Upregulation of microRNA-27a inhibits synovial angiogenesis and chondrocyte apoptosis in knee osteoarthritis rats through the inhibition of PLK2

The function of microRNA-27a (miR-27a) on synovial angiogenesis and chondrocyte apoptosis in rats with knee osteoarthritis (KOA) by targeting PLK2 is explored in this present study. The rat model of KOA was conducted by anterior cruciate ligament transection. Rats were injected with miR-27a mimics, mimics NC, pcDNA3.1-PLK2 pcDNA3.1, or RLK2 RNAi plasmid via tail vein. A series of assays were used to figure out the functions of miR-27a and PLK2 in synovial angiogenesis and chondrocyte apoptosis in rats with KOA. Furthermore, the putative binding site between miR-27a and PLK2 was determined. Downregulated miR-27a was found in synovial tissues and cartilage tissues of KOA rats. Upregulated miR-27a and downregulated PLK2 inhibited synovial injury and promoted apoptosis of synovial cells, inhibited synovial angiogenesis, inhibited cartilage injury and chondrocyte apoptosis, inhibited cartilage collagen destruction, and alleviates inflammatory injury of synovial tissue and cartilage tissue in KOA rats. Overexpression of PLK2 reverses the effect of upregulation of miR-27a on synovial angiogenesis and chondrocyte injury in KOA rats. Our study suggests that upregulation of miR-27a inhibits synovial angiogenesis and chondrocyte apoptosis in KOA rats through the inhibition of PLK2.     

Circular RNA circANKRD36 regulates Casz1 by targeting miR-599 to prevent osteoarthritis chondrocyte apoptosis and inflammation

Osteoarthritis (OA) is an ageing-related disease characterized by articular cartilage degradation and joint inflammation. circRNA has been known to involve in the regulation of multiple inflammatory diseases including OA. However, the mechanism underlying how circRNA regulates OA remains to be elucidated. Here, we report circANKRD36 prevents OA chondrocyte apoptosis and inflammation by targeting miR-599, which specifically degrades Casz1. We performed circRNA sequencing in normal and OA tissues and found the expression of circANKRD36 is decreased in OA tissues. circANKRD36 is also reduced in IL-1β–treated human chondrocytes. FACS analysis and Western blot showed that the knockdown of circANKRD36 promotes the apoptosis and inflammation of chondrocytes in IL-1β stress. We then found miR-599 to be the target of circANKRD36 and correlate well with circANKRD36 both in vitro and in vivo. By database analysis and luciferase assay, Casz1 was found to be the direct target of miR-599. Casz1 helps to prevent apoptosis and inflammation of chondrocytes in response to IL-1β. In conclusion, our results proved circANKRD36 sponge miR-599 to up-regulate the expression of Casz1 and thus prevent apoptosis and inflammation in OA.     

microRNA-15a模拟物对人关节软骨细胞增殖与凋亡的影响

目的：近年来研究表明,关节软骨细胞凋亡在骨关节炎发病过程中起到了重要的作用,本文旨在探讨microma-15a模拟物对于原代人膝关节软骨细胞增殖与凋亡的影响。方法：取人外伤性截肢后的膝关节软骨,采用双酶消化法分离获得人膝关节软骨细胞,并进行体外培养,通过甲苯胺蓝染色和II型胶原免疫细胞化学染色进行软骨细胞鉴定。将培养的软骨细胞传代后取第l代细胞,分为实验组和对照组,实验组采用mir．15a模拟物（has．mir-15amimics）转染软骨细胞,上调软骨细胞内mir-15a的表达量;对照组分为阴性对照组、空白对照组。采用MTT法测定细胞增殖曲线,流式细胞仪测定细胞凋亡率。结果：原代细胞中细胞呈多角形、圆形与梭型,贴壁生长;甲苯胺蓝染色胞质呈深蓝色,II型胶原染色胞质呈黄褐色,为特异性染色。经统计学分析,实验组与对照组相比增殖速率明显下降（P〈0．05）。实验组凋亡率（7．13％±0．57）与阴性对照组凋亡率（2．66％±0．15）相比明显增高（P〈0．05）。结论：采用双酶消化法成功分离并培养具有生物学特性的原代人膝关节软骨细胞,通过转染mir-15a模拟物外源性增加关节软骨细胞内mir．15a表达量可显著促进其凋亡并抑制其增殖,为阐明骨关节炎发病机制提供了新的理论依据,为’临床治疗提供了新的靶点。     

