Gene expression profile identifies potential biomarkers for human intervertebral disc degeneration

A discrimination of the highly specialised annulus fibrosus (AF) and nucleus pulposus (NP) cells in the mature human intervertebral disc (IVD) is thus far still not possible in a reliable way. The aim of this study was to identify molecular markers that distinguish AF and NP cells in human disc tissue using microarray analysis as a screening tool. AF and NP samples were obtained from 28 cervical discs. First, all samples underwent quality sorting using two novel scoring systems for small-sized disc tissue samples including macroscopic, haptic and histological evaluation. Subsequently, samples with clear disc characteristics of either AF or NP that were free from impurities of foreign tissue (IVD score) and with low signs of disc degeneration on cellular level (DD score) were selected for GeneChip analysis (HGU1332P). The 11 AF and 9 NP samples showed distinctly different genome-wide transcriptomes. The majority of differentially expressed genes (DEGs) could be specifically assigned to the AF, whereas no DEG was exclusively expressed in the NP. Nevertheless, we identified 11 novel marker genes that clearly distinguished AF and NP, as confirmed by quantitative gene expression analysis. The novel established scoring systems and molecular markers showed the identity of AF and NP in disc starting material and are thus of great importance in the quality assurance of cell-based therapeutics in regenerative treatment of disc degeneration.

Intervertebral disc generation tissue engineering treatment platform: gelatin-methacryloyl hydrogel composite system.

In chronic thromboembolic pulmonary hypertension (CTEPH), fibrosis of thrombi in the lumen of blood vessels and obstruction of blood vessels are important factors in the progression of the disease. Therefore, it is important to explore the key genes that lead to chronic thrombosis in order to understand the development of CTEPH, and at the same time, it is beneficial to provide new directions for early identification, disease prevention, clinical diagnosis and treatment, and development of novel therapeutic agents. The GSE130391 dataset was downloaded from the Gene Expression Omnibus (GEO) public database, which includes the full gene expression profiles of patients with CTEPH and Idiopathic Pulmonary Arterial Hypertension (IPAH). Differentially Expressed Genes (DEGs) of CTEPH and IPAH were screened, and then Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) functional enrichment analyses were performed on the DEGs; Weighted Gene Co-Expression Network Analysis (WGCNA) to screen the key gene modules and take the intersection genes of DEGs and the key module genes in WGCNA; STRING database was used to construct the protein-protein interaction (PPI) network; and cytoHubba analysis was performed to identify the hub genes. A total of 924 DEGs were screened, and the MEturquoise module with the strongest correlation was selected to take the intersection with DEGs A total of 757 intersecting genes were screened. The top ten hub genes were analyzed by cytoHubba: IL-1B, CXCL8, CCL22, CCL5, CCL20, TNF, IL-12B, JUN, EP300, and CCL4. IL-1B, CXCL8, CCL22, CCL5, CCL20, TNF, IL-12B, JUN, EP300, and CCL4 have diagnostic and therapeutic value in CTEPH disease, especially playing a role in chronic thrombosis. The discovery of NF-κB, AP-1 transcription factors, and TNF signaling pathway through pivotal genes may be involved in the disease progression process.

BackgroundLow back pain (LBP) has drawn much widespread attention and is a major global health concern. In this field, intervertebral disc degeneration (IVDD) is frequently the focus of classic studies. However, the mechanistic foundation of IVDD is unclear and has led to conflicting outcomes.MethodsGene expression profiles (GSE34095, GSE147383) of IVDD patients alongside control groups were analyzed to identify differentially expressed genes (DEGs) in the GEO database. GSE23130 and GSE70362 were applied to validate the yielded key genes from DEGs by means of a best subset selection regression. Four machine-learning models were established to assess their predictive ability. Single-sample gene set enrichment analysis (ssGSEA) was used to profile the correlation between overall immune infiltration levels with Thompson grades and key genes. The upstream targeting miRNAs of key genes (GSE63492) were also analyzed. A single-cell transcriptome sequencing data (GSE160756) was used to define several cell clusters of nucleus pulposus (NP), annulus fibrosus (AF), and cartilaginous endplate (CEP) of human intervertebral discs and the distribution of key genes in different cell clusters was yielded.ResultsBy developing appropriate p-values and logFC values, a total of 6 DEGs was obtained. 3 key genes (LRPPRC, GREM1, and SLC39A4) were validated by an externally validated predictive modeling method. The ssGSEA results indicated that key genes were correlated with the infiltration abundance of multiple immune cells, such as dendritic cells and macrophages. Accordingly, these 4 key miRNAs (miR-103a-3p, miR-484, miR-665, miR-107) were identified as upstream regulators targeting key genes using the miRNet database and external GEO datasets. Finally, the spatial distribution of key genes in AF, CEP, and NP was plotted. Pseudo-time series and GSEA analysis indicated that the expression level of GREM1 and the differentiation trajectory of NP chondrocytes are generally consistent. GREM1 may mainly exacerbate the degeneration of NP cells in IVDD.ConclusionsOur study gives a novel perspective for identifying reliable and effective gene therapy targets in IVDD.

