| Background:Osteoarthritis(OA)is a degenerative joint disease with articular cartilage degeneration,synovial inflammation,and osteophyte formation as the main pathological changes.Inhibition of chondrocyte autophagy in OA can promote chondrocyte apoptosis and increase degradation of extracellular matrix,thereby promoting the progress of OA.Ghrelin can promote cell autophagy and protect cartilage.However,in OA,how Ghrelin is regulated and the specific mechanism that affects autophagy is unclear.Circular RNA(circRNA)has been shown to participate in the regulation of gene expression.By targeting and binding microRNA(miRNA),miRNA inhibits the target gene.However,no studies have revealed the circRNA involved in the regulation of ghrelin expression.Obsjective:1.We establish OA rat model of chondrocyte and perform circRNA sequencing and mRNA sequencing.2.Based on ceRNA technology analysis for Ghrelin,we analyze and screen circRNAs related to Ghrelin and miRNAs with binding sites to circRNAs.3.Investigate the mechanism of circRNA/miRNA/Ghrelin regulatory network to regulate autophagy in osteoarthritis.Method:1.The chondrocytes of knee joint of SD rats were isolated and cultured,and the OA chondrocyte model was established by the intervention of 10 ng/ml IL-1β.2.The expression of ghrelin in chondrocytes was detected by qRT-PCR and Western blot,and the relationship between ghrelin and extracellular matrix Col2al,acan was also studied.3.The differentially expressed circRNAs and mRNAs were obtained by circRNA sequencing and mRNA sequencing.4.Based on the analysis of ceRNA technology targeting ghrelin,the circRNA and miRNA related to ghrelin were found.5.PCR experiments confirmed the target circRNA,target miRNA,and Sanger sequencing to verify the sequence of target circRNA.6.The double luciferase assay shown that the target miRNA was targeting to bind ghrelin mRNA and the target miRNA was targeting to bind the circRNA.7.The changes of Col2a1,Acan,ghrelin and autophagy related proteins in rat chondrocytes were measured by RNA transfection and co-overexpression of target circRNA and target miRNA.Result:1.After IL-1β interfered with chondrocytes for 12,24 hours,the expressions of Col2al,Acan,and Ghrelin decreased.2.CircRNA sequencing suggested that there were 107 circRNAs with up-regulated expression and 23 circRNAs with down-regulated expression in the rat chondrocytes of IL-1β intervention.3.mRNA sequencing indicated that there were 410 mRNAs with up-regulated expression and 322 mRNAs with down-regulated expression in the rat chondrocytes of IL-1β intervention.4.After rat chondrocytes treated with IL-1β for 12 and 24 hours,the expression of circSox6 and circPan3 was down-regulated by qRT-PCR detection.5.Bioinformatics analysis was used to predict the presence of binding sites between miR-667-5p and circPan3,and qRT-PCR was utilized to confirm that miR-667-5p was significantly up-regulated in OA chondrocytes.6.Through RNA transfection technology,it was proved that circPan3 can enhance the expression of Col2a1,Acan,Ghrelin,beclin1,LC3 Ⅱ and inhibit the expression of miR-667-5p,but miR-667-5p showed the opposite trend.7.During the dual luciferase reporter assay,it was confirmed that miR-667-5p can bind with the 3’ UTR(untranslated region)sequences of the Ghrl(Ghrelin)gene and the miR-667-5p was targeted by circPan3 in rat chondrocytes during OA development.Conclusion:This study shown that circPan3 was down-regulated in the OA rat chondrocytes intervened by IL-1β through RNA sequencing and bioinformatics analysis.And circPan3 promotes ghrelin synthesis and chondrocyte autophagy during rat osteoarthritis pathogenesis by sponging miR-667-5p. |
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