Long noncoding RNA MALAT-1 inhibits apoptosis and matrix metabolism disorder in interleukin-1β-induced inflammation in articular chondrocytes via the JNK signaling pathway

Proinflammatory cytokine such as interleukin (IL)-1β causes inflammation of articular cartilage. In this current study, we explored the chondroprotective effects of long noncoding RNA (lncRNA) MALAT-1 on cell proliferation, apoptosis, and matrix metabolism in IL-1β-induced inflammation in articular chondrocytes. Articular chondrocytes from knee joints of normal rats were isolated and cultured, followed by identification through observation of toluidine blue and COL II immunocytochemical stainings. The proliferation of chondrocytes at passage 2 was detected by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. The inflammatory chondrocytes induced by 10 ng/mL IL-1β were observed and identified by toluidine blue and COL II immunocytochemical stainings. pcDNA 3.1 and pcDNA-MALAT-1 were transfected in the chondrocytes. Ultrastructure of chondrocytes was observed by using a transmission electron microscope. The MTT assay was carried out to evaluate chondrocyte viability. Hoechst 33258 staining and flow cytometry were adopted to assess chondrocyte apoptosis. The chondrocytes at passage 2 with the biological characteristics of chondrocytes were used for subsequent experiments. In IL-1β-treated chondrocytes, the growth rate of chondrocytes slowed down, the cells became narrow and long, the vacuoles were seen in the cells, and the morphology of the chondrocytes was irregular. The toluidine blue staining and the immunohistochemical staining of COL II became weaker. In response to IL-1β induction, articular chondrocytes showed reduced MALAT-1 expression; moreover, obvious cartilage injury was observed with decreased chondrocyte viability and Col II expression and elevated chondrocyte apoptosis, MMP-13 expression, and p-JNK expression. With the treatment of pcDNA-MALAT-1, the cartilage injury was alleviated with increased chondrocyte viability and type II collagen (Col II) expression and reduced chondrocyte apoptosis, MMP-13 expression and p-JNK expression. Taken together these results, lncRNA MALAT-1 blocked the activation of the JNK signaling pathway; thereby, IL-1β-induced inflammation in articular chondrocytes was reduced with enhanced chondrocyte proliferation and suppressed chondrocyte apoptosis and extracellular matrix degradation.     

microRNA-206 is required for osteoarthritis development through its effect on apoptosis and autophagy of articular chondrocytes via modulating the phosphoinositide 3-kinase/protein kinase B-mTOR pathway by targeting insulin-like growth factor-1

microRNA (miR) has been shown to be involved in the treatment of diseases such as osteoarthritis (OA). This study aims to investigate the role of miR-206 in regulating insulin-like growth factor-1 (IGF-1) in chondrocyte autophagy and apoptosis in an OA rat model via the phosphoinositide 3-kinase (P13K)/protein kinase B (AKT)-mechanistic target of rapamycin (mTOR) signaling pathway. Wistar rats were used to establish the OA rat model, followed by the observation of histopathological changes, Mankin score, and the detection of IGF-1-positive expression and tissue apoptosis. The underlying regulatory mechanisms of miR-206 were analyzed in concert with treatment by an miR-206 mimic, an miR-206 inhibitor, or small interfering RNA against IGF-1 in chondrocytes isolated from OA rats. Then, the expression of miR-206, IGF-1, and related factors in the signaling pathway, cell cycle, and apoptosis, as well as inflammatory factors, were determined. Subsequently, chondrocyte proliferation, cell cycle distribution, apoptosis, autophagy, and autolysosome were measured. OA articular cartilage tissue exhibited a higher Mankin score, promoted cell apoptotic rate, increased expression of IGF-1, Beclin1, light chain 3 (LC3), Unc-51-like autophagy activating kinase 1 (ULK1), autophagy-related 5 (Atg5), caspase-3, and Bax, yet exhibited decreased expression of miR-206, P13K, AKT, mTOR, and Bcl-2. Besides, miR-206 downregulated the expression of IGF-1 and activated the P13K/AKT signaling pathway. Moreover, miR-206 overexpression and IGF-1 silencing inhibited the interleukins levels (IL-6, IL-17, and IL-18), cell apoptotic rate, the formation of autolysosome, and cell autophagy while promoting the expression of IL-1β and cell proliferation. The findings from our study provide a basis for the efficient treatment of OA by investigating the inhibitory effects of miR-206 on autophagy and apoptosis of articular cartilage in OA via activating the IGF-1-mediated PI3K/AKT-mTOR signaling pathway.     

