Proliferation Of Nucleus Pulposus Cells Research Articles

Downregulation of miR-760 Causes Human Intervertebral Disc Degeneration by Targeting the MyD88/Nuclear Factor-Kappa B Signaling Pathway.

Dysregulation of microRNAs (miRNAs) plays a critical role in the development of intervertebral disc degeneration (IDD). In this study, we present evidence from in vitro and in vivo research to elucidate the mechanism underlying the role of miR-760 in IDD. miRNA microarray and quantitative reverse transcription-polymerase chain reaction were used to determine the miRNA profiles in patients with IDD. Functional analysis was performed to evaluate the role of miR-760 in the pathogenesis of IDD. Luciferase reporter and western blotting assays were used to confirm the miRNA targets. The expression of miR-760 was significantly decreased in degenerative nucleus pulposus (NP) cells and negatively correlated with disc degeneration grade. Functional assays demonstrated that miR-760 delivery significantly increased NP cell proliferation and promoted the expression of collagen II and aggrecan. Moreover, MyD88 was identified as a target gene of miR-760. miR-760 effectively suppressed MyD88 expression by interacting with the 3′-untranslated region, which was abolished by miR-760 binding site mutations. An in vivo experiment using an IDD mouse model showed that the upregulation of miR-760 could effectively suspend IDD. Therefore, miR-760 was found to play an important role in IDD and can be used as a promising therapeutic target for the treatment of patients with IDD.

LncRNA LINC00284 promotes nucleus pulposus cell proliferation and ECM synthesis via regulation of the miR‑205‑3p/Wnt/β‑catenin axis.

Intervertebral disc degeneration (IDD) is a leading cause of degenerative spinal disease. Long non-coding RNA (lncRNA) LINC00284 is overexpressed in multiple types of cancer and promotes cancer cell proliferation and inhibits apoptosis; however, its role in human IDD and nucleus pulposus (NP) remain unclear. In the present study, intervertebral disc (IVD) tissues were collected from IDD patients for detection of LINC00284 expression using reverse transcription-quantitative PCR, the binding effect between miR-205-3p and LINC00284 was validated by dual-luciferase reporter assay. miR-205-3p and small interfering RNA (siRNA) was used for LINC00240 knockdown to investigate the proliferation, apoptosis of cells in the NP cells measured by Cell Counting Kit (CCK)-8 assay and Annexin V-FITC/Propidium Iodide (PI) staining with flow cytometry receptivity. IDD animal models were constructed for in vivo study of the role LINC00284 in IDD improvement. The results showed that LINC00284 expression was upregulated in IDD tissue and IL-1β-induced NP cells. LINC00284 knockdown resulted in an increase in IL-1β-induced NP cell proliferation, a decrease in apoptosis and matrix metalloproteinase-3 expression and an increase in expression of extracellular matrix (ECM) markers aggrecan and collagen II. In vivo experiments and histomorphometric analysis confirmed the protective effect of LINC00284 knockdown in IDD. LINC00284 was also shown to be a target of microRNA (miR)-205-3p, and there was a negative correlation between LINC00284 and miR-205-3p levels in IDD tissue. Additionally, LINC00284 knockdown or miR-205-3p upregulation resulted in inhibition of Wnt/β-catenin signaling and subsequent degradation of the ECM. The present study demonstrated that LINC00284 activated the Wnt/β-catenin signaling via sponging miR-205-3p, resulting in inhibition of NP cell proliferation and ECM synthesis. These results suggested that targeting LINC00284 to rescue miR-205-3p expression may be a potential method for IDD management.

Depleted Long Noncoding RNA GAS5 Relieves Intervertebral Disc Degeneration via microRNA-17-3p/Ang-2.

