived from the degenerativeintervertebral discs (IVD). According to the filtering criterion that genes have flags inpresent or marginal in at least5out of10samples were classified as meaningfullydetected genes, a total of18995LncRNAs and14635mRNAs were detected in the10samples. Quality assessment of the LncRNAand mRNAdata after filtering using box-plot,scatter-plot and hierarchical clustering suggested that the gene expression patterns ofnormal samples and degenerative samples were different, expression level of parts ofgenes in degenerative samples was different from that of the normal samples.Subsequently, we filtered the expression profile of the LncRNAs and mRNAs and identifydifferentially expressed LncRNAs and mRNAs with fold change of altered expressionlevels more than2and a p value of student’s t test less than0.05. According to the filteringcriterion, a total of2309LncRNAs and3017mRNAs were significantly differentiallyexpressed in degeneration group compared with the control group. Among them,1660LncRNAs were up-regulated with649LncRNAs down-regulated and2208mRNAs wereup-regulated with809mRNAs down-regulated.164LncRNAs (97up-regulated and67down-regulated) and265mRNAs (140up-regulated and125down-regulated) were highlydifferentially expressed with an absolute fold change greater than ten.271LncRNAs wereup-regulated shared by4of the5degenerative samples with the normalized intensitiesaltered more than2folds compared with all the normal specimens.631LncRNAs wereup-regulated in all degenerative specimens with a fold change greater than2comparedwith all the control samples. For the differentially down-regulated LncRNAs,51and147were down-regulated in4of the5degenerative samples and all degenerative samplesrespectively. We also detected that364and672mRNAs were over-expressed in4of the5and all degenerative samples respectively with a fold change more than2compared withall the normal samples, this number is92(4/5) and251(5/5) for the down-regulatedmRNAs. LncRNAs belonging to particular subgroups were identified in the current study, wedetected160HOX coding transcripts in all samples,22of which were differentiallyexpressed in degeneration group compared with the control group.292noncodingtranscripts were detected with37of them were differentially expressed in degenerationgroup. By filtering the array data, we detected951enhancer-like LncRNAs in allspecimens. Amongst them,112LncRNAs were differentially expressed in degenerationgroup compared with the control group,53of them have differentially expressed nearbycoding genes. In53pairs of differentially expressed LncRNAs and nearby coding genes,32pairs were altered in the same direction (up or down).Profiling data based on Rinn’slincRNAs indicated that267lincRNAs within2043lincRNAs detected were differentlyexpressed in degenerative group. Amongst them,133lincRNAs were expressed with adifferentially expressed nearby coding genes.To validate the microarray results, four LncRNAs were selected for the quantitative realtime PCR (qRT-PCR) analysis in fourteen human lumbar disc NP samples (8degenerativeand6normal samples). qRT-PCR results matched well with the microarray data,demonstrating a high credibility for the LncRNAs microarray analysis.The second part is the study of the bioinformatics prediction for the aberrantly expressedLncRNAs and mRNAs in degenerative samples. In this part series of bioinformaticsprediction methods, such as KEGG (the database of Kyoto Encyclopedia of Genes andGenomes) pathway, GO (the Gene Ontology project) analysis, DAVID (the databasefor annotation, visualization and integrated discovery) functional annotation clusteringwere applied for the functional annotation of the differentially expressed LncRNAs andmRNAs. KEGG biological pathways analysis for the differentially expressed mRNAsidentified several interesting pathways such as focal adhesion, ECM-receptor interaction,adherents junction, ubiquitin mediated proteolysis, other glycan degradation, antigenprocessing and presentation and TGF-beta signaling pathway. The DAVID functionalannotation clustering for the differentially expressed mRNAs showed terms of severalpathologic phenomena including extracellular matrix, cartilage development,vasculogenesis, apoptosis and negative regulation of gene expression，which weregenerally observed in the degenerative process of IVD. The DAVID functional annotationclustering was also applied to investigate the functional roles of the neighboring codinggenes of differentially expressed Rinn’s lincRNAs and enhancer-like LncRNAs. The annotation results indicated that these LncRNAs and coding genes were closelyrelated to the biological activities such as protein kinase and phosphorylation. Acoding-noncoding coexpression (CNC) network was constructed base on the Pearsoncorrelation analysis data of ten most differentially expressed LncRNAs and hundreds ofdifferentially expressed mRNAs. KEGG biological pathways analysis indicated that theaforementioned LncRNAs and mRNAs were mainly related to the pathway of ribosomeand antigen processing and presentation. Interestingly, the GO terms such as translationalelongation, negative regulation of cellular differentiation, cartilage development, negativeregulation of signal transduction, chondrocyte differentiation, collagen fibril organization,posttranscriptional regulation of gene expression, regulation of apoptosis and proteoglycanmetabolic process were found in the GO analysis results of the aforementioned LncRNAsand mRNAs.The final part is the studies of the expression of the certain pairs of LncRNA and codinggene in IDD. Two pairs of subtype LncRNAand its coding gene which were differentiallyexpressed in degenerated samples were selected for further investigation.In the first experiment, by means of qRT-PCR analysis for14NP samples we found thatthe expression of RP11-296A18.3, FAF1and CASP8were significantly increased indegenerative discs compared with the normal discs. Such results showed a highconsistency with the array results. Over-expression of FAF1can significantly potentiatesFas-mediated apoptosis and increase death of cells. Enhancer-like LncRNA, such asRP11-296A18.3, has been showed to amplify transcription of its nearby coding gene. Theup-regulated RP11-296A18.3highly likely caused the over-expression of its nearby geneFAF1and seemed to be involved in the aberrant apoptosis of NP cells in degeneration.In the second experiment, we aimed to investigate whether a close correlation existedbetween expression of lincRNA-SLC20A1-1and its nearby coding gene IL-37, and therole of the anti-inflammatory cytokine IL-37in IDD. The results showed thatlincRNA-SLC20A1-1had an upregulated expression in degenerative samples compared tothat in normal samples. Conversely, the expression of IL-37had a decreased expression indegenerative discs both at mRNAand protein level. Significantly elevated gene expressionof IL-1β, IL-16, TNF-α, TGF-β1and Smad3were detected in degenerative samples.Significantly negative correlation was observed between expression oflincRNA-SLC20A1-1and IL-37. In addition, negative correlations were also observed between IL-37, IL-1β, IL-16, TGF-β1, Smad3and degeneration degree of IVD.Downregulation of IL-37expression appeared to result in overexpression ofpro-inflammatory cytokines, such as IL-1β and IL-16, in degenerative IVD and may be acontributor involved in the progression of IDD.To our knowledge, this is the first study addressing LncRNAs expression in IDD. Ourfindings shed a novel light to the understanding of pathogenesis of disc degeneration.Further studies should be focused on the in-depth investigation of the function andpathogenic mechanisms of the certain LncRNAs involved in the IDD. Advance in theknowledge of the IDD related LncRNAs may be beneficial for the treatment andprevention of the diseases caused by IDD in the future.