| BackgroundThe SDF-1/CXCR4(stromal cell-derived factor-1/CXC motif chemokine receptor type 4)axis is closely related to the development of OA,more and more evidence indicates that miRNA abnormal expression plays a key role in the pathological process of OA regulatory role.Therefore exploring miRNAs which involved in regulating SDF-1/CXCR4 axis function is important to further study the occurrence and development of OA.Purpose:(1)Identify the differential expression of miRNAs in knee cartilage tissue of OA patients,and determine the miRNAs which significantly differently expressed and associated with the SDF-1/CXCR4 axis;(2)Investigate miRNAs that have a targeted relationship with CXCR4,and study the role of this target gene in the apoptosis and degeneration of articular chondrocytes;(3)To explore the downstream signaling pathway of target miRNA-mediated cartilage apoptosis and degeneration.Method:(1)miRNA differential expression analysis in knee joint cartilage tissue of OA patients:Adult knee cartilage tissue(n=9)amputated by traumatic surgery and knee cartilage tissue(n=9)of OA patients who underwent knee replacement surgery were selected,and knee joints were collected during knee replacement and amputation Cartilage tissue,chondrocytes are extracted by two-step enzymatic digestion and primary cell culture is used for later use.Subsequently,SDF-1 was added to primary OA chondrocytes to induce primary chondrocytes for 24 hours.Chondrocytes were immunohistochemically stained to detect the expression of CXCR4 in primary chondrocytes induced by SDF-1.The differences of miRNA expression between the two groups of patients were analyzed at the same time,the target genes were predicted and the candidate target genes were screened out.(2)Targeting CXCR4 to regulate apoptosis and degeneration of OA chondrocytes;① SDF-1 induced primary chondrocytes from normal adults and OA patients for 24 hours,and Q-PCR was used to verify the significant abnormal expression candidate gene screened by the gene chip in cartilage tissue in two groups of chondrocytes.Subsequently,a plasmid containing candidate genes and target gene suppressors was constructed,and plasmid transfection was performed in chondrocytes of primary OA patients.After plasmid transfection,chondrocytes were induced with SDF-1 for 24 hours,and then real-time PCR and Western blot were performed to detect the changes in CXCR4 gene and protein expression to further screen the target genes;② Construct wild-type and mutant CXCR4 3’-UTR luciferase reporter vector,detecting the effect of target gene on wild-type and mutant CXCR4 3’-UTR luciferase activity,and clarifying whether the target candidate target gene has a targeted binding relationship with CXCR4;The target,target gene inhibitor and CXCR4 plasmids,as well as blank plasmids containing no genes,were transfected into chondrocytes of OA patients as experimental and control groups,respectively.Chondrocytes were transfected 48h after induction with SDF-1 for 24h.Flow cytometry was used to detect chondrocyte apoptosis,and Western blot was used to detect apoptosis-related factor proteins(Bax,Bcl-2,cleaved caspase-3,cleaved PARP)expression;④ After transfection cells were induced by adding SDF-1 for 24 hours,the supernatant was taken,and the contents of IL-1β,IL-10 and TNF-α were detected by ELISA.Expression level of 13;⑤ After induction with SDF-1 for 24 hours in transfected cells,immunofluorescent labeling of collagen typeⅡ(Collagen)and aggrecan(Aggrecan)in chondrocytes,and fluorescence intensity detection using a fluorescence microplate reader,At the same time,the miRNA and protein expression levels were measured.(3)Analysis of downstream signaling pathways of target miRNA-mediated cartilage apoptosis and degeneration:The plasmids carrying target gene mimics,target gene inhibitors and CXCR4,and blank plasmids containing no genes were constructed and transfected into chondrocytes of OA patients as experimental and control groups,respectively.Chondrocytes were transfected 48 hours after SDF-1 was added to induce them for 24 hours.The KEGG pathway was used to analyze the target gene action pathway to find the signal pathway most closely related to the target miRNA.Western blot was used to detect changes in the phosphorylation level of the pathway proteinResults:(1)Compared with the normal group,a total of 70 miRNAs in knee joint cartilage tissues of OA patients showed significant differences.Among them,53 miRNAs were significantly down-regulated and 17 miRNAs were significantly up-regulated(multiple folds greater than 2,P<0.05)..A total of 4 genes(miR-150-5p,miR-142-5p,miR-513a-5p,miR-622)among the top 15 genes with differential expression changes were predicted to have a targeted relationship with CXCR4 through the target gene,and CXCR4 was in SDF Chondrocytes in the-1 induction group showed higher expression than the blank control group.(2)The results of PCR and Western verification of candidate target genes in OA chondrocytes are consistent with the results of gene chip detection in cartilage tissue of OA patients and normal adults.Among them,miR-150-5p,miR-142-5p,miR-513a-5p,miR-622 showed differential expression in chondrocytes induced by high concentration of SDF-1:miR-142-5p,miR-622 were significantly up-regulated(P<0.05),miR-150-5p,miR-513a-5p showed an up-regulation trend,but no statistical significance(P>0.05).MiR-142-5p over expression has a significant inhibitory effect on CXCR4 expression.The dual luciferase report shows that the mutant CXCR43’-UTR reporter is not sensitive to the interaction with miR-142-5p,while the wild-type CXCR4 3 ’-UTR reporter molecule can bind to miR-142-5p well,and the fluorescence intensity is obviously weakened.This result indicates that miR-142-5p and CXCR4 are target genes for each other.Flow cytometry showed that the miR-142-5p overexpression group significantly reduced the rate of chondrocyte apoptosis,lowered the mRNA and protein expression levels of apoptosis-related proteins Bax,cleaved caspase-3 and cleaved PARP,but decreased with chondrocytes.Bcl-2 expression that was negatively correlated was significantly up-regulated;ELISA results showed that the expression levels of IL-1β,TNF-α in miR-142-5p overexpression group decreased,IL-10 expression levels increased,and MMP-The expression level of 13 decreased significantly;the type II collagen and proteoglycan content in chondrocytes in the miR-142-5p overexpression group increased significantly,while the CXCR4 overexpression group had the opposite.When miR-142-5p and CXCR4 At the same time,overexpression of miR-142-5p reversed the negative regulation effect of CXCR4 overexpression on chondrocyte apoptosis and cartilage degeneration.(3)MiR-142-5p can significantly inhibit the activation of MAPK pathway in OA chondrocytes after SDF-1 induction after transfection into plasmids carrying miR-142-5p in OA chondrocytes.When CXCR4 and miR-142-5p were overexpressed in OA chondrocytes at the same time,the MAPK pathway activation caused by CXCR4 overexpression in OA chondrocytes was significantly inhibited by miR-142-5p.Conclusion:(1)The expression of CXCR4 in knee cartilage of OA patients was higher than that of normal people,and the expression of miR-142-5p was significantly increased.(2)miR-142-5p and CXCR4 are the target genes of each other.MiR-142-5p can inhibit the expression of CXCR4 in chondrocytes,induce apoptosis of chondrocytes and reduce the secretion of inflammation-related factors induced by high concentration of SDF-1.At the same time,it can reduce the degradation of cartilage matrix type II collagen and aggrecan.(3)miR-142-5p inhibits chondrocyte apoptosis and attenuates cartilage degeneration through MAPK pathway. |
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