Liang Kang

MicroRNA-23a-3p promotes the development of osteoarthritis by directly targeting SMAD3 in chondrocytes

Osteoarthritis (OA) is a common chronic degenerative joint disease. Progressive destruction of the integrity of articular cartilage is an important pathological feature, but treatment options that reverse this damage have not been developed. According to recent studies, microRNAs have important regulatory roles in the initiation and progression of OA. In the current study, the biological effects of miR-23a-3p and its expression in OA tissues were examined. We found that miR-23a-3p expression was obviously higher and SMAD3 expression was significantly lower in OA cartilage than in normal tissues. The hypomethylation status of CpG islands in the promoter region of miR-23a-3p was confirmed by methylation-specific polymerase chain reaction in OA cartilage tissues. Furthermore, a bioinformatics analysis and luciferase reporter assay identified SMAD3 as a target gene of miR-23a-3p and SMAD3 expression at both the protein and mRNA levels was inhibited by miR-23a-3p. A functional analysis demonstrated that miR-23a-3p overexpression suppresses type II collagen and aggrecan expression, while miR-23a-3p inhibition had the opposite effects. Small interfering RNA-mediated knockdown of SMAD3 reversed the effects of the miR-23a-3p inhibitor on the expression of type II collagen and aggrecan. Our results suggested that miR-23a-3p contributes to OA progression by directly targeting SMAD3, providing a potential therapeutic target for OA treatment.

Advanced glycation end products regulate anabolic and catabolic activities via NLRP3-inflammasome activation in human nucleus pulposus cells

Intervertebral disc degeneration is widely recognized as a cause of lower back pain, neurological dysfunction and other musculoskeletal disorders. The major inflammatory cytokine IL‐1β is associated with intervertebral disc degeneration; however, the molecular mechanisms that drive IL‐1β production in the intervertebral disc, especially in nucleus pulposus (NP) cells, are unknown. In some tissues, advanced glycation end products (AGEs), which accumulate in NP tissues and promote its degeneration, increase oxidative stress and IL‐1β secretion, resulting in disorders, such as obesity, diabetes mellitus and ageing. It remains unclear whether AGEs exhibit similar effects in NP cells. In this study, we observed significant activation of the NLRP3 inflammasome in NP tissues obtained from patients with degenerative disc disease compared to that with idiopathic scoliosis according to results detected by Western blot and immunofluorescence. Using NP cells established from healthy tissues, our in vitro study revealed that AGEs induced an inflammatory response in NP cells and a degenerative phenotype in a NLRP3‐inflammasome‐dependent manner related to the receptor for AGEs (RAGE)/NF‐κB pathway and mitochondrial damage induced by mitochondrial reactive oxygen species (mtROS) generation, mitochondrial permeability transition pore (mPTP) activation and calcium mobilization. Among these signals, both RAGE and mitochondrial damage primed NLRP3 and pro‐IL‐1β activation as upstream signals of NF‐κB activity, whereas mitochondrial damage was critical for the assembly of inflammasome components. These results revealed that accumulation of AGEs in NP tissue may initiate inflammation‐related degeneration of the intervertebral disc via activation of the NLRP3 inflammasome.

MicroRNA-494 promotes apoptosis and extracellular matrix degradation in degenerative human nucleus pulposus cells

Purpose This study investigated the expression and function of the microRNA-494 in intervertebral disc degeneration (IDD). Results MicroRNA-494 expression was upregulated during IDD progression; its overexpression increased the expression of ECM catabolic factors such as matrix metalloproteinase and A disintegrin and metalloproteinase with thrombospondin motif in NP cells while decreasing that of anabolic genes such as type II collagen and aggrecan; it also induced the apoptosis of NP cells, as determined by flow cytometry. These effects were reversed by microRNA-494 inhibitor treatment. SOX9 was identified as a target of negative regulation by microRNA-494. Promoter hypomethylation and NF-κB activation were associated with microRNA-494 upregulation in IDD. Materials and Methods MicroRNA-494 expression in degenerative nucleus pulposus (NP) tissue was assessed by quantitative real-time PCR. The effect of microRNA-494 on extracellular matrix (ECM) metabolism and NP cell apoptosis was evaluated by transfection of microRNA-494 mimic or inhibitor. The regulation of SRY-related high mobility group box (SOX)9 expression by microRNA-494 was assessed with the luciferase reporter assay, and the methylation status of the microRNA-494 promoter was evaluated by methylation-specific PCR and bisulfite sequencing PCR. The role of activated nuclear factor (NF)-κB in the regulation of microRNA-494 expression was evaluated using specific inhibitors. Conclusions MicroRNA-494 promotes ECM degradation and apoptosis of degenerative human NP cells by directly targeting SOX9.

