Intervertebral Disc Degeneration Tissues Research Articles

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.

Hsa_circ_0134111 promotes intervertebral disc degeneration via sponging miR-578

Intervertebral disc degeneration (IDD) is a chronic degenerative and age-dependent process characterized by aberrant apoptosis, proliferation, synthesis, and catabolism of the extracellular matrix of the nucleus pulposus (NP) cells. Recently, studies showed that circular RNAs play important roles in the development of many diseases. However, the role of circRNAs in IDD development remains unknown. We showed that circ_0134111 level was overexpressed in IDD tissue samples as compar-ed to control tissues. The upregulation of circ_0134111 was more drastic in the moderate and severe IDD cases than in those with mild IDD. In addition, we showed that interleukin-1β and tumor necrosis factor-α exposure significantly enhanced circ_0134111 expression in NP cells. Furthermore, ectopic expression of circ_0134111 induced proliferation, pro-inflammatory cytokine secretion, and ECM degradation in the NP cells. We also showed that circ_0134111 directly interacted with microRNA (miR)-578 in NP cells where elevated expression of circ_0134111 enhanced the ADAMTS-5 and MMP-9 expression. Moreover, miR-578 expression was significantly decreased in IDD patients and the miR-578 expression was negatively correlated with circ_0134111 expression in the IDD samples. Interleukin-1β and tumor necrosis factor-α exposure significantly decreased miR-578 levels in NP cells, in which ectopic miR-578 expression inhibited cell growth, pro-inflammatory cytokine expression, and ECM degradation. Finally, we showed that circ_0134111 overexpression induced the IDD-related phenotypic changes through inhibiting miR-578. These data suggested that circ_0134111 could promote the progression of IDD through enhancing aberrant NP cell growth, inflammation, and ECM degradation partly via regulating miR-578.

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.

MiR-98 Protects Nucleus Pulposus Cells against Apoptosis by Targeting TRAIL in Cervical Intervertebral Disc Degeneration.

The excessive apoptosis of nucleus pulposus (NP) cells is a major risk factor in the progress of cervical intervertebral disc degeneration (IVDD). In this study, we investigated the impact of miR-98 on apoptosis of NP cells and the potential molecular mechanisms. Lipopolysaccharide (LPS) was used to establish an NP cell IVDD model. The sponging effect of miR-98 on TRAIL 3′UTR was predicted by ENCORI and assessed by the dual-luciferase reporter gene system. The expression levels of miR-98, TRAIL, and TRAIL pathway-related genes were tested by qRT-PCR, Western blot, and immunofluorescence analysis. Cell apoptosis was analyzed by Hoechst 33258 staining and flow cytometry. Cell viability was analyzed by MTT assay. It was found that the expression level of miR-98 was downregulated, while the level of TRAIL was upregulated in IVDD tissues, and their levels were negatively and positively associated with the clinical MRI grade, respectively. The LPS treatment resulted in a significant decrease of the miR-98 expression level and an increase of the TRAIL expression level in NP cells. miR-98 reduced NP cell apoptosis under LPS treatment in vitro. miR-98 directly targeted TRAIL. Moreover, the mRNA and protein levels of DR5, FADD, cleaved caspase8, cleaved caspase3, and cleaved PARP were downregulated by miR-98 overexpression. Overexpression of TRAIL partially reversed the suppressive roles of miR-98 on cell apoptosis and activation of the TRAIL pathway. We concluded that miR-98 inhibited apoptosis of NP cells by inactivating the TRAIL pathway via targeting TRAIL in IVDD NP cells. These results indicated that miR-98 might be a therapeutic target for IVDD.

LncRNA OIP5-AS1 accelerates intervertebral disc degeneration by targeting miR-25-3p

