| Background:Osteoarthritis (OA) is a kind of degenerative joint disease, which seriously affects the health of the elderly, and is characterized by articular cartilage degradation. The main clinical manifestations includes recurrent joint pain and gradually increased joint movement disorder. Articular cartilage includes two components, articular cartilage chondrocytes and extracellular matrix (ECM). Overall, the main pathological features of cartilage degradation and OA are the apoptosis of chondrocytes and the imbalance of extracellular matrix synthesis and catabolic. ECM mainly contains water, collagen, proteoglycan and glycoproteins. Collagens form a fiber web to constitute the structural support of the articular cartilage matrix, and the predominantly component is collagen type Ⅱ, which is about 90-95% of the total amount of collagen. Proteoglycans play a role in cartilage tissue hydration with a huge expansion pressure. The integrity of ECM is a prerequisite for articular cartilage to exert physiological functions. As the only cell component in articular cartilage, the main function of chondrocytes is to maintain metabolic homeostasis of ECM. On the one hand, chondrocytes can continue to synthesize collagen, proteoglycan and other extracellular matrix component; on the other hand, chondrocytes also dynamically synthesize a large number of matrix-degrading enzymes, ADAMTS and other hydrolases, and then secrete to the extracellular to degrade the necrotic, dysfunction collagen or proteoglycan, so apoptosis or abnormal metabolism of chondrocytes seriously affect the metabolic homeostasis of ECM and the integrity of the cartilage tissue, easy to trigger OA. It can be seen that chondrocyte apoptosis is associated with ECM integrity, apoptosis can promote the degradation of ECM, and vice versa. As the main components of cartilage, chondrocyte and ECM regulate the normal physiological function of articular cartilage together. Currently, the pathogenesis of OA remains unclear, the study on cartilage damage is important to elucidate the pathogenesis of OA, providing a theoretical basis for OA prevention and intervention.MiRNAs are single-stranded RNA molecules that are widespread present in eukaryotes, containing about 17 to 25 nucleotides. MiRNA gene may be derived from various areas of genome, such as the spacer region of genome and the intron of encoding gene. Regardless of where it comes from, the processing mechanism is basically the same and mature miRNA formation need to undergo several steps. Firstly, miRNA genes are transcribed into the primary transcript, referred to as pri-miRNA. pri-miRNA is then processed to form about 70 nucleotides miRNA precursor, called pre-miRNA. After the second processing events, pre-miRNA is cut into 21～25 nt mature miRNA. MiRNA expression are under strictly regulation that are characterised with tissue-specific, spatial and temporal-specific, and their abnormal expression may cause a variety of diseases, such as cancer, cardiovascular disease. Little is known about the mechanisms leading to miRNA abnormal expression and transcriptional regulation of miRNA expression is very important. p53, as a transcription factor, has been reported that it can regulate the expression of miRNA. Mi RNA is highly conserved in evolution. By binding to the complementary base found in the target gene mRNA, miRNA directly degrades the target gene or inhibiting its translation process, thereby regulating the expression of the target gene. The human have about 20,000 genes, of which about a third of the genes are regulated by miRNA. One miRNA may regulate a plurality of target genes, a gene may also be regulated by a plurality of miRNA. microRNA plays a very important role in many life activities, such as cell proliferation, cell differentiation, apoptosis and organ development. The present study show that miRNA has important fuctions in the development process of OA.Purpose:This study was designed to investigate the differences of miR-502-5p expression levels between normal and OA cartilage tissue, and then studied the biological effects of miR-502-5p on IL-1β-induced chondrocyte injury and investigated the underlying mechanisms, exploring new therapeutic targets and providing a new theoretical basis for OA treatment.Methods:The first part of the work was to study the expression level and biological effects of miR-502-5p in OA cartilage tissues. First, RT-PCR assay was used to detect the expression levels of four predicted miRNAs in 20 groups of normal and OA cartilage, including miR-502-5p, miR-138, miR-205 and miR-146a. Chondrocytes were isolated from normal cartilage tissues by trypsin and collagenase type Ⅱ digestion and then according to different treatment, the cells were divided into four groups:control group, normally cultured chondrocytes; IL-1β group, chondrocytes treated with IL-1β; miR-502-5p mimic group, chondrocytes transfected with miR-502-5p mimic, then treated with IL-1β; negative control group, chondrocytes transfected with miR-502-5p negative control, and then treated with IL-1β. RT-PCR assay was used to detect the expression levels of miR-502-5p in different treatment groups. MTT method was used to detect cell proliferation rate in different treatment groups. Flow cytometry was used to measure the cell apoptosis level in different treatment groups. Western blot assay was used to detect the protein expression levels of apoptosis-related proteins Bcl-2, Bax and Caspase-3. RT-PCR and western blot were used to detect the expression levels of pro-inflammatory cytokine IL-6, TNFα and IL-8 in different treatment groups. RT-PCR and western