Intervertebral disc degeneration (IVDD) remains a clinical challenge and requires more effective therapeutic targets. Long noncoding RNAs (lncRNAs) have emerged as critical modulators of multiple biological processes, such as cell proliferation and extracellular matrix (ECM) remodeling. Accordingly, the current study sets out to explore the influence of the lncRNA growth arrest-specific 5 (GAS5) on IVDD and investigate the possible involvement of microRNA-17-3p (miR-17-3p)/Angiopoietin-2 (Ang-2) axis. Firstly, the expression patterns of GAS5, miR-17-3p, and Ang-2 were characterized by RNA quantification from the isolated human degenerative nucleus pulposus (NP) tissues. miR-17-3p was found to express at an abnormal low level while GAS5 and Ang-2 expressed at aberrant high level in the human degenerative NP tissues. Utilizing dual-luciferase reporter, RNA immunoprecipitation, and pull-down assays, GAS5 was found to competitively bound to miR-17-3p and further upregulate the expression of Ang-2, a target gene of miR-17-3p. Employing gain- and loss-of-function approaches, their expressions were altered in human degenerative nucleus pulposus cells (NPCs), followed by IL-1β treatment, in order to identify their roles in NP cell proliferation, apoptosis, and ECM metabolism. Silencing of GAS5 expression restrained the levels of cleaved caspase-3, cleaved caspase-7, cleaved caspase-9, MMP3, MMP13, ADAMTS4, and ADAMTS5 and increased collagen II and aggrecan levels. In vitro experiments also revealed that GAS5 depletion inhibited apoptosis and ECM degradation in HDNPCs, while elevating the proliferation through downregulation of Ang-2 by increasing miR-17-3p. Furthermore, in vivo data further validated that either GAS5 silencing or miR-17-3p reexpression alleviated IVDD degree with the help of IVDD mouse models. Altogether, our findings substantiated that downregulation of GAS5 reduced NPC apoptosis and promoted ECM remodeling, ultimately ameliorating the IVDD via miR-17-3p-dependent inhibition of Ang-2. We hope our discoveries offer a fresh molecular insight that can aid the development of novel therapies against IVDD.

Role of MiR-27a-3p in Intervertebral Disc Degeneration through Targeting RASSF5 via MST1/LATS1 and RAS/RAC1 Signaling Pathway.

Background The apoptosis of nucleus pulposus (NP) cells reduces the number of nucleus pulposus cells in intervertebral disc tissue, resulting in intervertebral disc degeneration (IDD). MicroRNAs (miRNAs) play an important regulatory role in abnormal cell proliferation and apoptosis. Methods The miR-27a-3p expressions in degenerative NP tissue and cells were measured via qPCR. The impacts of miR-27a-3p on the proliferation and apoptosis of human NP cells were evaluated by flow cytometry assays, MTT assays, and western blot analyses. In addition, target scan and luciferase reporter assay were applied to confirm that RASSF5 was directly binding to miR-27a-3p. Western blot was applied to assess the relationship between miR-27a-3p, RASSF5 and MST1/LATS1, and RAS/RAC1 signaling pathway. Results MiR-27a-3p was downregulated in degenerative NP tissues and cells by comparison with the control group. MiR-27a-3p overexpression enhanced cell proliferation and suppressed apoptosis of NP cells, while the above factors showed an opposite tendency after in the miR-27a-3p inhibitor group. The western blot experiment similarly suggested mir-27a-3p apparently downregulated apoptosis-related proteins (Bax and caspase-3) and upregulated antiapoptotic proteins (Bcl-2). In addition, RASSF5 was confirmed to be directly regulated by miR-27a-3p using the luciferase reporter assay. Overexpressed RASSF5 could reverse the effects caused by miR-27a-3p mimic. Finally, miR-27a-3p could downregulate RASSF5 and affected the MST1/LATS1 and RAS/RAC1 pathway. Conclusion MiR-27a-3p may target RASSF5 and enhance cell proliferation and imped cell apoptosis of the nucleus pulposus cells via the MST1/LATS1 and RAS/RAC1 pathway, lessening the degeneration of intervertebral discs.

Circular RNA hsa_circ_0083756 promotes intervertebral disc degeneration by sponging miR-558 and regulating TREM1 expression.