ABSTRACT It is obvious that epigenetic processes influence the evolution of intervertebral disc degeneration (IDD). However, its molecular mechanisms are poorly understood. Long noncoding RNAs (lncRNAs) have been validated to exert vital roles in IDD. Therefore, we tested the hypothesis that OIP5-AS1, a potential regulator of IDD, modulates IDD progression. RT-PCR was utilized to detect levels of OIP5-AS1, miR-25-3p, Collagen II and Aggrecan in IDD tissues and nucleus pulposus cells (NPCs). Immunofluorescence assay measured Collagen II expression. CCK-8, EdU, and flow cytometry estimated the levels of proliferation and apoptosis. Proteins were assessed via Western blot. The binding affinity of OIP5-AS1 with miR-25-3p was investigated by luciferase reporter assay. Enzyme-linked immunosorbent assay (ELISA) analyzed the levels of inflammatory factors. OIP5-AS1 was high expressed in IDD tissues and its expression gradually promoted with the increasing of Pfirrmann scores. The cell morphology of NPCs changed into spindle-shaped, and Collagen II expression was low. After OIP5-AS1 was silenced, cell proliferation was boosted whereas both apoptosis and extracellular matrix (ECM) degradation were restrained. In LPS-activated NPCs, OIP5-AS1 depletion also suppressed inflammation response. Further, miR-25-3p was a target of OIP5-AS1. The effects of OIP5-AS1 silence on proliferation, apoptosis, and ECM degradation were reversed upon miR-25-3p downregulation. Moreover, the inhibitory impact of OIP5-AS1 knockdown on the inflammation of LPS-treated NPCs was rescued with miR-25-3p inference. In general, lncRNA OIP5-AS1 exerted its effects in IDD by targeting miR-25-3p, implying the usage of OIP5-AS1/miR-25-3p as a novel regulatory axis for the molecular targets of IDD therapy.

Roles of circular RNAs in the pathogenesis of intervertebral disc degeneration (Review).

Lower back pain (LBP) is an extremely common symptom and is recognized as a leading contributor to disability and disease burden globally. Intervertebral disc degeneration (IDD) represents a major cause of LBP. However, the molecular mechanisms involved in the pathogenesis of IDD remain unclear, and currently available treatments, including conservative and surgical options, fail to effectively delay, stop or reverse the progression of IDD. Circular RNAs (circRNAs) are a newly discovered group of covalently closed, single-stranded and endogenous non-coding RNAs. A growing body of research has revealed that a number of circRNAs are widely and aberrantly expressed in IDD tissues. Furthermore, they play important roles in the pathogenesis of IDD, including proliferation, apoptosis, senescence, mitophagy, inflammation and extracellular matrix metabolism, mainly by acting as sponges for microRNAs. The present review aims to summarize the current understanding on the mechanisms of circRNA-mediated regulation in 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.

Correlational analysis of chemokine and inflammatory cytokine expression in the intervertebral disc and blood in patients with lumbar disc disease.

The involvement of intervertebral disc (IVD) tissues, whole blood (WB) cytokines, and chemokines in pain in patients with lumbar degenerative disc disease (LDD) is unknown. We investigated the relationships between inflammatory cytokines and chemokines in human IVD tissues and WB samples and their association with pain. Expression levels of chemokines and cytokine gene expression were measured in samples from 20 patients with LDD and compared between IVD tissues and WB samples. The associations between WB chemokine and cytokine gene expression levels and pain intensity (numeric rating scale) were also analyzed. The mRNA of C-C chemokine ligand 20 (CCL20), C-C chemokine receptor 6 (CCR6), interleukin-6 (IL-6), IL-1β, IL-17, and tumor necrosis factor-α (TNF-α) was expressed in degenerated IVD tissues. Pearson's product-moment correlation analysis produced positive correlations between CCR6 and IL-6 expression levels in IVD tissues (r = 0.845, p < 0.001) and WB samples (r = 0.963, p < 0.001). WB IL-6 and CCR6 mRNA expression levels correlated significantly with present pain, maximum pain, and average pain. By contrast, low back pain (LBP) did not correlate with serum chemokine/cytokine expression. This is the first study to report correlations between chemokine and inflammatory cytokine gene expression levels in IVD tissues and WB samples in patients with LDD in relation to pain intensity. WB CCR6 and IL-6 gene expression levels correlated significantly with present pain, maximum pain, and average pain, but not with LBP. These data provide a new understanding of the role of chemokines and inflammatory cytokines in patients with LDD and may lead to new treatment strategies for pain.

Role of the miR-133a-5p/FBXO6 axis in the regulation of intervertebral disc degeneration.