Downregulation of microRNA-23b protects against ischemia-reperfusion injury via p53 signaling pathway by upregulating MDM4 in rats

Total knee arthroplasty is a commonly performed safe procedure and typically executed in severe knee arthritis, but it also triggers ischemia-reperfusion injury (IRI). More recently, microRNAs (miRs) have been reported to play a contributory role in IRI through the key signaling pathway. Hence, the current study aimed to investigate the effect and specific mechanism of microRNA-23b (miR-23b), murine double minute 4 (MDM4), and the p53 signaling pathway in IRI rat models. First, the IRI model was established, and the expression pattern of miR-23b, MDM4, and the p53 signaling pathway-related genes was characterized in cartilaginous tissues. Then, miR-23b mimics or inhibitors were applied for the elevation or the depletion of the miR-23b expression and siRNA-MDM4 for the depletion of the MDM4 expression in the articular chondrocytes. By means of immunohistochemistry, quantitative real-time polymerase chain reaction, and Western blot analysis, IRI rats exhibited increased miR-23b expression, activated p53 signaling pathway, and decreased MDM4 expression. MDM4 was verified as a target gene of miR-23b through. Downregulated miR-23b increased the expression of MDM4, AKT, and Bcl-2, but decreased the expression of p53, p21, and Bax. In addition, a series of cell experiments demonstrated that downregulated miR-23b promoted articular chondrocyte proliferation and cell cycle entry, but inhibited articular chondrocyte apoptosis. The absence of the effects of miR-23b was observed after MDM4 knocked down. Our results indicate that silencing miR-23b could act to attenuate IRI and reduce the apoptosis of articular chondrocytes through inactivation of the p53 signaling pathway by upregulating MDM4, which provide basic therapeutic considerations for a novel target against IRI.     

Effects of long non-coding RNA LINC00667 on renal tubular epithelial cell proliferation,apoptosis and renal fibrosis via the miR-19b-3p/LINC00667/CTGF signaling pathway in chronic renal failure

The global prevalence of chronic renal failure (CRF) has significantly elevated with various reports indicating there to be a 10% worldwide rate. The functions of long non-coding RNAs (lncRNAs) and their deeper association with CRF at present remain poorly understood. Hence, the aim of the present study was to investigate the altered expressions of lncRNA LINC00667 in CRF and its associated effects on renal tubular epithelial cell proliferation, apoptosis and renal fibrosis through the microRNA-19b-3p (miR-19b-3p)/LINC00667/connective tissue growth factor (CTGF) signaling pathway. Initially, verification of the targeting relationship between LINC00667, CTGF and miR-19b-3p was performed, after which evidence was obtained indicating that miR-19b-3p could negatively regulate LINC00667 and CTGF. The expressions of CTGF in both the CRF and normal renal tissues were determined by immunohistochemistry means, with LINC00667 and CTGF determined to be highly expressed, while poor expression levels of miR-19b-3p were detected among the CRF tissues. The expressions of LINC00667, miR-19b-3p, fibrosis- and epithelial-mesenchymal transition (EMT)-related genes were also examined. The successfully established CRF rat models were treated with varying mimics, inhibitors, and siRNA. ELISA was applied to determine the renal function-related factors. Besides, the renal cell proliferation, migration and apoptosis were detected. In response to LINC00667 silencing, the renal tubular epithelial cells displayed increased proliferation and migration accompanied by reduced apoptosis based on upregulated miR-19b-3p, along with inhibited renal fibrosis and EMT detected. Taken together, the key findings of our study demonstrated that decreased lncRNA LINC00667 could promote renal tubular epithelial cell proliferation and ameliorate renal fibrosis in CRF via the miR-19b-3p/LINC00667/CTGF signaling pathway.     