ObjectivesIntervertebral disc degeneration (IVDD) is a leading cause of low back pain. Circular RNAs (circRNAs) have been demonstrated to exert vital functions in IVDD. However, the role and mechanism of hsa_circ_0083756 in the development of IVDD remain unclear.Materials and methodsRT‐qPCR was performed to detect expressions of hsa_circ_0083756, miR‐558 and TREM1 in nucleus pulposus (NP) tissues and cells. CCK8 assay, flow cytometry, TUNEL assay, RT‐qPCR and WB were used to clarify the roles of hsa_circ_0083756 in NP cells proliferation and extracellular matrix (ECM) formation. Bioinformatics analyses, dual‐luciferase reporter gene experiment, RNA immunoprecipitation (RIP) assay and FISH assay were performed to predict and verify the targeting relationship between hsa_circ_0083756 and miR‐558, as well as that between miR‐558 and TREM1. Ultimately, the effect of hsa_circ_0083756 on IVDD was tested through anterior disc‐puncture IVDD animal model in rats.Resultshsa_circ_0083756 was upregulated in degenerative NP tissues and cells. In vitro loss‐of‐function and gain‐of‐function studies suggested that hsa_circ_0083756 knockdown promoted, whereas hsa_circ_0083756 overexpression inhibited NP cells proliferation and ECM formation. Mechanistically, hsa_circ_0083756 acted as a sponge of miR‐558 and subsequently promoted the expression of TREM1. Furthermore, in vivo study indicated that silencing of hsa_circ_0083756 could alleviate IVDD in rats.Conclusionshsa_circ_0083756 promoted IVDD via targeting the miR‐558/TREM1 axis, and hsa_circ_0083756 may serve as a potential therapeutic target for the treatment of IVDD.

SERPINA1 is a hub gene associated with intervertebral disc degeneration grade and affects the nucleus pulposus cell phenotype through the ADIRF-AS1/miR-214-3p axis

Long noncoding RNAs (lncRNAs) and miRNAs have been reported to participate in intervertebral disc degeneration (IDD) progression. However, the key lncRNA-miRNA axis and its corresponding affected hub genes in IDD remain unknown. In this study, weighted gene coexpression network analysis (WGCNA) was first used to determine the key gene cluster and hub genes implicated in IDD progression. The expression levels of ADIRF-AS1, miR-214-3p, and SERPINA1 in nucleus pulposus (NP) tissues were detected. The ADIRF-AS1/miR-214-3p/SERPINA1 axis was identified, and its effects on the proliferation, senescence, and apoptosis of NP cells were investigated in vitro and in vivo. SERPINA1 overexpression in NP cells promoted cell viability and inhibited cell apoptosis and senescence. Moreover, SERPINA1 regulated the IDD grade in rat models. The lncRNA ADIRF-AS1 was downregulated in high-grade degeneration NP tissues and positively correlated with SERPINA1. ADIRF-AS1 overexpression attenuated cellular degenerative changes in NP cells. miR-214-3p directly bound to SERPINA1 and ADIRF-AS1 and negatively regulated ADIRF-AS1 expression. miR-214-3p inhibition exerted similar effects on cellular degenerative changes in NP cells to SERPINA1 or ADIRF-AS1 overexpression. Furthermore, miR-214-3p overexpression partially reversed the effects of ADIRF-AS1 overexpression. Collectively, these data suggest that ADIRF-AS1 overexpression could mitigate IDD by binding to miR-214-3p to upregulate SERPINA1. Additional studies (especially those using an axial loading-induced IDD animal model) will be needed to further validate the role of the ADIRF-AS1/miR-214-3p/SERPINA1 signaling axis in IDD progression.

Long intergenic non-coding RNA -00917 regulates the proliferation, inflammation, and pyroptosis of nucleus pulposus cells via targeting miR-149-5p/NOD-like receptor protein 1 axis