Simvastatin Inhibits IL-1β-Induced Apoptosis and Extracellular Matrix Degradation by Suppressing the NF-kB and MAPK Pathways in Nucleus Pulposus Cells

Statins are widely used hypocholesterolemic drugs that block the mevalonate pathway. Some studies have shown that statins may have the potential to inhibit intervertebral disk (IVD) degeneration (IDD). Interleukin (IL)-1β, a catabolic cytokine, is a key regulator of IDD. This study aimed to investigate the mechanism underlying the effect of simvastatin on IDD. The viability of nucleus pulposus (NP) cells was determined by the methyl-thiazolyl-tetrazolium (MTT) assay. The apoptosis of NP cells was measured by flow cytometric analysis, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), and western blotting of relevant apoptotic proteins. The protein levels of catabolic factors and anabolic factors were determined by western blotting. The cells were stimulated with IL-1β in the absence or presence of simvastatin to investigate the effects on matrix metalloproteinase (MMP)-3, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4, ADAMTS-5, type II collagen, and aggrecan expression. Our findings indicate that simvastatin considerably inhibited IL-1β-induced apoptosis in NP cells. We also found that simvastatin attenuated IL-1β-induced expression and MMP-3, MMP-13, ADAMTS-4, and ADAMTS-5 activities and also reduced the decrease in type II collagen and aggrecan expression. In addition, simvastatin considerably suppressed the nuclear translocation and activation of nuclear factor-kappa B (NF-KB) by inhibiting p65 phosphorylation and translocation and blocking inhibitor kB-α degradation. It also inhibited MAPK pathway activation by blocking c-Jun N-terminal kinase (JNK), p38, and ERK phosphorylation. The results of our study revealed that simvastatin is a potential agent for IDD prevention and treatment.

MicroRNA-132 upregulation promotes matrix degradation in intervertebral disc degeneration

&NA; MicroRNAs (miRNAs) have been shown to be involved in the pathogenesis of intervertebral disc degeneration (IDD). This experiment was designed to study the expression and role of the miRNA, miR‐132, in IDD. MiR‐132 expression in human nucleus pulposus (NP) tissue was assessed by quantitative real‐time PCR. The methylation status of the miR‐132 was assessed with methylation‐specific PCR and bisulfite sequencing PCR. The regulation of growth differentiation factor5 (GDF5) expression by miR‐132 was evaluated by luciferase reporter assay. Moreover, we investigated the function of miR‐132 on IDD in vivo using a classic needle‐punctured rat tail model. These results showed that miR‐132 expression was upregulated during IDD and this upregulation was associated with hypomethylation of its promoter. MiR‐132 overexpression led to increased expression of ECM catabolic factors, including MMP13 and ADAMTS4, in NP cells while levels of anabolic proteins, such as type II collagen and aggrecan, were diminished. GDF5 was identified as a direct target of negative regulation by miR‐132. MAPK/ERK signaling was also found to be associated with miR‐132‐induced ECM degradation. In addition, we showed that miR‐132 inhibition effectively attenuated NP ECM degradation in IDD in vivo. Our findings demonstrated that miR‐132 promotes ECM degradation by human NP cells by direct targeting of GDF5. Hence, miR‐132 represents a potential therapeutic target in the treatment of IDD. HighlightsGDF5 is a direct target of miR‐132.Aberrant miR‐132 expression in IDD is related to the hypomethylation of its promoter region.miR‐132 promotes NP ECM degradation in vitro and in vivo.miR‐132 exerts its function in human NP cells by targeting GDF5.

