Circ＿PDE1C Downregulated MiR-224-5p Mediates OA Chondrocytes Apoptosis And Degradation

Background Osteoarthritis（OA）is a chronic bone and joint disease that often occurs in the middle-aged and elderly.Its main characteristics are articular cartilage degeneration and joint inflammation.Clinically,chronic pain and joint activity disorder are the main manifestations,which aggravate year by year,and finally seriously affect the quality of life.The pathogenesis of OA is complex while its etiology is unclear.At present,the occurrence of OA is considered to be related to various risk factors,including mechanical,genetic and physical factors.In clinical practice,OA can only be alleviated and improved,but can not be completely cured.At present,the clinical effect of routine joint preservation therapy is poor,and the risk of side effects is high.Therefore,clarifying the pathological mechanism of osteoarthritis may help to find new specific biomarkers,so as to develop effective treatment methods to control the symptoms of osteoarthritis.Circular RNA（circrna）is a special class of noncoding RNA molecules,which is composed of 3 ’and 5’ phosphodiester bonds formed by covalent connection.It is stable in expression and resistant to RNA exonuclease mediated degradation.In addition,circular RNA is highly stable and sensitive in body fluids and can be used for biochemical testing.Functionally,circRNA can attach to microRNA（miRNA）,bind to miRNA as a molecular sponge,affecting the corresponding messenger RNA（mRNA）,and finally regulate the expression of target genes.Previous studies have shown that circRNAs are involved in the occurrence and development of OA,suggesting that they may be used as prospective diagnostic markers and therapeutic targets of OA.Recently,the mechanism of circrna as a microRNA（miRNA）sponge has become a growing interest in the field of RNA research,indicating that circRNA affects downstream mRNA expression and participates in various diseases by strongly binding and inhibiting miRNA transcription.Through the technology of in-silico analysis and microarray,we can find the differential genes between the articular chondrocytes of patients with OA and normal articular chondrocytes through clinical specimens,so as to study the mechanism of differentially expressed circrna in OA.Objective We aimed to investigate changes in the expression of a specific circRNA,hsa_circ₀134111（circ_PDE1C）and predict its functions in OA.Methods 1.We clinically collected cartilage tissues from the knee of 49 patients with OA who underwent arthroplasty from Feb.2019 to Feb.2020,and 13 amputation patients.Safranin O and fast green staining were performed to determine proteoglycan changes,and the histological data were further analyzed by measuring the Mankin scores.Then the tissue was collected for circRNA microarray sequencing,from which we selected adifferentially expressed circRNAs.We further analyzed the expression of the top 5 differentially expressed circRNAs in the cartilage tissues finding that the expression of circ₀134111（circ_PDE1C）was significantly higher in OA patients.A rat model of OA was constructed to detect circ_PDE1C expression in knee joint tissues.Subsequently,CHON-001 chondrocytes were treated with IL-1β to mimic OA in vitro.Infect cells using lentiviral vectors to knockdown or overexpress circ_PDE1C,the expression of cartilage degradation markers and apoptosis markers were detected using RT-q PCR,and the apoptosis of chondrocytes was detected by flow cytometry.Finally,FISH technology was used to Locate the circ_PDE1C in chondrocytes.2.We downloaded the miRNA expression microarray GSE105027 which contains knee tissues from 12 normal subjects and 24 OA patients who underwent meniscectomy,and the mRNA expression microarray GSE117999 which includes joint tissues from 12 patients who underwent meniscectomy and 12 OA patients from the Gene Expression Omnibus（GEO）database（https://www.ncbi.nlm.nih.gov/ geo/）.We screened differentially expressed miRNAs and mRNAs by setting Log Fold Change >1（for miRNA）or 2（for mRNA）and adj p value <0.05 as screening thresholds.Subsequently,we predicted downstream miRNAs of circ_PDE1C via circ Bank（http:// www.circbank.cn/）and