ABSTRACT Intervertebral disc degeneration (IDD) has caused great trouble in people’s lives. Dysregulated long noncoding RNAs (lncRNAs) are closely linked to IDD progression. Our study aims to analyze the role of LINC00917 in the progression of IDD. Forty nucleus pulposus (NP) IDD tissues and 40 NP tissues of intervertebral discs without apparent degeneration were collected. TBHP was used to induce IDD. Cell proliferation was measured using the MTT and EdU assays. Pyroptosis was detected using flow cytometry. RT-qPCR and Western blot assays were performed to determine mRNA, miRNA, and protein expression. Dual-luciferase reporter and RNA pull-down assays were performed to verify the relationship between LINC00917 or NLRP1 and miR-149-5p. LINC00917 expression was enhanced in TBHP-treated nucleus pulposus cells (NPCs). The knockdown of LINC00917 promoted proliferation and inhibited cytotoxicity, inflammatory response, and pyroptosis of NPCs. LINC00917 functions as a sponge for miR-149-5p. Having silenced miR-149-5p, the effects of LINC00917 knockdown on NPC proliferation and inflammation-induced pyroptosis were alleviated. NLRP1 overexpression induced cellular dysfunction and pyroptosis of NPCs. LINC00917 knockdown restored NPC cellular functions and inhibited IDD progression by modulating the miR-149-5p/NLRP1 axis.

MiR-1260b protects against LPS-induced degenerative changes in nucleus pulposus cells through targeting TCF7L2.

Nucleus pulposus (NP) cells play a critical role in maintaining intervertebral disc integrity through producing the components of extracellular matrix (ECM). NP cell dysfunction, including senescence and hyper-apoptosis, has been regarded as critical events during intervertebral disc degeneration development. In the present study, we found that Transcription Factor 7-Like 2 (TCF7L2) was overexpressed within degenerative intervertebral disc tissue samples, and TCF7L2 silencing improved lipopolysaccharide (LPS)-induced repression on NP cell proliferation, ECM synthesis, and LPS-induced NP cell senescence. miR-1260b directly targeted TCF7L2 and inhibited TCF7L2 expression. miR-1260b overexpression improved LPS-induced degenerative changes in NP cells; more importantly, TCF7L2 overexpression significantly reversed the effects of miR-1260b overexpression on LPS-stimulated degenerative changes within NP cells. For the first time, we demonstrated the function of the miR-1260b/TCF7L2 axis on the phenotypic maintenance of chondrocyte-like NP cells and ECM synthesis by NP cells under LPS stimulation.

IL-17 in intervertebral disc degeneration: Mechanistic insights and therapeutic implications.

Intervertebral disc degeneration (IDD) serves as an independent risk factor for lower back pain and is closely associated with spinal musculoskeletal disorders, including lumbar disc herniation, radiculopathy, and myelopathy. Interleukin-17 (IL-17), also named IL-17A, is a critical signature cytokine of T-helper 17 cells. Upon binding to the IL-17 receptor A/C heterodimeric complex, IL-17 can trigger multiple signal transduction pathways to stimulate gene transcription and increase messenger RNA stability. IL-17 expression is significantly increased in degenerative disc tissue and shows a positive correlation with disease severity. IL-17 has been shown to accelerate the development of IDD by promoting extracellular matrix degradation, enhancing inflammatory response, inducing neoangiogenesis, and inhibiting nucleus pulposus cell autophagy and proliferation. Targeting IL-17 represents a novel and promising approach for the therapeutic intervention of IDD. In this review, we summarized the recent progression about the role of IL-17 in IDD and highlighted its therapeutic implications.

EZH2 upregulates the expression of MAPK1 to promote intervertebral disc degeneration via suppression of miR-129-5p.

This study was designed to verify whether enhancer of zeste homolog 2 (EZH2) affects intervertebral disc degeneration (IVDD) development through regulation of microRNA (miR)-129-5p/MAPK1. Initially, we collected lumbar nucleus pulposus (NP) tissue samples from patients with juvenile idiopathic scoliosis (n = 14) and IVDD (n = 34). We measured the expression of related genes in clinical IVDD tissues and a lipopolysaccharide (LPS)-induced NP cell model. After loss- and gain-of-function assays, NP cell proliferation and senescence were examined. The targeting relationship between miR-129-5p and MAPK1 was explored by dual luciferase reporter gene and RNA immunoprecipitation (RIP) assays. The enrichment of EZH2 and H3K27me3 in miR-129-5p promoter was verified by chromatin immunoprecipitation (ChIP). Finally, an IVDD rat model was established to test the effects of transduction with lentiviral vector carrying miR-129-5p agomir and/or oe-EZH2 in vivo. miR-129-5p was underexpressed, and EZH2 and MAPK1 levels were overexpressed in lumbar nucleus pulposus from human IVDD patients and in LPS-induced NP cells. miR-129-5p overexpression or silencing of MAPK1 promoted proliferation of NP cells, while inhibiting their senescence. EZH2 inhibited miR-129-5p through H3K27me3 modification in the miR-129-5p promoter. miR-129-5p could target the downregulation of MAPK1 expression. EZH2 overexpression increased the release of inflammatory factors and cell senescence factors, which was reversed by miR-129-5p agomir in vivo. Taken together, EZH2 inhibits miR-129-5p through H3K27me3 modification, which upregulates MAPK1, thereby promoting the development of IVDD.