Methylation of microRNA-129-5P modulates nucleus pulposus cell autophagy by targeting Beclin-1 in intervertebral disc degeneration

MicroRNAs play an important role in the etiology and progression of many diseases, including intervertebral disc degeneration (IVDD). The miRNA miR-129-5P regulates autophagy in various cancers, but its role in human nucleus pulposus (NP) cells is unclear. The present study investigated whether miR-129-5p regulates the expression of Beclin-1 which is known to induce autophagy in NP cells by evaluating their levels in normal and degenerative disc tissues and human NP cells transfected with miR-129-5P mimic or inhibitor by quantitative real-time (qRT-)PCR, western blotting, flow cytometry, and immunofluorescence analysis. A bioinformatics analysis was used to predict the relationship between miR-129-5P and Beclin-1, which was confirmed by the dual luciferase assay. DNA methylation status was assessed by methylation-specific PCR, and the effect of demethylation on miR-129-5P level and autophagy was examined by qRT-PCR, western blotting, and flow cytometry. We found that miR-129-5P expression was downregulated while that of Beclin-1 and LC3-II was upregulated in degenerative disc NP cells. Meanwhile, autophagy was reduced in human NP cells transfected with miR-129-5P mimic, whereas the opposite result was observed upon treatment with miR-129-5P inhibitor. Bioinformatics analysis and the luciferase reporter assay revealed that Beclin-1 is a target of and is inhibited by miR-129-5P. We also found that CpG islands in the miR-129-5P promoter region were hypermethylated in degenerative as compared to normal disc tissue. Thus, miR-129-5P blocks NP cell autophagy by directly inhibiting Beclin-1, a process that is dependent on miR-129-5P promoter methylation.

Epigenetic silencing of miRNA-143 regulates apoptosis by targeting BCL2 in human intervertebral disc degeneration

Accumulating evidence indicates that microRNAs can regulate the apoptosis of various cells. Apoptosis of nucleus pulposus cells plays an important role in the progression of intervertebral disc degeneration. The aim of this study is to investigate whether microRNA-143 (miRNA-143) is involved in the progression of intervertebral disc degeneration. In this study, the expression of miRNA-143 and its biological modulatory effects were examined. Messenger RNA and protein expression of miRNA-143 and B-cell lymphoma-2 (BCL2) in both normal and degenerative disc tissues was determined by using RT-PCR and western-blot assays. After miRNA-143 transfection, BCL2 expression and NP cell apoptosis were assessed by using RT-PCR, western-blot, and flow cytometry. The relationship between miRNA-143 and BCL2 was assessed by bioinformatics and dual luciferase assays. Epigenetic regulation of miRNA-143 was determined by methylation-specific PCR and the effect of hypomethylation using 5-AZA. In this study, miRNA-143 expression significantly increased, while that of BCL2 decreased in degenerative disc specimens. In addition, CpG islands in the promoter region of miRNA-143 were hypomethylated in degenerative disc tissues. Furthermore, bioinformatics analysis and luciferase reporter assay indicated that BCL2 was a target gene of miRNA-143, and miRNA-143 suppressed BCL2 messenger RNA (mRNA) and protein expression. MiRNAmiRNA-143 overexpression enhances apoptosis of nucleus pulposus cells, while miRNA-143 inhibitor had the opposite effect. BCL2 knockdown reversed the effects of the miRNA-143 inhibitor on nucleus pulposus apoptosis. Our results suggest that miRNA-143 promotes the progression of nucleus pulposus apoptosis by directly targeting BCL2, providing a potential therapeutic target for the treatment of intervertebral disc degeneration disease.

Icariin Attenuates Interleukin-1β-Induced Inflammatory Response in Human Nucleus Pulposus Cells