ObjectiveLow back pain is a leading cause of disabilities worldwide, and intervertebral disc degeneration (IVDD)-related disorders have been recognised as one of the main contributors. Nevertheless, the underlying mechanism has not yet been fully understood. The aim of this study was to investigate the role of the miR-133a-5p/FBXO6 axis in the regulation of IVDD.MethodsRT-qPCR, WB and IHC were performed to assess the expression of FBXO6 in human IVD tissues. Nucleus pulposus (NP) cells were treated with IL-1β to induce IVDD cellular model. Silence of FBXO6 was achieved using specific siRNAs. CCK-8 assay, flow cytometry, TUNEL assay, RT-qPCR and WB were used to evaluate the role and mechanism of FBXO6 in the process of IVDD. Online tools, GSE datasets and RT-qPCR were used to search the candidate miRNAs targeting FBXO6. The direct binding sites between FBXO6 and miR-133a-5p were further verified by a dual luciferase assay. RT-qPCR, WB and rescue experiments were conducted to identify the regulatory function of miR-133a-5p on the expression of aggrecan, collagen Ⅱ, MMP3, ADAMTS5, IL-6 and COX2. In addition, the role of the NF-κB pathway in regulating miR-133a-5p was studied using lentiviral shRNA, WB and RT-qPCR.ResultsResults showed that FBXO6 mainly expressed in the NP tissue of IVD and the expression of FBXO6 decreased with the process of IVDD as well as under IL-1β stimulation. The silence of FBXO6 led to the decreased expression of aggrecan and collagen Ⅱ and the increased expression of MMP3, ADAMTS5, IL-6 and COX2, which further induced the degeneration of NP cells. The bioinformatic analysis showed that miR-133a-5p was the candidate miRNA targeting FBXO6. miR-133a-5p was upregulated in IVDD tissues and significantly inhibited the expression of FBXO6. The inhibition of miR-133a-5p ameliorated the acceleration of IVDD induced by the silence of FBXO6 in vitro. Moreover, it was demonstrated that IL-1β regulated the expression of the miR-133a-5p/FBXO6 axis via the NF-κB pathway in NP cells.ConclusionmiR-133a-5p was upregulated by IL-1β to aggravate intervertebral disc degeneration via sponging FBXO6. Inhibiting miR-133a-5p expression or rescuing FBXO6 expression may be promising strategies for the treatment of IVDD.The translational potential of this articleThis study suggests that the miR-133a-5p/FBXO6 axis could regulate NP cells proliferation, apoptosis, synthesis and degradation of extracellular matrix, which provides a promising therapeutic target and strategy for the treatment of IVDD.

Experimental study on the effect of zinc finger protein A20 on lumbar intervertebral disc degeneration in rabbits

To investigate the effect of zinc finger protein A20 on lumbar intervertebral disc degeneration in rabbits. Twenty-six 3-month-old New Zealand rabbits, 2.0-2.5 kg in weight, were used to establish the model of intervertebral disc degeneration at L 3, 4, L 4, 5, and L 5, 6 by transabdominal needle puncture. At 4 weeks after operation, the 24 rabbits were randomly divided into 4 groups after successful modeling, which checked by MRI. The target intervertebral discs of each group were injected with zinc finger protein A20 overexpressed adenovirus (Ov-A20 group), empty carrier adenovirus (NC group), phosphate buffer saline (control group), and shRNA-A20 adenovirus (Sh-A20 group). The biological responses of animals in each group were comprehensive scored before 1 day of injection and after 1, 2, 3, and 6 days of injection. At 2, 4, and 8 weeks after injection, the animals in each group were observed by MRI to obtain the exact T2 relaxation time (T2 signal value). After MRI examination, the animals were killed to take the degenerative intervertebral disc tissue; and the tissue was detected by Alcian blue staining to observed the intervertebral disc degeneration. The expressions of zinc finger protein A20, collagen Ⅱ, and aggrecan were detected by immunohistochemistry staining. The expressions of zinc finger protein A20, nuclear factor κB binding protein [P65, phosphate P65 (P-P65), collagen Ⅱ, aggrecan], inflammatory factors [tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β)], autophagy-related protein [LC3 (LC3Ⅱ/LC3Ⅰ) and P62] were detected by Western blot. The comprehensive score of biological response in each group after injection was significantly lower than that before injection ( P<0.05). At 6 days after injection, the comprehensive score of biological response in the Sh-A20 group was significantly lower than that in other groups ( P<0.05), and there was no significant difference among other groups ( P>0.05). The detection of MRI showed that the T2 signal value in the Ov-A20 group was the highest at 2, 4, and 8 weeks after injection ( P<0.05), and the T2 signal value in the Sh-A20 group was the lowest at 2 and 4 weeks after injection ( P<0.05). There was no significant difference between other groups ( P>0.05). Alcian blue staining showed that the expression of aggrecan was the highest in Ov-A20 group and the lowest in Sh-A20 group at 4 weeks ( P<0.05); the expression of aggrecan in Ov-A20 group was the highest at 8 weeks ( P<0.05), and there was no significant difference between other groups ( P>0.05). Immunohistochemical staining showed that the expressions of zinc finger protein A20, collagen Ⅱ, and aggrecan were the highest in Ov-A20 group and lowest in Sh-A20 group ( P<0.05). Western blot showed that the expressions of zinc finger protein A20, collagen Ⅱ, aggrecan, and LC3 (LC3Ⅱ/LC3Ⅰ) proteins were the highest in the Ov-A20 group and the lowest in Sh-A20 group ( P<0.05), while the expressions of P-P65, TNF-α, IL-1β, and P62 proteins were the lowest in Ov-A20 group and the highest in Sh-A20 group ( P<0.05). There was no significant difference in the expression of p65 protein between groups ( P>0.05). Zinc finger protein A20 can effectively regulate the process of lumbar intervertebral disc degeneration in rabbits by inhibiting inflammation.