blot method were used to detect the expression levels of ECM structural proteins proteoglycans and collagen type Ⅱ, metalloproteinases MMP-3, MMP-9 and MMP-13.The second part of the work is about the regulation relations between Wnt/β-catenin signaling pathway and miR-502-5p-p53 negative feedback loop. RT-PCR and western blot method were used to detect the expression level of p53 in 20 groups normal and OA cartilage tissues. Chondrocytes were isolated from normal cartilage tissues by trypsin and collagenase type Ⅱ digestion. To explore the regulation effect of p53 on miR-502-5p, the cells were treated differently and were divided into four groups:control group, normally cultured chondrocytes; IL-1β group, chondrocytes treated with IL-1β; si-p53+IL-1β group, chondrocytes transfected with p53 siRNA, then treated with IL-1β; negative control group, chondrocytes transfected with p53 siRNA negative control, and then treated with IL-1β. Western blot assay was used to detect p53 protein expression level in different treatment groups. RT-PCR assay was used to detect the expression levels of miR-502-5p in different treatment groups. Then we constructed miR-502-5p promoter luciferase reporter vector, and luciferase reporter experiment was performed to study the p53 transcriptional regulation to miR-502-5p. Then we explored the regulation effect of miR-502-5p on p53, the cells were divided into four groups:control group, IL-1β group, miR-502-5p mimic group and negative control group. RT-PCR and western blot method were used to detect p53 protein expression levels in different groups. Luciferase reporter vectors of p53 3’-UTR was constructed, performing luciferase reporter assay to study the regulation between miR-502-5p and p53. Furthermore, to explore the regulation effect of Wnt/β-catenin signaling pathway on p53, RT-PCR and western blot method were used to detect the Wntl, Wnt3a and P-catenin expression level in 20 groups of normal and OA cartilage tissues. Chondrocytes were isolated from normal cartilage tissues by trypsin and collagenase type Ⅱ digestion and then according to different treatment, the cells were divided into three groups:control group, normally cultured chondrocytes; IL-1β group, chondrocytes treated with IL-1β; Wnt3a inhibition agent group, chondrocytes treated with sFRP-3 and Dkk-1, then treated with IL-1β. Western blot assay was used to detect the protein level of Wnt3a, β-catenin and p53. RT-PCR assay was used to detect the expression level of miR-502-5p in different treatment groups.The third part of the work was to study the target genes of miR-502-5p and associated pathways. RT-PCR and western blot method were used to detect the expression levels of TRAF2 in 20 group of normal and OA cartilage tissues. Luciferase reporter vectors of TRAF2 3’-UTR was constructed, and luciferase reporter experimental was performed to study the regulation effect of miR-502-5p on TRAF2. Chondrocytes were isolated from normal cartilage tissues by trypsin and collagenase type Ⅱ digestion and divided into four groups:control group, IL-1β group, miR-502-5p mimic group and negative control group. RT-PCR and western blot method were used to detect TRAF2 protein expression levels in different treatment groups. Then to investigate whether miR-502-5p influence NF-κB signaling pathway mediated by TRAF2, chondrocytes were isolated from normal cartilage tissues by trypsin and collagenase type Ⅱ digestion and according to different treatments, the cells were divided into five groups:control group, normally cultured chondrocytes; IL-1β group, chondrocytes treated with IL-1β; PDTC group, chondrocytes treated with NF-κB inhibitor PDTC, and then treated with IL-1β; si-TRAF2+IL-1β group, chondrocytes transfected with TRAF2 siRNA, then treated with IL-1β; miR-502-5p mimic group, chondrocytes transfected miR-502-5p mimic, then treated with IL-1β. Western blot method was used to detect the protein levels of NF-κB and IicB-α in different treatment groups. MTT method was used to detect cell proliferation in different treatment groups. Flow cytometry was used to measure the cell apoptosis in different treatment groups. Western blot method was used to detect the expression levels of pro-inflammatory cytokine IL-6, TNFa and IL-8 in different groups. Western blot method was used to detect the protein expression levels of ECM structural proteins proteoglycans and type Ⅱ collagen, metalloproteinases MMP-3, MMP-9 and MMP-13 in different treatment groups.Results:The first result was that the expression of miR-502-5p was down-regulated in OA cartilage tissue, and miR-502-5p played an important biological role in chondrocytes. Compared with normal control group, the expression of miR-502-5p was significantly reduced in OA cartilage tissues. IL-1β treatment induced significantly reduced expression of miR-502-5p in chondrocytes. And transfected with miR-502-5p mimic caused the overexpression of miR-502-5p. Compared with the control group, the cell proliferation rate was decreased, the apoptosis rate was increased, the protein expression level of anti-apoptotic protein Bcl-2 was significantly reduced, and the expression levels of apoptotic protein Bax and Caspase-3 were significantly increased, the mRNA and protein expression levels of IL-6, IL-8 and TNFa were all increased significantly, the mRNA and protein expression levels of collagen type Ⅱ and aggrecan were significantly lower, and the mRNA and protein expression levels of MMP-3,MMP-9 and MMP-13 were significantly upregulated in IL-1β group. Compared with IL-1β group, the cell proliferation rate was significantly increased, the apoptosis rate