RNA hybrid website（http://alk.ibms.sinica.edu.tw/cgi-bin /RNAhybrid/RNAhybrid.cgi）and screened against differentially expressed miRNAs in the GSE105027 microarray.We then performed an RNA pull-down analysis using biotinylation-labeled circ_PDE1C probes and found significant differences among three miRNAs（miR-103a-3p,miR-224-5p,and miR-320-3p）,Subsequently,we further analyzed the expression of miR-103a-3p,miR-224-5p,and miR-320-3p in OA patients.The expression of miR-224-5p had the highest correlation with the Mankin score of OA patients.Thus,we investigated the binding relationship between miR-224-5p and circ_PDE1C.The mRNA downstream of miR-224-5p was then predicted by Target Scan（http://www.targetscan.org/vert₇2/）and RNA Hybrid website and screened against the differentially expressed genes in the GSE117999 microarray.Finally,the signaling pathways enriched in the differentially expressed genes in the GSE117999 microarray were analyzed by gene set enrichment analysis（GSEA）.3.Dual-Luciferase reporter assay was used to test the binding relationship between circ_PDE1C and miR-224-5p and CCL2.we further transfected miR-224 inhibitor into CHON-001 cells with shcirc_PDE1C and miR-224 mimic into CHON-001 cells with circ_PDE1C-Oe.The effect of miR-224-5p expression on CCL2 and IL-1 β Induced chondrocyte degradation and apoptosis was deceted by RT-q PCR.Finally,the effect of CCL2 expression on JAK2 and STAT3 phosphorylation was studied,and the proportion of phos-stat3 in necleus was detected by immunofluorescence.Results 1.Through microarray analysis of clinical samples,different circRNAs were found.Compared with amputated patients,circ_PDE1C was significantly overexpressed in knee cartilage of OA patients（P < 0.05）,and its expression level was positively correlated with Mankin score.At the same time,animal experiments found that circ_PDE1C was also significantly overexpressed in the cartilage of OA rats.IL-1 β was used to induce CHON001 cells to construct in vitromodel of OA chondrocytes.we used Alcian blue and Toluidine blue staining and found a significant increase in glycosaminoglycan content in cells after knocking down the expression of circ_PDE1C.Loss of circ_PDE1C significantly inhibited ECM degradation after IL-1β induction by upregulating Aggrecan and Collagen II levels and downregulating MMP-9 and MMP-13 levels.Caspase-3 activity was also significantly inhibited and the proportion of apoptosis was similarly reduced after circ_PDE1C knockdown.However,the exact opposite results were observed in cells overexpressing circ_PDE1C.2.We intersected regulatory miRNAs predicted by circ Bank and RNA Hybrid with miRNAs downregulated in GSE105027,and we screened eight miRNAs.We then performed an RNA pull-down analysis using biotinylation-labeled circ_PDE1C probes and found significant differences among three miRNAs（miR-103a-3p,miR-224-5p,and miR-320-3p）.Subsequently,we further analyzed the expression of miR-103a-3p,miR-224-5p,and miR-320-3p in OA patients.Their expressions in OA patients were much lower than those in non-OA subjects.However,the expression of miR-224-5p had the highest correlation with the Mankin score of OA patients.Same way of using Bioinformatics analysis to find the target mRNA CCL2 and JAK/STAT signaling pathway.3.A significant decline in luciferase activity in cells co-transfected with miR-224 and circ_PDE1C-wt was noticed,as well as in cells co-transfected with miR-224 and CCL2-wt.Cell experiment in vitroshowed that circ_PDE1C can target mir-224-5p,overexpression of mir-224-5p can inhibit the degradation and apoptosis of chondrocytes.Mir-224-5p also targets CCL2,which activates JAK2 / STAT signaling pathway,thereby promoting cartilage degradation and apoptosis.Conclusion circ_PDE1C participates in the mediation of chondrocyte degradation and apoptosis by directly interacting with the miR-224-5p/CCL2 axis and also indirectly by activating the JAK/STAT3 pathway.The present study,therefore,suggests that circ_PDE1C is a key modulator in maintaining the chondrocyte phenotype.However,OA still represents a multifactorial condition,and more researches are needed to explore other possible molecular mechanisms.