MicroRNA-146a is linked to pain-related pathophysiology of osteoarthritis

Because miR-146a is linked to osteoarthritis (OA) and cartilage degeneration is associated with pain, we have characterized the functional role of miR-146a in the regulation of human articular cartilage homeostasis and pain-related factors. Expression of miRNA 146a was analyzed in human articular cartilage and synovium, as well as in dorsal root ganglia (DRG) and spinal cord from a rat model for OA-related pain assessment. The functional effects of miR-146a on human chondrocytic, synovial, and microglia cells were studied in cells transfected with miR-146a. Using real-time PCR, we assessed the expression of chondrocyte metabolism-related genes in chondrocytes, genes for inflammatory factors in synovial cells, as well as pain-related proteins and ion channels in microglial cells. Previous studies showed that miR-146a is significantly upregulated in human peripheral knee OA joint tissues. Transfection of synthetic miR-146a significantly suppresses extracellular matrix-associated proteins (e.g., Aggrecan, MMP-13, ADAMTS-5, collagen II) in human knee joint chondrocytes and regulates inflammatory cytokines in synovial cells from human knee joints. In contrast, miR-146a is expressed at reduced levels in DRGs and dorsal horn of the spinal cords isolated from rats experiencing OA-induced pain. Exogenous supplementation of synthetic miR-146a significantly modulates inflammatory cytokines and pain-related molecules (e.g., TNFα, COX-2, iNOS, IL-6, IL8, RANTS and ion channel, TRPV1) in human glial cells. Our findings suggest that miR-146a controls knee joint homeostasis and OA-associated algesia by balancing inflammatory responses in cartilage and synovium with pain-related factors in glial cells. Hence, miR-146a may be useful for the treatment of both cartilage regeneration and pain symptoms caused by OA.     

The lncRNA MEG3 downregulation leads to osteoarthritis progression via miR-16/SMAD7 axis

Background

Osteoarthritis (OA) is a chronic joint disease and there is no a definitive cure at present. Long non-coding RNAs (lncRNAs) have been confirmed to play important roles in the development of OA. However, the underlying mechanism of lncRNA maternally expressed gene 3 (MEG3) in OA has not been well elucidated.

Methods

The rat OA model and interleukin-1β (IL-1β)-induced rat chondrocytes were constructed. The expression pattern of lncRNA MEG3 and miR-16 was detected by RT-qPCR assay in cartilage tissues of rat OA model. The effect of MEG3 and miR-16 on IL-1β-induced chondrocytes was evaluated on the basis of cell viability and apoptosis. Then, the interaction among MEG3, miR-16 SMAD7 was explored by dual-luciferase reporter assay and RIP assay.

Results

It is found that lncRNA MEG3 was down-regulated and miR-16 was up-regulated in rat OA cartilage tissues. MEG3 knockdown promoted proliferation and inhibited apoptosis, while miR-16 knockdown suppressed proliferation and promoted apoptosis in IL-1β-induced rat chondrocytes. Moreover, MEG3 was involved in miR-16 pathway and MEG3 suppressed miR-16 expression. Additionally, SMAD7 was a target gene of miR-16 and miR-16 suppressed SMAD7 expression in IL-1β-induced chondrocytes. Moreover, the expression of SMAD7 induced by MEG3 or si-MEG3 was markedly reversed by the introduction of miR-16 or anti-miR-16. Furthermore, MEG3 exerted its anti-proliferation and pro-apoptosis by regulating miR-16 and SMAD7.

Conclusion

MEG3 was down-regulated and miR-16 was up-regulated in cartilage tissues of rat OA model. MEG3 knockdown might lead to the progression of OA through miR-16/SMAD7 axis.