BackgroundFerroptosis was involved in the pathogenesis of intervertebral disc degeneration (IVDD). However, the exact mechanism of IVDD associated with ferroptosis still required deeper studies.MethodThe differentially expressed genes (DEGs) in rat lumbar disc tissue between the control and IVDD group treated with IL-1β were detected by RNA sequencing (RNA-seq). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were performed on DEGs. We further screened the differential expressed ferroptosis-related genes (DEFRGs). Besides, a protein-protein interaction (PPI) network of DEFRGs was constructed by STRING database. The Cytoscape database identified significant modules and the hub genes. The loss function of PDK4 by siRNA inference was investigated in NPCs by CCK8 assay, ELISA assay, and the analysis of ferroptosis indicators.ResultDEGs were identified using RNA-seq. KEGG pathway analysis showed that these genes were mainly involved in Parkinson’s disease, oxytocin signaling pathway, calcium ion signaling pathway, AMPK signaling pathway, and glucagon signaling pathway. Eight hub genes (including LDHA, PKM, EP300, EGFR, EGLN1, SCD, PDK4, and FABP4) were found by the PPI network and Cytoscape on a total of 25 ferroptosis-related genes that were identified in rat lumbar disc tissue after IVDD treatment. PDK4 silencing promoted NPCS proliferation, decreased the levels of the proinflammatory factors, and suppressed ferroptosis.ConclusionThe study suggested the potential roles of ferroptosis-related genes in IVDD and further revealed the role of PDK4 in the progression of IVDD.

The risk of PCOS is significantly increased in obese women, and studies have shown that weight loss can improve symptoms of PCOS. Coffee has been shown to be effective in reducing body weight. In this study, we focused on the SLC16A6 gene using bioinformatics and searched for coffee and its monomers using reverse network pharmacology. The gene expression omnibus (GEO) database was searched to screen for differentially expressed genes (DEGs) in PCOS patients. Gene ontology (GO) functional enrichment analysis and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis were then performed. The effects of caffeine on body weight, estrous cycle, ovarian pathology, serum insulin concentration and insulin resistance index, and SLC16A6 transporter gene expression in ovarian tissue of obese PCOS rats were monitored. The common differentially expressed gene SLC16A6 was identified in this study, and animal experiments confirmed the efficacy of caffeine in the treatment of obese PCOS rats. Caffeine can effectively improve the symptoms of obese PCOS rats. The mechanism by which caffeine can treat obese patients with PCOS is related to increasing the expression of the SLC16A6 gene.