Circular RNAs in Intervertebral Disc Degeneration: An Updated Review

Low back pain, a common medical condition, could result in severe disability and inflict huge economical and public health burden. Its pathogenesis is attributed to multiple etiological factors, including intervertebral disc degeneration (IDD). Emerging evidence suggests that circular RNAs (circRNAs), a major type of regulatory non-coding RNA, play critical roles in cellular processes that are pertinent to IDD development, including nucleus pulposus cell proliferation and apoptosis as well as extracellular matrix deposition. Increasing number of translational studies also indicated that circRNAs could serve as novel biomarkers for the diagnosis of IDD and/or predicting its clinical outcomes. Our review aims to discuss the recent progress in the functions and mechanisms of newly discovered IDD-related circRNAs.

LncRNA HOTAIR influences cell proliferation via miR-130b/PTEN/AKT axis in IDD

ABSTRACT Intervertebral disc degeneration (IDD) constitutes the pathological foundation of most musculoskeletal disorders of the spine. Previous studies have noted that cell proliferation is a common feature of IDD. Bioinformatics indicated that aberrantly expressed long non-coding RNAs (lncRNAs) were involved in the development of IDD. In this study, we aimed to investigate the function of lncRNA HOTAIR in the proliferation of human nucleus pulposus (NP) cells of IDD in vitro and further clarified its mechanism. The expression of HOTAIR and miR-130b was quantified by qRT-PCR in nucleus pulposus (NP) tissues. Furthermore, NP cells proliferation were assayed by CCK8 and Immunostaining. Dual-luciferase reporter and RIP assay were used to examine the expression of HOTAIR, PTEN, and their co-target gene miR-130b. Western blotting was used to test AKT expression. Our in vitro experiments on human normal NP cells observed that HOTAIR was significantly dysregulated in IDD. Further, HOTAIR can suppress proliferation by directly targeting miR-130b. In addition, Both HOTAIR and PTEN were confirmed to target miR-130b, and miR-130b upregulation reversed the phenomenon of ectopic expression of HOTAIR. More importantly, HOTAIR upregulation significantly reduced CyclinD1 protein expression by PTEN/AKT signaling pathway. Our findings suggest that HOTAIR may bind to miR-130b and subsequently increased CyclinD1 expression via PTEN/Akt pathway. Thereby, HOTAIR could become a potential target for the treatment of IDD. Abbreviations : IDD; intervertebral disc degenerationncRNAs; non-coding RNAslncRNAs; long non-coding RNAsmiRNAs; microRNAsNP; nucleus pulposusqRT-PCR; quantitative reverse transcription–PCR LBP; Low back pain ORF; open reading frame HOTAIR; Hox transcript antisense intergenic RNA FAF1; Fas-associated protein factor-1Erk; extracellular signal-regulated kinase TUG1; Taurine Up-regulated Gene 1 HIF1A hypoxia-inducible factor 1-alpha PI3K; phosphoinositide-3 kinase AIS; adolescent idiopathic scoliosis ECM; extracellular matrix LN;lupus nephritis CT;computed tomography MRI; magnetic resonance imaging PBS; phosphate-buffered salin PBS; phosphate-buffered salin PVDF; polyvinylidene fluoride TBST; Tris-buffered saline Tween ECL; enhanced chemiluminescenceRIP; RNA immunoprecipitation

Small extracellular vesicles from hypoxic mesenchymal stem cells alleviate intervertebral disc degeneration by delivering miR-17-5p