     

MicroRNA-186 ameliorates Knee osteoarthritis via regulation of P2X7-mediated Cathepsin-K/Runx2/ADAMTS5 signalling axis in articular chondrocytes

Knee osteoarthritis (KOA) is a chronic joint disorder involving the articular cartilage and tissues around the synovial joint. The key objective of this study was to determine the effect of miR-186-5p administration on the expression of pathogenic signalling in the chondrocytes using a surgical destabilization of the medial meniscus (DMM) model of KOA, and to testify the mechanism of P2X7-mediated regulation of RUNX2/ADAMTS5 axis by miR-186 in the KOA rats. After eight weeks of intra-articular injection of the miR-186-5p and negative control lentivirus samples, the knee cartilage tissues were subjected to histopathological analysis Safranin-O/Fast green staining. Further, the articular chondrocytes were separated and analysed for various proteins including P2X7, cathepsin-K, RUNX2 and ADAMTS5 using Western blotting method. We observed that the protein expressions of P2X7, cathepsin-K/RUNX2/ADAMTS5, and also MMP-13 were upmodulated in the KOA rats, while intra-articular miR-186-5p lentivirus administration prevented these aberrations.Hence, the study concludes that miR-186 orchestrates P2X7 expression and the P2X7-mediated cathepsin-K/RUNX2/ADAMTS5 axis and regulates the pathogenesis of KOA. In light of this evidence, we propose that molecular therapeutic interventions targeting miR-186 activation might attenuate osteoarthritic cartilage degeneration.     

Exosomes from human umbilical cord mesenchymal stem cells inhibit ROS production and cell apoptosis in human articular chondrocytes via the miR-100-5p/NOX4 axis

Cyclic strain-induced chondrocyte damage is actively involved in the pathogenesis of osteoarthritis and arthritis. MicroRNAs (miRNAs) carried by exosomes have been implicated in various diseases. However, the role of miR-100-5p in cyclic strain-induced chondrocyte damage remains to be elucidated. miR-100-5p and NADPH oxidase 4 (NOX4) were silenced or overexpressed in human primary articular chondrocytes. PKH-67 Dye was used to trace exosome endocytosis. Reactive oxygen species (ROS) production was monitored using DCFH-DA. Cell apoptosis was measured using a flow cytometer. Quantitative RT-PCR and Western blots were used to evaluate gene expression. Cyclic strain promoted ROS production and apoptosis in primary articular chondrocytes in a time-dependent manner. HucMSCs-derived exosomal miR-100-5p inhibited cyclic strain-induced ROS production and apoptosis in primary articular chondrocytes. miR-100-5p directly targeted NOX4. Overexpressing NOX4 attenuated hucMSCs-derived exosomes-mediated protective effects in primary articular chondrocytes. Cyclic strain promotes ROS production and apoptosis in primary articular chondrocytes, which was abolished by hucMSCs-derived exosomal miR-100-5p through its target NOX4. The findings highlight the importance of miR-100-5p/NOX4 axis in primary articular chondrocytes injury and provide new insights into therapeutic strategies for articular chondrocytes injury and osteoarthritis.     

Overexpression of microRNA-202-3p protects against myocardial ischemia-reperfusion injury through activation of TGF-β1/Smads signaling pathway by targeting TRPM6

MicroRNAs (miRNAs) have been found to act as key regulators in the pathogenesis of myocardial ischemic-reperfusion (I/R) injury. In this study, we explore the role and mechanism of microRNA-202-3p (miR-202-3p) in regulating cardiomyocyte apoptosis, in respective of the TGF-β1/Smads signaling pathway by targeting the transient receptor potential cation channel, subfamily M, member 6 (TRPM6). The targeting relationship between miR-202-3p and TRPM6 was verified by a dual-luciferase reporter gene assay. Sprague-Dawley rat models of myocardial I/R injury were initially established and treated with different mimics, inhibitors and siRNAs to test the effects of miR-202-3p and TRPM6 on myocardial I/R injury. The levels of inflammatory factors; IL-1β, IL-6, TNF-α as well as the degree of myocardial fibrosis and cardiomyocyte apoptosis were determined in rats transfected with different plasmids. TRPM6 was found to be the target of miR-202-3p. Up-regulated miR-202-3p or knockdown of TRPM-6 alleviated oxidative stress and inflammatory response, reduced ventricular mass, altered cardiac hemodynamics, suppressed myocardial infarction, attenuated cell apoptosis, and inhibited myocardial fibrosis. MiR-202-3p overexpression activates the TGF-β1/Smads signaling pathway by negatively regulating TRPM6 expression. Taken together, these findings suggest that miR-202-3p offers protection against ventricular remodeling after myocardial I/R injury via activation of the TGF-β1/Smads signaling pathway.     