BACKGROUND Low back pain is a common problem, mainly caused by intervertebral disc degeneration (IDD). An important pathophysiological characteristic of IDD is the loss of homeostatic balance of the extracellular matrix metabolism. Interleukin-1β (IL-1β) is one of the inflammatory mediators stimulating the degradation of extracellular matrix in the nucleus pulposus (NP) and contributing to IDD pathogenesis. Icariin, which is isolated from Epimedium brevicornum, acts as an anti-inflammatory drug. OBJECTIVE This study aimed to explore the pharmacological effects of icariin in IDD by simulating NP inflammation in vitro. METHOD Human NP cells were isolated and cultured in vitro. NP cells were pretreated with icariin (0.1, 1 and 10 µM) and stimulated by IL-1β (10 ng/ml). The concentration of Prostaglandin E2 was determined by enzymelinked immunosorbent assay. Nitric oxide was determined by Griess reagent assay. The expression of cyclooxygenase- 2 (COX-2), inducible nitric oxide synthase (iNOS), degrading enzymes, collagen II, aggrecan, mitogenactivated protein kinase (MAPK), and nuclear factor-kappa B (NF-κB)-related signaling molecules was assessed via western blotting. RESULTS IL-1β induced pronounced expression of COX-2 and iNOS, and stimulated production of prostaglandin E2 and nitric oxide. Icariin exhibited significant anti-inflammatory effect, inhibiting IL-1β-induced production of degrading enzymes, as well as extracellular matrix reduction. Finally, icariin suppressed IL-1β-induced activation of MAPK- and NF-κB-related signaling pathways. CONCLUSION The present findings suggest that icariin may have a protective effect on NP cells. The antiinflammatory effect may contribute to the therapeutic action of icariin in IDD.

Dysregulated miR-127-5p contributes to type II collagen degradation by targeting matrix metalloproteinase-13 in human intervertebral disc degeneration.

BACKGROUND Intervertebral disc degeneration (IDD) is a chronic disease associated with the degradation of extracellular matrix (ECM). Matrix metalloproteinase (MMP)-13 is a major enzyme that mediates the degradation of ECM components. MMP-13 has been predicted to be a potential target of miR-127-5p. However, the exact function of miR-127-5p in IDD is still unclear. OBJECTIVE We designed this study to evaluate the correlation between miR-127-5p level and the degeneration of human intervertebral discs and explore the potential mechanisms. METHODS miR-127-5p levels and MMP-13 mRNA levels were detected by quantitative real-time polymerase chain reaction (qPCR). To determine whether MMP-13 is a target of miR-127-5p, dual luciferase reporter assays were performed. miR-127-5p mimic and miR-127-5p inhibitor were used to overexpress or downregulate miR-127-5p expression in human NP cells, respectively. Small interfering RNA (siRNA) was used to knock down MMP-13 expression in human NP cells. Type II collagen expression in human NP cells was detected by qPCR, western blotting, and immunofluorescence staining. RESULTS We confirmed that miR-127-5p was significantly downregulated in nucleus pulposus (NP) tissue of degenerative discs and its expression was inversely correlated with MMP-13 mRNA levels. We reveal that MMP-13 may act as a target of miR-127-5p. Expression of miR-127-5p was inversely correlated with type II collagen expression in human NP cells. Moreover, suppression of MMP-13 expression by siRNA blocked downstream signaling and increased type II collagen expression. CONCLUSION Dysregulated miR-127-5p contributed to the degradation of type II collagen by targeting MMP-13 in human IDD. Our findings highlight that miR-127-5p may serve as a new therapeutic target in IDD.

MicroRNA-15b silencing inhibits IL-1β-induced extracellular matrix degradation by targeting SMAD3 in human nucleus pulposus cells

ObjectivesTo determine the role of microRNA-15b (miR-15b) in interleukin-1 beta (IL-1β)-induced extracellular matrix (ECM) degradation in the nucleus pulposus (NP).ResultsMiR-15b was up-regulated in degenerative NP tissues and in IL-1β-stimulated NP cells, as compared to the levels in normal controls (normal tissue specimens from patients with idiopathic scoliosis). Bioinformatics and luciferase activity analyses showed that mothers against decapentaplegic homolog 3 (SMAD3), a key mediator of the transforming growth factor-β signaling pathway, was directly targeted by miR-15b. Functional analysis demonstrated that miR-15b overexpression aggravated IL-1β-induced ECM degradation in NP cells, while miR-15b inhibition had the opposite effects. Prevention of IL-1β-induced NP ECM degeneration by the miR-15b inhibitor was attenuated by small-interfering-RNA-mediated knockdown of SMAD3. In addition, activation of MAP kinase and nuclear factor-κB up-regulated miR-15b expression and down-regulated SMAD3 expression in IL-1β-stimulated NP cells.ConclusionsMiR-15b contributes to ECM degradation in intervertebral disc degeneration (IDD) via targeting of SMAD3, thus providing a novel therapeutic target for IDD treatment.