Minimally invasive repair strategies are a very promising approach for the treatment of intervertebral disc degeneration (IDD). In recent years, small extracellular vesicles (sEVs) secreted from mesenchymal stem cells (MSCs) have been shown great potential in alleviating IDD. However, in vitro experiments, MSCs are usually exposed to a normoxic micro-environment, which differs greatly from the hypoxic micro-environment in vivo. The primary purpose of our research was to determine whether sEVs isolated from MSCs under hypoxic status (H-sEVs) exhibit a more beneficial effect on protecting IDD compared with sEVs derived from MSCs under normoxic status (N-sEVs). A tail IDD rat model and a series of experiments in vitro were conducted to compare the beneficial effects of PBS, N-sEVs, and H-sEVs treatment. Then, to validate the role of sEVs miRNAs in IDD, a miRNA microarray sequencing analysis and a series of rescue experiments were conducted. Luciferase activity, RNA-ChIP and western blot were performed to explore the potential mechanisms. The results indicate that sEVs alleviate IDD by ameliorating the homeostatic imbalance between anabolism and catabolism in vivo and in vitro. Microarray sequencing result shows that miR-17-5p is maximally enriched in H-sEVs. Toll-like receptor 4 (TLR4) was determined to be a target downstream gene of miR-17-5p. Finally, it was found that H-sEVs miR-17-5p may modulate proliferation and synthesis of human nucleus pulposus cells (HNPCs) matrix via TLR4 pathway. In conclusion, H-sEVs miR-17-5p alleviate IDD via promoting HNPCs matrix proliferation and synthesis, providing new therapeutic targets for IDD. Statement of significanceIntervertebral disc degeneration (IDD) is the primary cause of low back pain (LBP), which is a huge burden to society. Our research demonstrates for the first time that hypoxic pretreatment of small extracellular vesicles (H-sEVs) effectively alleviated the progress of IDD. In short, in the present research, we found that H-sEVs miR-17-5p could modulate proliferation and synthesis of nucleus pulposus cells (NPCs) matrix via TLR4/PI3K/AKT pathway. Therefore, hypoxic pre-treatment is a prospective and efficient method to optimize the therapeutic effect of MSCs-derived sEVs. miRNA and MSCs-derived sEVs combination may be a promising therapeutic approach for IDD.

Hydrogen sulfide regulates autophagy in nucleus pulposus cells under hypoxia.

ObjectiveHydrogen sulfide (H2S) has been found to act as an important gasotransmitter to regulate cell activities. This study aimed to investigate the effect of H2S on autophagy of nucleus pulposus (NP) cells under hypoxia and possible mechanism.Materials and MethodsNP cells were isolated from rat caudal discs. Cobalt chloride was used to mimic hypoxia, sodium hydrosulfide was used to emulate exogenous H2S and 3‐methyladenine was used to block cell autophagy. Cell viability was assessed by phase contrast microscope and Cell Counting Kit‐8 method. Moreover, expression of key autophagic proteins was analyzed via western blotting, and transmission electron microscopy was performed to detect autophagosomes.ResultsHypoxia markedly impaired NP cell proliferation compared with control. Whereas H2S provided pro‐proliferation and pro‐autophagy effects on hypoxic NP cells. However, these beneficial impact of H2S on hypoxic NP cells were reversed by autophagy inhibitor.ConclusionsOur results showed that H2S played a cytoprotective role in NP cells exposed to hypoxia in an autophagy‐dependent manner.

Influence of extracellular nanovesicles derived from adipose-derived stem cells on nucleus pulposus cell from patients with intervertebral disc degeneration.

An increasing number of individuals are suffering from lower back and neck pain caused by intervertebral disc degeneration each year. Although the application of mesenchymal stem cells (MSCs) has provided desirable results in the treatment of intervertebral disc degeneration, there are multiple risks associated with the directed application of MSCs. An increasing number of studies have suggested that stem cells, through the release of extracellular nanovesicles, have vital functions in tissue regeneration and repair with low risk. The present study investigated the effect of extracellular nanovesicles derived from adipose-derived stem cells (ADSCs) on nucleus pulposus (NP) cells from patients with intervertebral disc degeneration. Human NP cells were obtained from patients with intervertebral disc degeneration undergoing surgical procedures in addition to ADSCs from liposuction patients. ADSC-derived extracellular nanovesicles were isolated and characterized. The differentiation and biological activity of NP cells cultured with or without ADSC-derived extracellular nanovesicles were assessed and inflammatory factors and intervertebral disc degeneration-associated markers were also measured. The results indicated that extracellular nanovesicles derived from ADSCs increased the migration and proliferation of NP cells and inhibited inflammatory activity, suggesting their utility for the treatment of intervertebral disc degeneration.