A20 regulates inflammation through autophagy mediated by NF-κB pathway in human nucleus pulposus cells and ameliorates disc degeneration in vivo

Intervertebral disc degeneration (IDD) is closely related to loss of the extracellular matrix (ECM), apoptosis and inflammation in nucleus pulposus cells (NPCs). It has been reported that Zinc finger protein A20/TNFAIP3 (A20) can inhibit the activity of the NF-κB pathway and promote autophagy. Therefore, we speculated that A20 can regulate inflammation and ameliorate IDD through autophagy mediated by NF-κB in human NPCs. Our results indicated that the expression of A20 and inflammatory factors in IDD tissues was increased. A20 is an essential negative regulator in the NF-κB pathway. Constructed adenoviral shRNA and overexpression vectors for A20 could effectively regulate the inflammation, autophagy, and activity of NF-κB, which in turn affected the progression of IDD. Inhibition of NF-κB on the basis of knocking down A20 results in increased autophagy, suggesting that A20-regulated autophagy was mediated by NF-κB. In vivo, A20 overexpression could ameliorate the progression of IDD and promote autophagy at the same time, while deletion of A20 leads to low levels of autophagy and severe degeneration. In summary, A20 plays an important role in inhibiting inflammation through autophagy mediated by NF-κB in NPCs and ameliorating IDD.

LncRNA NEAT1 promotes nucleus pulposus cell matrix degradation through regulating Nrf2/ARE axis

BackgroundThis study aimed to assess the role and mechanism of lncRNA NEAT1 in intervertebral disc degeneration (IVD).MethodsLncRNA profile (GSE56081) between IVD and healthy control was downloaded from the Gene Expression Omnibus (GEO) database and analyzes differential lncRNA expression. Expression of lncRNA NEAT1 in IVD tissue and TNF-α/IL-1β-stimulated nucleus pulposus cells were further measured by RT-PCR. The lncRNA NEAT1 overexpression plasmids (pcDNA-NEAT1) were constructed and transfected into nucleus pulposus cells. Catabolic biomarkers (MMP-3 and MMP-13), anabolic biomarkers (Col II and Aggrecan) and Nrf2 expression were further measured. To further investigate the function of Nrf2, nucleus pulposus cells were pretreated with or without 25 μM tert-Butylhydroquinone (TBHQ), a Nrf2 activator, for 18 h and subsequently cotreated with pcDNA-NEAT1.ResultsA total of 1432 lncRNAs were differentially expressed in GSE56081. Bioinformatic analysis found that these lncRNAs mainly enriched in Nrf2/ARE signaling pathway. LncRNA NEAT1 was highly expressed in IVD tissues than that of healthy control. Moreover, TNF-α/IL-1β induced a time- and dose-dependent increase in the mRNA expression of lncRNA NEAT1 in the nucleus pulposus cells. Overexpression of lncRNA NEAT1 abates promotes nucleus pulposus cells proliferation but induces matrix degradation. Meanwhile, nucleus and cytoplasm Nrf2 expression was significantly down-regulated by lncRNA NEAT1 upregulation. Nrf2 activator (TBHQ) could partially reverse the inhibitory effects of overexpression of lncRNA NEAT1 on matrix degradation.ConclusionCollectively, our data unveiled the lncRNA NEAT1 promotes matrix degradation by regulating Nrf2/ARE signaling pathway, suggesting a potential therapeutic for IVD in the future.

