The Role And Mechanism Of Circular RNA CircCDH13 As A Mediator In Osteoarthritis By Targeting MicroRNAs

Background:Osteoarthritis(OA),characterized by chronic inflammation,progressive destruction of articular cartilage,subchondral bone sclerosis and osteophyte formation,is one of the most prevalent age-related joint disorder in the elderly.Pain,stiffness and decreased mobility of the affected joints are early symptoms of OA,while OA at a late stage may result in loss of joint function and even disability.Consequently,OA greatly affects the physical health and quality of life of patients,and continuously increases the financial burden on the family and the society.Therefore,the prevention and treatment of OA has gradually become an urgent medical problem.Currently,there are no effective disease-modifying therapies available for OA in clinic,and most of them are symptomatic.Thus,joint replacement remains the primary treatment for patients with advanced OA.Thus,there are important clinical value and social significance to have an in-depth study of the pathological process of OA and its regulatory mechanism for the prevention and treatment of OA.Circular RNAs(circ RNAs)are a class of endogenous RNAs characterized by covalently closed loop structures with neither 5' to 3' polarity nor a polyadenylated tail.They are mainly produced by precursor m RNA back-splicing and widely expressed in mammals with stable,and tissue-specific patterns.Studies have shown that circ RNAs are critically involved in transcriptional and posttranscriptional gene expression regulation,and there has been accumulating evidence suggesting that circ RNAs have great potential in regulating normal cell growth and development,maintain stem cell characteristics,and play an important role in the development of various diseases,such as heart failure,disc degeneration,and carcinomas.However,although the role of circ RNA in the physiological functions and a variety of diseases has been revealed by a number of studies,little is known about their functions in the development and progression of OA.Objective and Methods:In our study,the expression of circ RNA in OA cartilage and normal cartilage was detected by microarray.The expression of Circ CDH13 were determined by PCR,and the cyclization of Circ CDH13 were verified by Sanger sequencing and Northern blot.Gain-of-function and loss-of-function approaches in vitro was used to demonstrate the participation of Circ CDH13 in OA.Apoptosis of chondrocytes was detected by Annexin V-FITC flow cytometry and TUNEL staining,and expression of MMP13,Col2a1,ADAMTS-5 and Aggrecan was detected by Western blot and immunofluorescence.The target relationship between Circ CDH13 and mi R-296-3p,mi R-296-3p and PTEN was predicted by bioinformatics,and verified by RNA pulldown and luciferase assay.To determine whether Circ CDH13 plays a role in OA progression in vivo,adeno-associated virus(AAV)was used to silence Circ CDH13 expression,and intra-articularly administered to destabilization of medial meniscus(DMM)-induced OA mice.Cartilage destruction was detected by safranin O and Fast Green staining,and all joints were evaluated by X-ray and micro-CT.The expressions of MMP13,Col2a1,ADAMTS-5 and Aggrecan in cartilage were detected by Western blot and immunohistochemical analysis.Results:Our study found that,compared with normal cartilage,a large number of circ RNAs were differentially expressed in human OA cartilage.The expression level of Circ CDH13 in OA cartilage and IL-1?-induced human chondrocytes was significantly up-regulated.The back-splice junction of Circ CDH13 identified by Sanger sequencing was consistent with that of the Circ Base.Northern blot confirmed that Circ CDH13 could be amplified by convergent primers and divergent primers in c DNA,and Circ CDH13 could resist digestion of RNase R.Thus,suggesting the circular structure of Circ CDH13.Gain-of-function and loss-of-function by RNA interference and overexpression was used to demonstrate the effect of Circ CDH13 on chondrocytes in OA using IL-1?-induced human chondrocytes.Compared with IL-1?-induced group,down-regulation of Circ CDH13 significantly decreased chondrocyte apoptosis rate,increased expression of Col2a1 and Aggrecan and decreased expression of MMP13 and ADAMTS-5.However,Circ CDH13 overexpressed in chondrocytes significantly increased the apoptosis rate of chondrocytes and expression levels of MMP13 and ADAMTS-5,while decreased Col2a1 and Aggrecan expression.Nuclear-cytoplasmic fractionation showed that Circ CDH13 was mainly localized in the cytoplasm of human chondrocyte.mi R-296-3p was predicted as target mi RNA of Circ CDH13 by Circ CDH13 pulldown,mi Randa and Target Scan,and luciferase assay showed that a significant decrease in fluorescence intensity in Circ CDH13-wt and mi R-296-3p co-transfected group,indicating that mi R-296-3p can directly bind to Circ CDH13.PCR showed that expression of mi R-296-3p was significantly down-regulated in OA cartilage.Further experiments showed that overexpression of mi R-296-3p in vitro inhibited IL-1? and Circ CDH13-induced apoptosis of chondrocytes,degradation of Col2a1 and Aggrecan,and synthesis of MMP13 and ADAMTS-5.PTEN was predicted as the target gene of mi R-296-3p by Target Scan,and luciferase assay found that the fluorescence intensity of PTEN 3'UTR wt and mi R-296-3p co-transfected group was significantly decreased,conforming that mi R-296-3p could directly bind to PTEN.Western blot analysis showed that PTEN was highly expressed in OA.Further experiments found that PTEN overexpression significantly increased the apoptosis rate of chondrocytes,expression of MMP13 and ADAMTS-5,and decreased the expression of Col2a1 and Aggrecan.However,mi R-296-3p could directly inhibit the expression of PTEN in chondrocytes,and subsequently inhibit PTEN-induced chondrocyte apoptosis,Col2a1 and Aggrecan degradation,as well as synthesis of MMP13 and ADAMTS-5.Moreover,we found that Circ CDH13 had a mouse homologous circ RNA(mmu?circ?0014630),and the sequences of Circ CDH13 in human was 89% the same as Circ CDH13 in mice.Circ CDH13 expression was inhibited by adeno-associated virus(AAV),and intra-articularly administered to DMM-induced OA mice.The results showed that,compared with the DMM group,cartilage destruction and osteophyte formation were reduced in the knee joint of mice from the Circ CDH13 silencing group.Western blot and immunohistochemical analysis showed that silencing of Circ CDH13 significantly increased the synthesis of extracellular matrix Col2a1 and Aggrecan,while decreased the secretion of matrix degrading enzymes MMP13 and ADAMTS-5 in chondrocytes.Conclusions:In summary,our study identified that a large number of circ RNAs were differentially expressed in OA cartilage tissue,among which Circ CDH13 was significantly up-regulated.Circ CDH13 could induce chondrocyte apoptosis,promote degradation of the extracellular matrix and synthesis of its degrading enzymes in human chondrocytes in vitro.Circ CDH3 is mainly located in the cytoplasm of chondrocytes,and can act as mi R-296-3p sponge to up-regulate the expression of PTEN,thus promoting the development of OA.Circ CDH13 is highly homologous in humans and mice.In vivo silencing of Circ CDH13 in mice can effectively alleviate DMM-induced cartilage destruction,osteophyte formation,extracellular matrix degradation and secretion of matrix degrading enzymes,and play a protective role in DMM-induced knee osteoarthritis in mice.