Calcium induces differentiation of primary human salivary acinar cells

We previously reported that connective tissue growth factor/hypertrophic chondrocyte-specific gene product 24 (CTGF/Hcs24) stimulated the proliferation and differentiation of rabbit growth cartilage (RGC) cells in vitro. In this study, we investigated the effects of CTGF/Hcs24 on the proliferation and differentiation of rabbit articular cartilage (RAC) cells in vitro. RAC cells transduced by recombinant adenoviruses generating mRNA for CTGF/Hcs24 synthesized more proteoglycan than the control cells. Also, treatment of RAC cells with recombinant CTGF/Hcs24 (rCTGF/Hcs24) increased DNA and proteoglycan syntheses in a dose-dependent manner. Northern blot analysis revealed that the rCTGF/Hcs24 stimulated the gene expression of type II collagen and aggrecan core protein, which are markers of chondrocyte maturation, in both RGC and RAC cells. However, the gene expression of type X collagen, a marker of hypertrophic chondrocytes, was stimulated by rCTGF/Hcs24 only in RGC cells, but not in RAC cells. Oppositely, gene expression of tenascin-C, a marker of articular chondrocytes, was stimulated by rCTGF/Hcs24 in RAC cells, but not in RGC cells. Moreover, rCTGF/Hcs24 effectively increased both alkaline phosphatase (ALPase) activity and matrix calcification of RGC cells, but not of RAC cells. These results indicate that CTGF/Hcs24 promotes the proliferation and differentiation of articular chondrocytes, but does not promote their hypertrophy or calcification. Taken together, the data show that CTGF/Hcs24 is a direct growth and differentiation factor for articular cartilage, and suggest that it may be useful for the repair of articular cartilage.     

Kartogenin prevents cartilage degradation and alleviates osteoarthritis progression in mice via the miR-146a/NRF2 axis

Osteoarthritis (OA) is a common articular degenerative disease characterized by loss of cartilage matrix and subchondral bone sclerosis. Kartogenin (KGN) has been reported to improve chondrogenic differentiation of mesenchymal stem cells. However, the therapeutic effect of KGN on OA-induced cartilage degeneration was still unclear. This study aimed to explore the protective effects and underlying mechanisms of KGN on articular cartilage degradation using mice with post-traumatic OA. To mimic the in vivo arthritic environment, in vitro cultured chondrocytes were exposed to interleukin-1β (IL-1β). We found that KGN barely affected the cell proliferation of chondrocytes; however, KGN significantly enhanced the synthesis of cartilage matrix components such as type II collagen and aggrecan in a dose-dependent manner. Meanwhile, KGN markedly suppressed the expression of matrix degradation enzymes such as MMP13 and ADAMTS5. In vivo experiments showed that intra-articular administration of KGN ameliorated cartilage degeneration and inhibited subchondral bone sclerosis in an experimental OA mouse model. Molecular biology experiments revealed that KGN modulated intracellular reactive oxygen species in IL-1β-stimulated chondrocytes by up-regulating nuclear factor erythroid 2-related factor 2 (NRF2), while barely affecting its mRNA expression. Microarray analysis further revealed that IL-1β significantly up-regulated miR-146a that played a critical role in regulating the protein levels of NRF2. KGN treatment showed a strong inhibitory effect on the expression of miR-146a in IL-1β-stimulated chondrocytes. Over-expression of miR-146a abolished the anti-arthritic effects of KGN not only by down-regulating the protein levels of NRF2 but also by up-regulating the expression of matrix degradation enzymes. Our findings demonstrate, for the first time, that KGN exerts anti-arthritic effects via activation of the miR-146a-NRF2 axis and KGN is a promising heterocyclic molecule to prevent OA-induced cartilage degeneration.Subject terms: Osteoarthritis, Drug development     