The distinct roles of myosin IIA and IIB under compression stress in nucleus pulposus cells.

ObjectivesInappropriate or excessive compression applied to intervertebral disc (IVD) contributes substantially to IVD degeneration. The actomyosin system plays a leading role in responding to mechanical stimuli. In the present study, we investigated the roles of myosin II isoforms in the compression stress‐induced senescence of nucleus pulposus (NP) cells.Material and methodsNucleus pulposus cells were exposed to 1.0 MPa compression for 0, 12, 24 or 36 hours. Immunofluorescence and co‐immunoprecipitation analysis were used to measure the interaction of myosin IIA and IIB with actin. Western blot analysis and immunofluorescence staining were used to detect nuclear expression and nuclear localization of MRTF‐A. In addition, the expression levels of p‐RhoA/RhoA, ROCK1/2 and p‐MLC/MLC were measured in human NP cells under compression stress and in degenerative IVD tissues.ResultsCompression stress increased the interaction of myosin IIA and actin, while the interaction of myosin IIB and actin was reduced. The actomyosin cytoskeleton remodelling was involved in the compression stress‐induced fibrotic phenotype mediated by MRTF‐A nuclear translocation and inhibition of proliferation in NP cells. Furthermore, RhoA/ROCK1 pathway activation mediated compression stress‐induced human NP cells senescence by regulating the interaction of myosin IIA and IIB with actin.ConclusionsWe for the first time investigated the regulation of actomyosin cytoskeleton in human NP cells under compression stress. It provided new insights into the development of therapy for effectively inhibiting IVD degeneration.

Bleomycin induces fibrotic transformation of bone marrow stromal cells to treat height loss of intervertebral disc through the TGF\u03b2R1/Smad2/3 pathway

BackgroundLower back pain is often accredited to loss of intervertebral disc (IVD) height and compromised spine stability as a result of intervertebral disc degeneration (IVDD). We aim to locally use bleomycin to induce the fibrotic transformation of bone marrow stromal cells (BMSCs) as a means to induce reparative fibrosis to slow down the height loss.MethodsIVDs from patients were gathered for histological examination. The expression of the transforming growth factor beta 1 (TGF-β) signaling pathway was determined by qPCR and western blotting. Nucleus pulposus (NP) cells, annulus fibrosus (AF) cells, and the rats’ bone marrow stromal cells (BMSC) were cultured and their responsiveness to bleomycin was evaluated by Cell Counting Kit-8, comet assay, transwell migration, and wound healing assays. Rat IVDD models were created by puncture and rescued by bleomycin injection, and the effectiveness was evaluated by images (X-ray and MRI) and atomic force microscope.ResultsHistological examination showed increased levels of pro-fibrotic markers in IVDD tissues from patients. AF cells and BMSC cells were induced to adopt a pro-fibrotic phenotype with increased expression fibrotic markers Col1a1, Col3a1, and FSP1. The pro-fibrotic effect of bleomycin on AF cells and BMSCs was in part due to the activation of the TGFβ-TGFβR1-SMAD2/3 signaling pathway. Pharmacological inhibition or gene knock-down of TGFβR1 could mitigate the pro-fibrotic effects.ConclusionLocally, injection of bleomycin in rats’ IVD induced rapid fibrosis and maintained its height through the TGFβ-TGFβR1-SMAD2/3 signaling pathway.

Mir-573 regulates cell proliferation and apoptosis by targeting Bax in human degenerative disc cells following hyperbaric oxygen treatment

