Dataset Information

Single-Cell RNA-Seq Analysis of Cells from Degenerating and Non-Degenerating Intervertebral Discs from the Same Individual Reveals New Biomarkers for Intervertebral Disc Degeneration.

ABSTRACT: In this study, we used single-cell transcriptomic analysis to identify new specific biomarkers for nucleus pulposus (NP) and inner annulus fibrosis (iAF) cells, and to define cell populations within non-degenerating (nD) and degenerating (D) human intervertebral discs (IVD) of the same individual. Cluster analysis based on differential gene expression delineated 14 cell clusters. Gene expression profiles at single-cell resolution revealed the potential functional differences linked to degeneration, and among NP and iAF subpopulations. GO and KEGG analyses discovered molecular functions, biological processes, and transcription factors linked to cell type and degeneration state. We propose two lists of biomarkers, one as specific cell type, including C2orf40, MGP, MSMP, CD44, EIF1, LGALS1, RGCC, EPYC, HILPDA, ACAN, MT1F, CHI3L1, ID1, ID3 and TMED2. The second list proposes predictive IVD degeneration genes, including MT1G, SPP1, HMGA1, FN1, FBXO2, SPARC, VIM, CTGF, MGST1, TAF1D, CAPS, SPTSSB, S100A1, CHI3L2, PLA2G2A, TNRSF11B, FGFBP2, MGP, SLPI, DCN, MT-ND2, MTCYB, ADIRF, FRZB, CLEC3A, UPP1, S100A2, PRG4, COL2A1, SOD2 and MT2A. Protein and mRNA expression of MGST1, vimentin, SOD2 and SYF2 (p29) genes validated our scRNA-seq findings. Our data provide new insights into disc cells phenotypes and biomarkers of IVD degeneration that could improve diagnostic and therapeutic options.

SUBMITTER: Cherif H

PROVIDER: S-EPMC8999935 | biostudies-literature | 2022 Apr

REPOSITORIES: biostudies-literature

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Single-Cell RNA-Seq Analysis of Cells from Degenerating and Non-Degenerating Intervertebral Discs from the Same Individual Reveals New Biomarkers for Intervertebral Disc Degeneration.

Cherif Hosni H Mannarino Matthew M Pacis Alain Sarabia AS Ragoussis Jiannis J Rabau Oded O Ouellet Jean A JA Haglund Lisbet L

International journal of molecular sciences 20220403 7

In this study, we used single-cell transcriptomic analysis to identify new specific biomarkers for nucleus pulposus (NP) and inner annulus fibrosis (iAF) cells, and to define cell populations within non-degenerating (nD) and degenerating (D) human intervertebral discs (IVD) of the same individual. Cluster analysis based on differential gene expression delineated 14 cell clusters. Gene expression profiles at single-cell resolution revealed the potential functional differences linked to degenerati ...[more]

PMID: 35409356

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Project description:BackgroundNucleus pulposus (NP) deterioration plays a significant role in the development of intervertebral disc degeneration (IVDD) and low back pain (LBP). This paper aims to identify potential genes within degenerated NP tissue and elucidate the pathogenesis of IVDD through bioinformatics analysis.MethodsWe conducted a transcriptomic analysis of patient's degenerative NP tissue employing advanced bioinformatics techniques and machine learning algorithms. Utilizing hdWGCNA, we successfully acquired WGCNA single-cell sequencing data and pinpointed crucial genes implicated in IVDD. Subsequently, we employed the Monocle3 package to perform pseudotime sequence analysis, enabling the identification of genes associated with the differentiation and developmental processes of NP tissue. Following this, normalized and logarithmically transformed the bulk sequencing data. Subsequently, we conducted preliminary screening using single-factor logistic regression on the genes derived from single-cell sequencing. Next, we applied two machine learning techniques, namely, SVM-RFE and random forest, to discern pivotal pathogenic genes. Finally, we used validation sets to verify trends and qualitativeness and performed in vitro and in vivo validation analyses of normal and degenerative NP tissues.Results909 genes associated with IVDD were identified through hdWGCNA, while pseudotime sequence analysis uncovered 1964 genes related to differentiation and developmental processes. The two had 208 genes in common. Subsequently, we conducted an initial screening of single-cell genes by integrating the bulk database with single logistic regression. Next, we utilized machine learning techniques to identify the IVDD genes CDH, DPH5, and SELENOF. PCR analysis confirmed that the expression of CDH and DPH5 in degraded nucleus pulposus cells (NPCs) was decreased by 31% and 28% in vivo, and 36% and 29% in vitro, respectively, while SELENOF showed the opposite trend. Furthermore, IVDD was validated through imaging and histological staining.ConclusionAs pathogenic genes in IVDD, our findings indicate that CTH, DPH5, and SELENOF are important players and might be promising therapeutic targets for IVDD treatment.