was significantly reduced, the anti-apoptotic protein Bcl-2 expression was significantly increased, while the expression of apoptotic protein Bax and Caspase-3 were significantly reduced, the mRNA and protein expression levels of IL-6, IL-8 and TNFα were decreased obviously, the mRNA and protein expression levels of collagen type Ⅱ and aggrecan were significantly increased, and the mRNA and protein expression levels of MMP-3, MMP-9 and MMP-13 were decreased significantly in miR-502-5p mimic group.The second part was that Wnt/β-catenin signaling pathway regulated the expression of p53, thereby regulating the expression of miR-502-5p. Compared with normal control group, the mRNA and protein expression levels of p53 were significantly up-regulated in OA cartilage tissue, and the expression of p53 and miRNA-502-5p had a negative correlation. Compared with the control group, the protein expression level of p53 was significantly increased; the expression level of miR-502-5p in IL-1β group was decreased significantly, the promoter activity of miR-502-5p was significantly reduced in IL-1β group; compared with IL-1β group, the expression level of p53 was significantly decreased, the expression level of miR-502-5p was significantly increased, the promoter activity of miR-502-5p was increased significantly in si-p53group. Compared with the control group, the mRNA and protein levels of p53 were significantly increased, the luciferase activity of pGL3-p53 3’-UTR did not change significantly in IL-1β group; compared with IL-1β group, the protein level of p53 was significantly reduced, while no significant differences were found in mRNA levels, and the fluorescence luciferase activity of pGL3-p53 3’-UTR did not change significantly in miR-502-5p mimic group. Compared with normal control group, the protein expression levels of Wnt protein Wntl and Wnt3a were significantly up-regulated in OA cartilage tissues, and the β-catenin expression level was also significantly raised. Compared with the control group, the expression levels of Wnt3a, β-catenin and p53 were upregulated, the expression level of miR-502-5p was decreased significantly in IL-1β group; compared with IL-1β group, the expression levels of Wnt3a, β-catenin and p53 were significantly reduced, and the expression level of miR-502-5p was significantly increased in Wnt3a inhibitor group.The third part was the study on the target gene TRAF2 of miR-502-5p and TRAF2 mediated NF-κB signal pathway. Compared with normal control group, the mRNA and protein expression levels of TRAF2 were significantly upregulated in OA cartilage tissues, and the expression of miRNA-502-5p and TRAF2 had a negative correlation. Software predicted that miR-502-5p targeted on TRAF2 3’-UTR. Compared with miR-502-5p negative control group, luciferase activity of pGL3-TRAF2 3’-UTR vector was significantly decreased in miR-502-5p mimic group, while luciferase activity of pGL3- TRAF2 3’-UTR mut vector had no significant change. Compared with the control group, the mRNA and protein expression levels of TRAF2 in IL-1β group were significantly increased; compared with IL-1β group, the mRNA and protein expression levels of TRAF2 were significantly reduced in miR-502-5p mimic group.Compared with the control group, IL-1β induced activation of NF-κB pathway, so that NF-κB protein expression level was significantly up-regulated, while IκB-α protein level was significantly reduced, the cell proliferation rate was decreased, the apoptosis rate was increased significantly, the protein levels of IL-6, IL-8 and TNFa were significantly increased, the type Ⅱ collagen and proteoglycan protein levels were significantly reduced, and the expression of MMP-3,MMP-9 and MMP-13 were significantly increased in IL-1β group; compared with IL-1β group, PDTC group, miR-502-5p mimic group and si-TRAF2 group can inhibit the activation of NF-κB signaling pathway, NF-κB expression was significantly decreased, while hcB-α expression was significantly increased, the cell proliferation rate was significantly increased, the apoptosis rate was significantly decreased, the protein levels of IL-6, IL-8 and TNFa were significantly reduced, the protein levels of collagen type II and aggrecan in PDTC group were significantly increased, while the expression levels of MMP-3, MMP-9 and MMP-13 were reduced.Conclusion:In summary, this study showed that miR-502-5p expression levels in OA cartilage tissues and IL-1β-induced chondrocytes were both significantly reduced, and miR-502-5p had a protective effect on IL-1β-induced chondrocyte injury, including promoting chondrocyte proliferation, reducing chondrocyte apoptosis, reducing inflammatory cytokines production and promoting metabolic balance of ECM. p53 could regulate the transcription of miR-502-5p, thereby affecting its expression. And miR-502-5p could also inhibit the expression of p53, suggesting that miR-502-5p and p53 formed a negative feedback regulation loop. Wnt/β-catenin signaling pathway positively regulated the expression of p53, in other words,Wnt/β-catenin regulated p53 expression and thus regulated miR-502-5p-p53 negative feedback loop. Further mechanism analysis showed that miR-502-5p targeted TRAF23’-UTR to reduce its expression, thereby inhibiting the TRAF2-mediated NF-κB signaling pathway, thus reducing the IL-1β-induced chondrocyte injury, exerting anti-apoptotic, anti-inflammatory and anti-matrix degradation function. Our study shows that miR-502-5p is expected to become a new target for OA therapy, providing new research directions for the prevention and treatment of OA. |
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