BackgroundMicroRNA (miRNA) plays a vital role in the intervertebral disc (IVD) degeneration. The expression level of miR-573 was downregulated whereas Bax was upregulated notably in human degenerative nucleus pulposus cells. In this study, we aimed to investigate the role of miR-573 in human degenerative nucleus pulposus (NP) cells following hyperbaric oxygen (HBO) treatment.MethodsNP cells were separated from human degenerated IVD tissues. The control cells were maintained in 5% CO2/95% air and the hyperoxic cells were exposed to 100% O2 at 2.5 atmospheres absolute. MiRNA expression profiling was performed via microarray and confirmed by real-time PCR, and miRNA target genes were identified using bioinformatics and luciferase reporter assays. The mRNA and protein levels of Bax were measured. The proliferation of NPCs was detected using MTT assay. The protein expression levels of Bax, cleaved caspase 9, cleaved caspase 3, pro-caspase 9, and pro-caspase 3 were examined.ResultsBioinformatics analysis indicated that the 3′ untranslated region (UTR) of the Bax mRNA contained the “seed-matched-sequence” for hsa-miR-573, which was validated via reporter assays. MiR-573 was induced by HBO and simultaneous suppression of Bax was observed in NP cells. Knockdown of miR-573 resulted in upregulation of Bax expression in HBO-treated cells. In addition, overexpression of miR-573 by HBO increased cell proliferation and coupled with inhibition of cell apoptosis. The cleavage of pro-caspase 9 and pro-caspase 3 was suppressed while the levels of cleaved caspase 9 and caspase 3 were decreased in HBO-treated cells. Transfection with anti-miR-573 partly suppressed the effects of HBO.ConclusionMir-573 regulates cell proliferation and apoptosis by targeting Bax in human degenerative NP cells following HBO treatment.

The miR-623/CXCL12 axis inhibits LPS-induced nucleus pulposus cell apoptosis and senescence

Nucleus pulposus cell (NPC) is the major cell type maintaining the physiological function of intervertebral discs by producing extracellular matrix (ECM). NPC apoptosis and senescence together contribute to NPC loss, finally leading to intervertebral disc degeneration (IDD). Herein, miR-623 showed to be downregulated within IDD tissue samples according to both bioinformatics and experimental analyses. In LPS-injured NPCs, miR-623 overexpression promoted LPS-suppressed cell proliferation; moreover, miR-623 overexpression inhibited cell apoptosis and senescence, increased ECM secretion, and reduced levels of inflammatory factors. In contrast to miR-623, CXCL12 expression was significantly upregulated in IDD tissues; miR-623 directly bound CXCL12 to inhibit its expression. In LPS-stimulated NPCs, CXCL12 silencing also LPS-induced changes in cell proliferation, cell senescence, ECM secretion, and inflammatory factor levels. More importantly, CXCL12 overexpression aggravated LPS-induced changes and significantly reversed the protective effects of miR-623 overexpression. In conclusion, the miR-623/CXCL12 axis could affect NPC apoptosis and senescence, ECM deposition, and inflammatory factor levels under LPS stimulation in vitro. The p65 signaling might be involved.

Upregulated lnc‑HRK‑2:1 prompts nucleus pulposus cell senescence in intervertebral disc degeneration.

Intervertebral disc (IVD) degeneration is a complicated physiological change involving cellular senescence, inflammation and the degradation of the extracellular matrix. Long non-coding RNAs (lncRNAs) have been identified as new players in IVD degeneration. The present study aimed to identify lncRNAs implicated in IVD degeneration via the regulation of cellular senescence. In the present study, nucleus pulposus (NP) cells isolated from moderately degenerated IVD tissues exhibited a senescent phenotype with increased senescence rates, detected by senescence-associated β-galactosidase (SA-β-gal) staining, and reduced growth and migratory abilities. Microarray and target prediction analyses identified 353 differentially expressed lncRNAs, and 251 cis- and 2,170 trans-acting targets in degenerated NP cells. Bioinformatic analyses revealed that these predicted targets were enriched in the regulation of response to DNA damage stimulus, positive regulation of cell cycle processes and interferon-β production. In addition, a network of the top 10 upregulated and top 10 downregulated lncRNA targets was constructed, and two trans-acting targets, C-C motif chemokine ligand 5 (CCL5) and polyribonucleotide nucleotidyltransferase 1 (PNPT1) involved in aging or senescence, and their corresponding lncRNAs, lnc-ST8SIA5-1:2 and lnc-HRK-2:1, were identified. Reverse transcription-quantitative PCR validation demonstrated that the two targets and two candidate lncRNAs were significantly upregulated in degenerated NP cells. Overexpression of lnc-HRK-2:1, with validated higher expression levels, in normal NP cells induced a senescent phenotype, with enhanced rates of senescence detected by SA-β-gal staining in cells, decreased growth and migratory abilities and improved expression levels of CCL5 and PNPT1. Collectively, these results suggested that upregulation of lnc-HRK-2:1 prompted NP cell senescence in IVD degeneration, which may be associated with increased expression levels of CCL5 and PNPT1.

Knockdown of hsa_circ_0059955 Induces Apoptosis and Cell Cycle Arrest in Nucleus Pulposus Cells via Inhibiting Itchy E3 Ubiquitin Protein Ligase.