Effects of autophagy on apoptosis of articular chondrocytes in adjuvant arthritis rats

Rheumatoid arthritis (RA) is a chronic, systemic autoimmune disease that eventually leads to joint deformities and loss of joint function. Previous studies have demonstrated a close relationship between autophagy and the development of RA. Although autophagy and apoptosis are two different forms of programmed death, the relationship between them in relation to RA remains unclear. In this study, we explored the effect of autophagy on apoptosis of articular chondrocytes in vivo and in vitro. Adjuvant arthritis (AA) and acid‐induced primary articular chondrocyte apoptosis were used as in vivo and in vitro models, respectively. Articular chondrocyte autophagy and apoptosis were both observed dynamically in AA rat articular cartilage at different stages (15 days, 25 days and 35 days). Moreover, chondrocyte apoptosis and articular cartilage injury in AA rats were increased by the autophagy inhibitor 3‐methyladenine (3‐MA) and decreased by the autophagy activator rapamycin. In addition, pre‐treatment with 3‐MA increased acid‐induced chondrocyte apoptosis, while pre‐treatment with rapamycin reduced acid‐induced chondrocyte apoptosis in vitro. These results suggest that autophagy might be a potential target for the treatment of RA.     

MicroRNA-384 regulates cell proliferation and apoptosis through directly targeting WISP1 in laryngeal cancer

Laryngeal cancer (LC) is an increasingly common malignant tumors of head and neck cancer. Aberrant expression of microRNA (miRNA) is closely related with LC development. In the current study, we investigated the biological function and underlying molecular mechanism of miR-384 in LC. The results showed that the miR-384 expression was markedly downregulated in LC tissue and cell lines (TU212 and TU686) as compared with that of adjacent nontumor tissues and a normal human bronchial epithelial cell line. Next, we performed gain-of-function and loss-of-function experiments in the TU212 and TU686 cells by transfecting the cells with miR-384 mimics, miR-384 inhibitor, or miRNA control. Moreover, results showed that miR-384 mimic remarkably inhibited LC cell proliferation, which was notably decreased by miR-384 inhibitor. Furthermore, miR-384 mimics notably increased the amounts of DNA fragmentation from the apoptotic cells (a hallmark of apoptosis) and the caspase-3 activity, whereas miR-384 inhibitor resulted in a decline of DNA fragmentation and the caspase-3 activity compared with its control. In addition, a dual-luciferase reporter assay confirmed that Wnt-induced secreted protein-1 (WISP1) gene was a direct target of miR-384. MiR-384 mimic remarkably inhibited the messenger RNA and protein expression of WISP1, which was upregulated by miR-384 inhibitor as compared to its control. WISP1 knockdown by small interfering RNA inhibited LC cell proliferation and promoted cell apoptosis. WISP1 overexpression partly abrogates the effect of miR-384 overexpression. Taken together, these data indicate that miR-384 regulates LC cell proliferation and apoptosis through targeting WISP1 signaling pathway, providing a novel insight into the LC treatment.     

Anaphylatoxin receptors and complement regulatory proteins in human articular and non-articular chondrocytes: interrelation with cytokines