Previous studies have shown that asthma is a risk factor for lung cancer, while the mechanisms involved remain unclear. We attempted to further explore the association between asthma and non-small cell lung cancer (NSCLC) via bioinformatics analysis. We obtained GSE143303 and GSE18842 from the GEO database. Lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) groups were downloaded from the TCGA database. Based on the results of differentially expressed genes (DEGs) between asthma and NSCLC, we determined common DEGs by constructing a Venn diagram. Enrichment analysis was used to explore the common pathways of asthma and NSCLC. A protein-protein interaction (PPI) network was constructed to screen hub genes. KM survival analysis was performed to screen prognostic genes in the LUAD and LUSC groups. A Cox model was constructed based on hub genes and validated internally and externally. Tumor Immune Estimation Resource (TIMER) was used to evaluate the association of prognostic gene models with the tumor microenvironment (TME) and immune cell infiltration. Nomogram model was constructed by combining prognostic genes and clinical features. 114 common DEGs were obtained based on asthma and NSCLC data, and enrichment analysis showed that significant enrichment pathways mainly focused on inflammatory pathways. Screening of 5 hub genes as a key prognostic gene model for asthma progression to LUAD, and internal and external validation led to consistent conclusions. In addition, the risk score of the 5 hub genes could be used as a tool to assess the TME and immune cell infiltration. The nomogram model constructed by combining the 5 hub genes with clinical features was accurate for LUAD. Five-hub genes enrich our understanding of the potential mechanisms by which asthma contributes to the increased risk of lung cancer.

Pulp capping material should facilitate hard tissue regeneration on the injured pulp tissue. TheraCal LC (TC) was recently developed. Although TC has shown reliable clinical outcomes after direct pulp capping, there are still remaining concerns regarding its detrimental effect on pulp cells. Therefore, this study aimed to identify the gene expression of human deciduous tooth-derived dental pulp cells exposed to TC compared to mineral trioxide aggregate (MTA). The cells were cultured and exposed to TC and MTA for 24 and 72 h. Next, total RNA was isolated. QuantSeq 3′ mRNA-sequencing was used to examine differentially expressed genes (DEGs) in exposed to TC and MTA. Functional analysis of DEGs was performed using bioinformatics analysis. In gene ontology (GO) functional enrichment analysis, cells in TC for 24 h presented significantly enriched immune response (p < 0.001) and inflammatory response (p < 0.01) compared to MTA. TC showed enriched positive regulation of cell migration at 72 h (p < 0.001). In Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, neuroactive ligand–receptor interaction (p = 1.19 × 10−7) and calcium signaling pathway (p = 2.96 × 10−5) were confirmed in the shared DEGs in TC. In conclusion, DEGs in TC may be involved in pathways associated with osteoclastogenesis and osteoclastic differentiation.

Polymerase I and transcript release factor (PTRF) plays a role in the regulation of gene expression and the release of RNA transcripts during transcription, which have been associated with various human diseases. However, the role of PTRF in glioma remains unclear. In this study, RNA sequencing (RNA-seq) data (n = 1022 cases) and whole-exome sequencing (WES) data (n = 286 cases) were used to characterize the PTRF expression features. Gene ontology (GO) functional enrichment analysis was used to assess the biological implication of changes in PTRF expression. As a result, the expression of PTRF was associated with malignant progression in gliomas. Meanwhile, somatic mutational profiles and copy number variations (CNV) revealed the glioma subtypes classified by PTRF expression showed distinct genomic alteration. Furthermore, GO functional enrichment analysis suggested that PTRF expression was associated with cell migration and angiogenesis, particularly during an immune response. Survival analysis confirmed that a high expression of PTRF is associated with a poor prognosis. In summary, PTRF may be a valuable factor for the diagnosis and treatment target of glioma.

Intervertebral disc (IVD) degeneration is the major cause of back pain. Notch signaling is activated in annulus fibrosus (AF) and nucleus pulposus (NP) tissues of degenerated IVDs, and induced by IL1-β and TNF-α in NP cells. However, the role of Notch activatin in the pathogenesis of IVD degeneration is largely unknown. In this study, we overexpressed the Notch1 intracellular domain (NICD1) in AF, NP, and chondrogenic ATDC5 cells via adenoviruses. Overexpression of NICD1 activated transcription of Notch signaling target genes in AF, NP, and ATDC5 cells, and caused cell type-specific effects on expression of matrix anabolic and catabolic genes. Activation of Notch signaling promoted expression of matrix catabolic genes and inhibited expression of matrix anabolic genes in both AF and ATDC5 cells, whereas its activation suppressed expression of matrix catabolic genes (including Mmp3, Mmp13, Adamts4, and Adamts5) and attenuated TNF-α and inflammatory macrophage-induced Mmp13 expression in NP cells. Consistently, sustained activation of Notch1 signaling in postnatal IVDs in mice severely disrupted growth plate and endplate cartilage tissues, but did not overly affect NP tissues. Together, these data indicated that activation of Notch signaling exerted differential and cell type-specific effects in intervertebral discs, and specific Notch signaling regulation may be considered during the treatment of IVD degeneration.