BackgroundCircular RNAs (circRNAs) play an important role in the progression of intervertebral disc (IVD) degeneration (IVDD). Using bioinformatics analysis, we have found that the expression of circRNA hsa_circ_0059955 was significantly downregulated in IVDD tissues. However, the relevant mechanism of hsa_circ_0059955 in the progression of IVDD remains unclear.MethodsCCK-8 and flow cytometry assays were used to evaluate cell proliferation and apoptosis. In addition, Western blot assay was used to detect the expressions of ITCH, p73, CDK2 in nucleus pulposus (NP) cells. Moreover, a puncture-induced IVDD rat model was established to explore the role of hsa_circ_0059955 in IVDD.ResultsThe level of hsa_circ_0059955 was significantly decreased in IVDD tissues from IVDD patients. Itchy E3 ubiquitin protein ligase (ITCH) is the host gene of hsa_circ_0059955, and downregulation of hsa_circ_0059955 significantly decreased the expression of ITCH in NP cells. In addition, downregulation of hsa_circ_0059955 markedly inhibited proliferation and induced apoptosis and cell cycle arrest in NP cells. Moreover, in vivo study illustrated that overexpression of hsa_circ_0059955 ameliorated IVDD in rats.ConclusionDownregulation of hsa_circ_0059955 could induce apoptosis and cell cycle arrest in NP cells in vitro, while overexpression of hsa_circ_0059955 attenuated the IVDD in a puncture-induced rat model in vivo. Therefore, hsa_circ_0059955 might serve as a therapeutic target for the treatment of IVDD.

MiR-424-5p regulates apoptosis and cell proliferation via targeting Bcl2 in nucleus pulposus cells

ABSTRACT miRNAs play an important role in the pathogenesis of intervertebral disc degeneration (IDD). The role and the underlying mechanism of miR-424-5p in human nucleus pulposus (NP) are still unknown. We aimed to explore the role of miR-424-5p in IDD. Real-time PCR was used to detect the expression of miR-424-5p and Bcl2 in IDD tissues and idiopathic scoliosis tissues. Human NP cells were used in our study. MTT and Hoechst apoptosis assays were used to detect the proliferation and apoptosis of NP cells, respectively. Western blotting assays were used to detect the expression levels of Bcl-2, cleaved caspase-3, cleaved caspase-9, caspase-3 and caspase-9 in degenerative NP cells. A luciferase reporter assay was applied to confirm the relationship between miR-424-5p and Bcl2. Our results showed that the expression of miR-424-5p was increased and Bcl2 was decreased in degenerative NP cells. miR-425-5p expression was negatively correlated with Bcl2 expression in IDD tissues. Suppression of miR-424-5p using an inhibitor increased Bcl2 expression at both the mRNA and protein levels, and it promoted cell viability and inhibited apoptosis. Furthermore, the levels of cleaved caspase-3 and cleaved caspase-9 were downregulated in miR-424-5p-silenced NP cells. Interestingly, we found that silencing miR-424-5p increased p62 expression at both the mRNA and protein levels. Finally, a luciferase reporter assay verified the binding of the miR-424-5p and the 3’UTR of Bcl2. These results suggested that silencing miR-424-5p suppressed NP cell apoptosis by upregulating Bcl2. Therefore, miR-424-5p might be a novel target for IDD therapies.

HO-1 overexpression alleviates senescence by inducing autophagy via the mitochondrial routein human nucleus pulposus cells.

Intervertebral disc degeneration (IDD) is closely associated with aging. Our previous studies have confirmed that heme oxygenase-1 (HO-1) can inhibit nucleus pulposus (NP) cell apoptosis. However, whether or not HO-1 is involved in NP cell senescence and autophagy is unclear. Our results indicated that HO-1 expression was reduced in IDD tissues and replicative senescent NP cells. HO-1 overexpression using a lentiviral vector reduced the NP cell senescence level, protected mitochondrial function, and promoted NP cell autophagy through the mitochondrial pathway. Autophagy inhibitor 3-MA pretreatment reversed the anti-senescent and protective effects on the mitochondrial function of HO-1, which promoted the degradation of the extracellular matrix (ECM) in the intervertebral disc. In vivo, HO-1 overexpression inhibited IDD and enhanced autophagy. In summary, these results suggested that HO-1 overexpression alleviates NP cell senescence by inducing autophagy via the mitochondrial route.