Project description:BackgroundIntervertebral disc degeneration (IDD) is a leading cause of disability with limited treatment strategies. A better understanding of the mechanism of IDD might enable less invasive and more targeted treatments. This study aimed to identify the circular RNA (circRNA)-microRNA (miRNA)-messenger RNA (mRNA) competing endogenous RNA (ceRNA) regulatory mechanisms in IDD. METHODS : The GSE67567 microarray dataset was downloaded from the Gene Expression Omnibus database. After data preprocessing, differentially expressed circRNAs, miRNAs and mRNAs between IDD and controls were identified. A ceRNA network was constructed on the basis of the interaction between circRNAs and miRNAs, and miRNAs and mRNAs. Pathway enrichment analysis was performed on the mRNAs in the ceRNA network. Then, with 'intervertebral disc degeneration' as keywords, IDD-related Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were searched for in the Comparative Toxicogenomics Database.ResultsA total of 105 differentially expressed circRNAs, 84 miRNAs and 967 mRNAs were identified. After analysis, 86 circRNA-miRNA, and 126 miRNA-mRNA regulatory relationship pairs were obtained to construct a ceRNA network. The mRNAs were enriched in six KEGG signalling pathways, and four were associated with IDD: the hsa04350: TGF-beta signalling pathway, hsa04068: FoxO signalling pathway, hsa05142: Chagas disease (American trypanosomiasis) and hsa04380: Osteoclast differentiation. An IDD-related ceRNA network was constructed involving four circRNAs, three miRNAs and 11 mRNAs. Auxiliary validation showed that the expression levels of miR-185-5p, miR-486-5p, ACVR1B, FOXO1, SMAD2 and TGFB1 were consistent in different databases.ConclusionsOur study identified some circRNA-miRNA-mRNA interaction axes potentially associated with the progression of IDD, viz.: circRNA_100086-miR-509-3p-MAPK1, circRNA_000200-miR-185-5p-TGFB1, circRNA_104308-miR-185-5p-TGFB1, circRNA_400090-miR-486-5p-FOXO1 and circRNA_400090-miR-486-5p-SMAD2.

Project description:BackgroundDuring the intervertebral disc (IVD) degeneration process, initial degenerative events occur at the extracellular matrix level, with the appearance of neoepitope peptides formed by the cleavage of aggrecan and collagen. This study aims to elucidate the spatial and temporal alterations of aggrecan and collagen neoepitope level during IVD degeneration.MethodsBovine caudal IVDs were cultured under four different conditions to mimic different degenerative situations. Samples cultured after 1- or 8-days were collected for analysis. Human IVD samples were obtained from patients diagnosed with lumbar disc herniation (LDH) or adolescent idiopathic scoliosis (AIS). After immunohistochemical (IHC) staining of Aggrecanase Cleaved C-terminus Aggrecan Neoepitope (NB100), MMP Cleaved C-terminus Aggrecan Neoepitope (MMPCC), Collagen Type 1α1 1/4 fragment (C1α1) and Collagenase Cleaved Type I and II Collagen Neoepitope (C1,2C), staining optical density (OD)/area in extracellular matrix (OECM) and pericellular zone (OPCZ) were analyzed. Conditioned media of the bovine IVD was collected to measure protein level of inflammatory cytokines and C1,2C.ResultsFor the bovine IVD sections, the aggrecan MMPCC neoepitope was accumulated in nucleus pulposus (NP) and cartilage endplate (EP) regions following mechanical overload in the one strike model after long-term culture; as for the TNF-α induced degeneration, the OECM and OPCZ of collagen C1,2C neoepitope was significantly increased in the outer AF region after long-term culture; moreover, the C1,2C was only detected in conditioned medium from TNF-α injection + Degenerative loading group after 8 days of culture. LDH patients showed higher MMPCC OECM in NP and higher C1,2C OECM in AF region compared with AIS patients.ConclusionsIn summary, aggrecan and collagen neoepitope profiles showed degeneration induction trigger- and region-specific differences in the IVD organ culture models. Different IVD degeneration types are correlated with specific neoepitope expression profiles. These neoepitopes may be helpful as biomarkers of ECM degradation in early IVD degeneration and indicators of different degeneration phenotypes.

Dataset Information

Single-Cell RNA-Seq Analysis of Cells from Degenerating and Non-Degenerating Intervertebral Discs from the Same Individual Reveals New Biomarkers for Intervertebral Disc Degeneration.

Publications

Single-Cell RNA-Seq Analysis of Cells from Degenerating and Non-Degenerating Intervertebral Discs from the Same Individual Reveals New Biomarkers for Intervertebral Disc Degeneration.

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