mRNA analysis. The aim of this study was to identify differentially expressed genes (DEGs) in disc degeneration, analyze the potential biological functions of DEGs, and screen for a new target to prevent the degeneration. Intervertebral disc degeneration (IDD) is an irreversible process and causes long-term heavy socioeconomic burdens. Existing and therapies under development are unable to prevent disc degeneration in a safe and effective manner. Therefore, elucidating the potential mechanism underlying degeneration and the development of new targets for IDD therapy are urgently required. Nucleus pulposus (NP) cells from mild and severe IDD (Ctrl and IDD groups) were separated, and DEGs of the two groups were identified with mRNA microarray analysis, followed by bioinformatics analysis.Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was performed to verify the microarray results. Gene over-expression and silencing technologies were used to study the role of plant homeodomain finger protein 6 (PHF6). qRT-PCR and western blot analyses were used to detect the expressions of collagen II (COL2), matrix metalloproteinases 13 (MMP13), and ADAM metallopeptidase with thrombospondin type 1 motif 4 (ADAMTS4). The study identified 377 up- and 116 downregulated DEGs in NP cells from two groups. These DEGs were mainly involved in cellular and metabolic processes and enriched in immune system and nucleotide metabolism pathways. Upregulated PHF6, with the highest verified fold change, was significantly increased in the IDD group. Over-expressing PHF6 in Ctrl NP cells significantly inhibited the expression of COL2 and enhanced the expressions of MMP13 and ADAMTS4, whereas silencing PHF6 in IDD NP cells reversed such expression alterations. Upregulated PHF6 caused IDD by promoting extracellular matrix degradation; therefore, PHF6 could be developed as a potential novel target to prevent the degeneration. Our DEG profiling of NP cells from IDD patients provided a database to identify the key genes involved in IDD. N/A.

Aims This study is aimed at investigating the pathogenesis of rheumatoid arthritis (RA) by identifying key biomarkers, associated immune infiltration, and small-molecule compounds using bioinformatic analysis. Methods Six datasets were obtained from the Gene Expression Omnibus database, and the batch effect was adjusted. Functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) were used to analyse differentially expressed genes (DEGs). Furthermore, candidate small-molecule drugs associated with RA were selected from the Connectivity Map (CMap) database. The least absolute shrinkage and selection operator regression, support vector machine recursive feature elimination, and multivariate logistic regression analyses were performed on DEGs to screen for RA diagnostic markers. The receiver operating characteristic curve, concordance index, and GiViTi calibration band were the metrics used to assess the diagnostic markers of RA identified in this analysis. The single-sample gene set enrichment analysis was performed to calculate the scores of infiltrating immune cells and evaluate the activities of immune-related pathways. Finally, the correlation between screening markers and RA diagnosis was determined. Results A total of 227 DEGs were identified. Functional enrichment analysis and KEGG revealed that DEGs were enriched by the immune response. CMap analysis identified 11 small-molecule compounds with therapeutic potential for RA. In gene expression, the activities of 13 immune cells and 12 immune-related pathways significantly differed between patients with RA and healthy controls. DPYSL3 and SPP1 had the potential to diagnose RA. SPP1 expression was positively correlated with DPYSL3 in 11 immune cells and 10 immune-related pathways. Conclusion This study comprehensively analysed DEGs and immune infiltration and screened for potential diagnostic markers and small-molecule compounds of RA.

