Knockout Of MiR-21-5p Alleviates Osteoarthritis And Regulates Interleukin-6-mediated Matrix Degradation In Mice Chondrocytes Through Enhancing Growth Differentiation Factor 5 Expression

Objective:Temporomandibular joint osteoarthritis(TMJ-OA)is characterized by the disruption of extracellular matrix homeostasis.The specific regulatory mechanism of MiR-21-5p,which is abnormally expressed in chondrocytes of osteoarthritis patients,remains unclear.The aim of the study was to determine whether MiR-21-5p participates in temporomandibular joint(TMJ-OA)pathogenesis by targeting GDF5.Methods:(1)The vivo experimentsThe disease state of TMJ-OA was simulated by the animal model of unilateral anterior crossbite(UAC)for 3 weeks.MiR-21-5p knockdown mice and wild-type mice were randomly divided into four groups:normal group(WT),knockout group(KO),normal intervention group(WT+OA)and knockout intervention group(KO+OA).The TMJ tissue samples were collected and observed to exhibit histopathological changes compared with control groups by immunohistochemistry,Safranin O staining and Toluidine blue staining,and the degree of cartilage lesions was quantified by the evaluation standard system of OARSI.When measuring the thickness of cartilage,the middle zone of cartilage was divided into five zones,and calculate the thickness of cartilage in each zone,and finally calculate the average value of five zones as the thickness of each group.The expression of type ? collagen and GDF-5 were observed by immunohistochemistry.(2)The vitro experimentsThe mandibular condylar chondrocytes(MCCs)were extracted from 3-4 weeks old C57 mice.MCCs were randomly divided into groups and transfected with MiR-21-5p(mimic),anti-MiR-21-5p(inhibitor),GDF5 siRNA,GV141-GDF5 plasmid and their negative control,and intervened with IL-6.The relationships among MiR-21,GDF-5 and TMJ-OA related gene expression were studied by immunofluorescence,Western blot,RT-PCR and Alcian Blue cell staining respectively.At the same time,luciferase test was performed to further prove the targeting relationship between MiR-21 and GDF-5.Results:(1)Compared with WT+OA group,the level of pathological changes was superior to that of KO+OA group;As for NC and KO group,there was no statistical difference between them.In WT+OA group,however,the score of cartilage lesions was significantly higher than that in KO+OA group.The results of cartilage staining showed that the optical density and cartilage thickness of WT+OA group were significantly lower than those of WT group;compared with KO group,the IOD and cartilage thickness of KO-OA group were significantly lower.Compared with WT-OA group,however,the cartilage thickness and IOD of KO-OA group were significantly higher.(2)The results of cell experiment showed that when the expression of MiR-21 in MCCs was up-regulated,the expression of GDF5,col-2 and AGG protein decreased significantly,while the expression of MMP3,9 and 13 protein increased significantly compared with that of the control group.When the expression of MiR-21 in MCCs was down regulated,it was reverse to the above trend.The results of luciferase showed that GDF-5 was the target gene of MiR-21.Furthermore,MMP-13 expression could be down regulated when GDF-5 was up-regulated;however,the opposite result would be produced when GDF-5 was silenced.When above groups were intervened with IL-6,the same trend appeared.Conclusions:MiR-21-5p,which serves as a critical regulator of GDF5 in chondrocytes,regulates MMP13 expression and is involved in matrix degradation,contributing to the progression of TMJ OA.