| Objective:To investigate the expression of miRNA-330-3p in cartilage degeneration of temporomandibular joint osteoarthritis(TMJOA),and its effect on cartilage metabolism and downstream target regulation.Method:1.TMJOA modeling was performed in rats,and the expression of miRNA-330-3p in cartilage tissues was detected by real-time PCR.The mouse chondrocyte cell line ATDC5 was subjected to inflammation/stress stimulation,and the expression changes of miRNA-330-3p were detected by real-time PCR.2.After transfection of ATDC5 and human chondrocyte cell line SW1353 with mimics or inhibitors of miRNA-330-3p,the effects of miRNA-330-3p on chondrocyte proliferation and metabolism were explored by fluorescence staining as well as immunoblotting experiments.TMJOA was modeled in rats,and after intra-articular injection of miRNA-330-3p,changes in chondrocyte number and metabolism were detected by HE and immunohistochemical experiments to explore the role of miRNA-330-3p in the development of TMJOA.3.Bioinformatics analysis was used to predict and screen downstream targets that may be regulated by miRNA-330-3p.After inflammatory/stress stimulation of SW1353 and establishment of a rat TMJOA model,the expression changes of downstream target in chondrocytes and tissues were detected by Western blot and immunohistochemical experiments.After overexpression and inhibition of the target,its effect on chondrocyte metabolism was studied by Western blot.The expression as well as the regulatory relationship between miRNA-330-3p and downstream targets were investigated by transfection and real-time PCR assay as well as dual luciferase assay.Results:1.The expression of miRNA-330-3p in cartilage tissue of TMJOA rats decreased by about 3.7-fold(p=0.035).MiRNA-330-3p expression was significantly decreased in ATDC5 under inflammation/stress stimulation(p=0.001,p=0.000028).2.Overexpression of miRNA-330-3p can promote ATDC5 proliferation.In ATDC5 and SW1353,miRNA-330-3p can promote the increase of SOX9 protein,an important anabolic molecular marker,and the decrease of MMP13,a catabolic molecular marker.Inhibition of miRNA-330-3p showed the opposite result.After intra-articular injection of miRNA-330-3p agomir in TMJOA rats,the number of chondrocytes increased.Meanwhile SOX9 expression increased,and MMP13 and MMP3 expression decreased in the group,showing relatively mild cartilage destruction.3.Four prediction websites jointly predicted 388 miRNA-330-3p downstream target genes.KEGG pathway analysis showed that multiple predicted targets were involved in the WNT pathway,of which CTNNB1 was a classically involved molecule in this pathway.CTNNB1 expression was elevated in SW1353 in response to inflammatory/stress stimuli.In cartilage tissue from TMJOA rats,CTNNB1 expression was also elevated.After overexpression of CTNNB1 in SW1353,SOX9 decreased and MMP13 increased,which promoted the imbalance of chondrocyte metabolism.Inhibition of CTNNB1 had the opposite result.After overexpression of miRNA-330-3p in SW1353,CTNNB1 was significantly decreased at both mRNA and protein levels(P<0.05),and correspondingly,after inhibition of miRNA-330-3p,the expression of CTNNB1 was increased(P<0.05).There was a negative relationship between the expression levels of the two,however,there was not a direct binding relationship between miRNA-330-3p and CTNNB1.The regulatory mechanism of CTNNB1 by miRNA-330-3p still needs further study.Conclusions:1.The expression of miRNA-330-3p in TMJOA cartilage is decreased compared with normal cartilage.2.MiRNA-330-3p promotes SOX9 expression and inhibits MMP13 expression in chondrocytes.Intra-articular injection of miRNA-330-3p during the development of TMJOA in rats can reduce the destruction of cartilage.3.CTNNB1 is a downstream target of miRNA-330-3p.CTNNB1 inhibits the synthesis and promotes catabolism of chondrocytes.Its expression is regulated by miRNA-330-3p,but there is not a direct binding relationship,and the specific regulatory pathway still needs further study... |
PDF Full Text Request