Upregulation of FoxO6 in nucleus pulposus cells promotes DNA damage repair via activation of RAD51.

DNA damage is an essential risk for intervertebral disc degeneration (IDD). Here, we attempted to uncover the effect of FoxO6 and RAD51 on the DNA damage repair of nucleus pulposus (NP) cells in IDD. We collected the human NP tissues of different degeneration degrees and tested the collagen II, FoxO6, and RAD51 expression. Besides, the IL-1β induced NP cell model was also used to elucidate the degenerative progress in vitro. We used Chromatin immunoprecipitation (ChIP) and luciferase reporter assay to confirm whether the FoxO6 protein could enhance the RAD51 expression by binding to its promoter. The FoxO6 gene was upregulated in NP cells by vectors transfection. Immunofluorescence staining was used to measure the RAD51 and γH2AX foci formation. Besides, the typical NP cell gene expression was analyzed by RT-PCR. Cell proliferation was determined by CCK-8, and the cell cycle distribution was determined by flow cytometry. Like collagen II, FoxO6 and RAD51 expression were all decreased both in the severe degenerated NP tissue and in the IL-1β treated NP cells. Upregulation of FoxO6 gene in NP cells enhanced the RAD51 expression via activating the promoter region and inhibited the DNA damage marker γH2AX formation. FoxO6 upregulation alleviated the loss of collagen II, aggrecan, SOD1, and CAT, and suppressed the increase of collagen I/X, TNF-α, and IL-1β expression, which was affected by IL-1β. Besides, FoxO6 also helped the proliferation and cell cycle of NP cells with the activation of RAD51. Upregulation of FoxO6 promotes the DNA repair and maintains the typical phenotype of NP cells, via somehow the mediation of RAD51.

MicroRNA-338-3p as a novel therapeutic target for intervertebral disc degeneration

Recent studies have demonstrated the pivotal role played by microRNAs (miRNAs) in the etiopathogenesis of intervertebral disc degeneration (IDD). The study of miRNA intervention in IDD models may promote the advancement of miRNA-based therapeutic strategies. The aim of the current study was to investigate whether intradiscal delivery of miRNA can attenuate IDD development. Our results showed that miR-338-3p expression was significantly increased in the nucleus pulposus (NP) of patients with IDD. Moreover, there was a statistically significant positive correlation between the expression level of miR-338-3p and the severity of IDD. Our functional studies showed that miR-338-3p significantly influenced the expression of extracellular matrix synthesis genes, as well as the proliferation and apoptosis of NP cells. Mechanistically, miR-338-3p aggravated IDD progression by directly targeting SIRT6, a negative regulator of the MAPK/ERK pathway. Intradiscal injection of antagomir-338-3p significantly decelerated IDD development in mouse models. Our study is the first to identify miR-338-3p as a mediator of IDD and thus may be a promising target for rescuing IDD.

DNMT3B decreases extracellular matrix degradation and alleviates intervertebral disc degeneration through TRPA1 methylation to inhibit the COX2/YAP axis.