Tissue trauma induces an inflammatory response associated with a cytokine release that may engage complement pathways. Cytokine-mediated complement expression may contribute to cartilage degradation. Hence, we analysed the complement expression profile in primary articular and non-articular chondrocytes and its interrelation with cytokines. The expression of the anaphylatoxin receptors (C3aR and C5aR) and the complement regulatory proteins (CPRs) CD35, CD46, CD55 and CD59 was studied in cultured articular, auricular and nasoseptal chondrocytes using RTD-PCR and immunofluorescence labelling. The complement profile of peripheral blood mononuclear cells (PBMCs) was opposed to the expression in articular chondrocytes. The time-dependent regulation (6 and 24?h) of these complement factors was assessed in articular chondrocytes in response to the cytokines TNF??, IL-10 or TNF?? combined with IL-10 (each 10?ng/mL). C3aR, C5aR, CD46, CD55 and CD59 but almost no CD35 mRNA was expressed in any of chondrocyte types studied. The anaphylatoxin receptor expression was lower and that of the CRPs was higher in chondrocytes when compared with PBMCs. The majority of the studied complement factors were expressed at a significantly lower level in non-articular chondrocytes compared with the articular chondrocytes. TNF?? significantly increased the C3aR expression in chondrocytes after 6 and 24?h. TNF?? + IL-10 significantly downregulated C5aR and IL-10 significantly inhibited the CD46 and CD55 gene expression after 24?h. C5aR and CD55 could be localised in cartilage in situ. Anaphylatoxin receptors and CRPs are regulated differentially by TNF?? and IL-10. Whether cytokine-induced complement activation occurs in response to cartilage trauma has to be further identified.     

CTGF facilitates cell‐cell communication in chondrocytes via PI3K/Akt signalling pathway

PurposesGap junction intercellular communication (GJIC) is essential for articular cartilage to respond appropriately to physical or biological stimuli and maintain homeostasis. Connective tissue growth factor (CTGF), identified as an endochondral ossification genetic factor, plays a vital role in cell proliferation, migration and adhesion. However, how CTGF regulates GJIC in chondrocytes is still unknown. This study aims to explore the effects of CTGF on GJIC in chondrocytes and its potential biomechanism.Materials and methodsqPCR was performed to determine the expression of gene profile in the CCN family in chondrocytes. After CTGF treatment, CCK‐8 assay and scratch assay were performed to explore cell proliferation and migration. A scrape loading/dye transfer assay was adopted to visualize GJIC in living chondrocytes. Western blot analysis was done to detect the expression of Cx43 and PI3K/Akt signalling. Immunofluorescence staining was used to show protein distribution. siRNA targeting CTGF was used to detect the influence on cell‐cell communication.ResultsThe CTGF (CCN2) was shown to be the highest expressed member of the CCN family in chondrocytes. CTGF facilitated functional gap junction intercellular communication in chondrocytes through up‐regulation of Cx43 expressions. CTGF activated PI3K/Akt signalling to promote Akt phosphorylation and translocation. Suppressing CTGF also reduced the expression of Cx43. The inhibition of PI3K/Akt signalling decreased the expressions of Cx43 and thus impaired gap junction intercellular communication enhanced by CTGF.ConclusionsFor the first time, we provide evidence to show CTGF facilitates cell communication in chondrocytes via PI3K/Akt signalling pathway.     

Circular RNA SLTM as a miR-421-competing endogenous RNA to mediate HMGB2 expression stimulates apoptosis and inflammation in arthritic chondrocytes

Osteoarthritis (OA) is the most common age-related joint disease characterized by chronic inflammation, progressive articular cartilage destruction, and subchondral sclerosis. Accumulating evidence suggests that circular RNAs (circRNAs) play key roles in OA, but the function of circSLTM in OA remains greatly unknown. Therefore, this study focused on interleukin-1β (IL-1β)-treated primary human chondrocytes as well as a rat model to investigate the expression pattern and functional role of circSLTM in OA in vitro and in vivo. CircSLTM and high mobility group protein B2 (HMGB2) were upregulated in IL-1β-induced chondrocytes, whereas miR-421 was downregulated. Knockdown of circSLTM or overexpression of miR-421 ameliorated IL-1β-induced chondrocyte apoptosis and inflammation. The regulatory relationship between circSLTM and miR-421, as well as that between miR-421 and HMGB2, was predicted by bioinformatics and then verified by the RNA immunoprecipitation experiment and dual-luciferase reporter gene assay. Furthermore, silencing of circSLTM increased cartilage destruction and decreased cartilage tissue apoptosis rate and inflammation in a rat model of OA. Taken together, our findings demonstrate the fundamental role of circSLTM in OA progression and provide a potential molecular target for OA therapy.