We intend to explore potential mechanisms of Tripterygium wilfordii Hook.f (TwHF) induced kidney injury (KI) using the methods of network toxicology and molecular docking. We determined TwHF potential compounds with its targets and KI targets, obtained the TwHF induced KI targets after intersecting targets of TwHF and KI. Then we conducted protein-protein interaction (PPI) network, gene expression analysis, gene ontology (GO) function and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis to explore the mechanism of TwHF-induced KI. Finally we conducted molecular docking to verify the core toxic compounds and the targets. We obtained 12 TwHF toxic compounds and 62 TwHF-induced KI targets. PPI network, gene expression analysis and GO function enrichment analysis unveiled the key biological process and suggested the mechanism of TwHF-induced KI might be associated with inflammation, immune response, hypoxia as well as oxidative stress. KEGG pathway enrichment analysis indicated PI3K-Akt signaling pathway, HIF-1 signaling pathway and TNF signaling pathway were key signaling pathways of TwHF induced KI. Molecular docking showed that the binding energy of core targets and toxic compounds was all less than -6.5 kcal/mol that verified the screening ability of network pharmacology and provided evidence for modifying TwHF toxic compounds structure. Through the study, we unveiled the mechanism of TwHF induce KI that TwHF might activate PI3K-Akt signaling pathway as well as TNF signaling pathway to progress renal inflammation, mediate hypoxia via HIF-1 signaling pathway to accelerate inflammatory processes, and also provided a theoretical basis for modifying TwHF toxic compounds structure as well as supported the follow-up research.

Intervertebral disc degeneration (IDD) is widely known as the principal cause of low back pain, diminishing patients' quality of life and imposing a huge economic burden on healthcare systems worldwide. However, the underlying mechanisms of IDD remain to be determined. This study aimed to scrutinize data sets via bioinformatics to identify microRNAs (miRNAs)/genes and pathways associated with IDD. The array profiling of patients with IDD and individuals without IDD was acquired from the Gene Expression Omnibus (GEO) database (viz., GSE19943, GSE63492, and GSE34095). The expression profiles of miRNAs and genes with differential patterns were analyzed using GEO2R. The target genes of the chosen miRNA were then examined, and in silico functional analyses were performed on the signaling pathways and biological processes of the differentially expressed genes. Three human miRNAs were up and downregulated in IDD patients in the examined data sets. Among them, hsa-miR-508-5p had a significant differential expression in the IDD group, and SEC11A, IPO5, FN1, and MRPS10, as the targets of hsa-miR-508-5p, were upregulated in the IDD group. Furthermore, extracellular matrix-receptor interactions, focal adhesion, and actin cytoskeleton regulation were important pathways involved in IDD. Our analysis identified hsa-miR-508-5p as a novel miRNA involved in IDD pathogenies. Our findings not only further confirmed the significant role of miRNAs in IDD pathogenesis but also extended the spectrum of the miRNAs and genes involved in IDD. Though, still, further experimental investigations are needed to confirm our findings.

Animal experimental study. To establish an early and reproducible intervertebral disc (IVD) degeneration model to provide a basis for studying IVD degeneration. The pathophysiology and pathogenesis of IVD degeneration, a common condition that causes low back pain, are not clearly understood. An experimental animal model of human IVD degeneration is needed. A total of 91 IVDs were grouped as follows: group 1, percutaneous puncture using a 20-gauge needle; group 2, percutaneous puncture using a 15-gauge needle; and group 3, nonpuncture. The pre- and postpunctured IVDs were examined and graded. Hematoxylin and eosin, Masson trichrome, safranin O staining methods, and immunohistochemistry for type II collagen were performed. In group 1, the magnetic resonance imaging (MRI) signal intensity was grade I for all IVDs before puncture, and at the different time points after puncture. In group 2, the MRI signal intensity was grade III in the 4th week and grade IV in the 8th week after puncture. In group 3, the MRI signal intensity was grade I at all time points. In groups 1 and 2, a marked reduction in nucleus pulposus cells was observed in the 12th week. Fissures in the annulus fibrosus were observed in the 8th week. Decreased proteoglycans in the model discs was noted in the 8th and 12th week, and collagen II significantly decreased in the 12th week after puncture (P < 0.05). In group 2, the number of cells and fissures in the IVDs were decreased, with decreased proteoglycan and collagen II content in the 8th week. No changes were found in group 3. Slowly progressive and mild disc degeneration can be induced by puncture using 20-gauge needles through the IVDs, which might be suitable for investigating novel therapies for disc degeneration/pain. 2.