Intervertebral disc degeneration (IVDD) is a main cause of low back pain that is associated with extracellular matrix (ECM) degradation and inflammation. This study aims to investigate the role of DNMT3B and its regulatory mechanisms in IVDD. IVDD rat models were constructed followed by transfections with oe-DNMT3B or oe-YAP in order to explore the role of DNMT3B in the development of IVDD. After that transfection, nucleus pulposus (NP) cells were isolated and transfected with oe-DNMT3B, oe-TRPA1, si-YAP, oe-YAP or oe-COX2 in order to investigate the functions of DNMT3B in NP cells. DNMT3B was poorly expressed in IVDD tissues and NP cells whereas TRPA1, COX2, and YAP were highly expressed. The proliferation or apoptosis of NP cells was detected through CCK-8 assay or flow cytometry, respectively. Overexpression of DNMT3B promoted the proliferation of NP cells, inhibited their apoptosis, as well as increasing the expression of collagen II and aggrecan and decreasing expression of MMP3 and MMP9. Besides, DNMT3B suppressed inflammation and alleviated IVDD. Mechanistically, DNMT3B modified the TRPA1 promoter by methylation to inhibit the expression of COX2. Overexpression of COX2 promoted the apoptosis of NP cells and decreased the expression of YAP, which was reversed by upregulating DNMT3B. DNMT3B may promote the proliferation of NP cells and prevent their ECM degradation through the TRPA1/COX2/YAP axis, thereby alleviating IVDD in rats.

MicroRNA‑25 protects nucleus pulposus cells against apoptosis via targeting SUMO2 in intervertebral disc degeneration.

It has been reported that microRNA (miRNA/miR)‑25 is downregulated in patients with intervertebral disc degeneration (IVDD). However, the potential role of miR‑25 in IVDD remains unclear. Therefore, the present study aimed to investigate the effects of miR‑25 on human intervertebral disc nucleus pulposus cells (NPCs). The expression levels of miR‑25 and those of small ubiquitin‑related modifier2(SUMO2) were determined in human nucleus pulposus (NP) tissues by reverse transcription‑quantitative PCR (RT‑qPCR) and western blot analyses. Subsequently, the potential interaction between miR‑25 and SUMO2 was validated via dual‑luciferase reporter assay and RNA pull‑down assay with biotinylated miRNA. The effects of miR‑25 on NPC proliferation and apoptosis were evaluated using Cell Counting Kit‑8 assay, 5‑ethynyl‑2'‑deoxyuridine incorporation assay, and flow cytometry. The results showed that miR‑25 was downregulated in patients with IVDD. In addition, miR‑25 increased the proliferation of NPCs and inhibited their apoptosis. Furthermore, the current study verified that miR‑25 could directly target SUMO2 and regulate its expression via the p53 signaling pathway. Additionally, the effects of miR‑25 on NPCs were abrogated following SUMO2 overexpression. Overall, the results of the present study demonstrated that miR‑25 could promote the proliferation and inhibit the apoptosis of NPCs via targeting SUMO2, suggesting that miR‑25 may be a potential target in the treatment of IVDD.

MiR-532-3p Suppresses Nucleus Pulposus Cells Proliferation and Extracellular Matrix Production, Promotes Cell Apoptosis via Targeting High Mobility Group AT-Hook 2

The present study aimed to investigate the function and mechanism of microRNA (miR)-532-3p in intervertebral disc degeneration (IDD). Further, whether miR-532-3p regulates HMGA2 in nucleus pulposus (NP) cells was explored. We collected human nucleus pulposus (NP) tissues from the patients with IDD, and detected miR-532-3p in NP tissues using RT-qPCR. MiR-532-3p mimic and inhibitor were constructed, and they were transfected into the human nucleus pulposus cells (HNPCs) by Lipofectamine 3000. MTT assay was conducted to determine cell proliferation. Cell apoptosis and extracellular matrix remodeling were examined by flow cytometric, Caspase 3/8 Assay Kits and Western blot. A dual-luciferase reporter assay was applied to investigate whether miR-532-3p targets High mobility group AT-hook 2 (HMGA2). We found miR-532-3p expression level was significantly increased in NP tissues of IDD patients, comparing with the controls. MiR-532-3p exerted an inhibitory effect on HNPCs proliferation; however, cell apoptosis and the degradation of extracellular matrix were induced by miR-532-3p. MiR-532-3p directly targets HMGA2, and HMGA2 overexpression reversed the role of miR-532-3p mimic in HNPCs proliferation, apoptosis, and extracellular matrix remodeling. Our study is the first to report that miR-532-3p might suppress NP cell proliferation, promote cell apoptosis and inhibit ECM production of NP cells via targeting HMGA2, thus facilitating the progression of IDD. MiR-532-3p was supposed to be a novel target for the treatment of IDD.