The Role Of Circ?0001103 In Suppressing The Development Of Osteoarthritis By Regulating The MiR-375/SIRT1 Signal Axis

Osteoarthritis(OA)is a kind of complex joint degenerative disease and is a major cause of joint dysfunction.OA is characterized by progressive destruction of articular cartilage,attributed to a variety of factors,including abnormal gene expression,oxidative stress,abnormal chondrocyte apoptosis,inflammation,and cartilage matrix degradation,etc.OA is a heterogeneous disease with a variety of molecular and clinical phenotypes.Currently,the pathogenesis of OA has not been fully clarified.Therefore,it is of great significance to explore the key molecules and signal pathway in the development of OA to offer insight into OA mechanismCircRNAs is a type of noncoding RNAs.Previous studies have shown that circRNAs were abnormally expressed in OA articular cartilage and played a role in the progression of OA by regulating cartilage matrix degradation,inflammation,and chondrocyte apoptosis.It is believed that circRNAs could be used as new diagnostic markers and therapeutic targets for OA.Expression profile of circRNA in OA cartilage tissues is different from normal tissues.Among these circRNA,circ₀001103 was down-regulated in OA articular cartilage,suggesting that circ₀001103 might be involved in the regulation of OA pathological mechanism.However,the mechanism of circ₀001103 in OA is not illustrated,so this study intends to explore the function and regulation mechanism of circ₀001103 in the occurrence and development of OA.Previous studies have shown that circRNA can act as microRNAs(miRNAs)sponges and down-regulate miRNAs expression,thus engaging in a variety of pathogenesis.MiRNAs is another type of noncoding RNAs with extensive regulatory roles in human diseases.Previous studies reported that miR-375 may be an important inflammatory mediator,because miR-375 plays an important role in a variety of inflammatory diseases,such as inflammatory bowel disease,fatty liver,myocardial infarction,etc.However,the mechanism of miR-375 in OA development has not been studied.In addition,Sirtuin 1(SIRT1)belongs to the Sirtuin family of nicotinamide adenine dinucleotide(NAD)dependent deacetylases.It has been reported that SIRT1 can interact with NF-?B,p53 and other transcription factors to participate in chondrogenesis and chondrogenic differentiation.The mechanism of SIRT1 in OA is still unclear so far and needs further study.This study aims to study the role of circ₀001103 in the development of OA and whether participating in regulating the miR-375/SIRT1 signal axis,providing new ideas for elucidating the OA mechanism.This study is divided into three parts:the first part is to study the correlation analysis between circ₀001103 and inflammatory response and cartilage metabolism in OA articular cartilage;the second part is to study the function of circ₀001103 in OA cell model;in the third part,we study the role of circ₀001103 in suppressing the development of osteoarthritis by regulating the miR375/SIRT1 signal axis.Part ?:Correlation analysis of circ₀001103 with inflammation and cartilage matrix metabolism in OA articular cartilageObjective:To investigate the expression of circ₀001103 in OA articular cartilage and its correlation with inflammatory response and cartilage matrix metabolism.Methods:(1)The articular cartilage tissues by 30 OA patients in our hospital were selected as the experimental group and 10 normal articular cartilage tissues were selected as the control group.The expression of inflammatory factors(IL-8,IL-6,TNFa)and matrix degradation factors(COL2A1,matrix degradation enzyme ADAMTS4)in normal articular cartilage and OA articular cartilage were detected by qRT-PCR and Western blot respectively.(2)qRT-PCR was used to detect the expression of circ₀001103 in normal articular cartilage and OA articular cartilage.The correlation between the expression of circ₀001103 and IL-8,IL-6,TNF-?,COL2A1 and ADAMTS4 in OA articular cartilage tissues was analyzed by Pearson method.Results:(1)The expression of inflammatory factors including IL-8,IL-6 and TNF-? in OA articular cartilage was increased,and the expression of ADAMTS4(matrix degradation enzyme encoding gene)was increased,while the expression of COL2A1(encoding gene of type ? collagen)was decreased(P<0.05).(2)The expression of circ₀001103 in OA articular cartilage was down-regulated in comparison with normal cartilage(P<0.05).(3)the expression of circ₀001103 was negatively correlated with the expression of inflammatory factors and matrix degradation enzymes,but positively correlated with the gene encoding type ? collagen:circ₀001103 was negatively correlated with IL-8(r=-0.5743,P=0.009);circ₀001103 was negatively correlated with IL-6(r=-0.5414,P=0.0020).circ₀001103 was negatively correlated with TNF-?(r=-0.6014,P=0.0004).circ₀001103 was negatively correlated with ADAMTS4(r=-0.5709,P=0.0010).circ₀001103 was positively correlated with COL2A1(r=0.5018,P=0.0047).Conclusion:Circ₀001103 was down-regulated in OA articular cartilage tissue and was closely related to the inflammation and cartilage matrix metabolism.Part ?:The function study of circ₀001103 in OA cell modelObjectives:To investigate the effects of circ₀001103 on chondrocyte proliferation,apoptosis,inflammation,and cartilage matrix metabolism in in IL-1?treated chondrocytes.Methods:(1)Establishment of OA cell model:Primary chondrocytes were treated with medium containing 10 ng/mL IL-1? for 24 h,and were denoted as IL-1? group(experimental group).Chondrocytes cultured in ordinary medium were taken as control group.(2)Establishment of cell model with circ₀001103 overexpression:The pCD5ciR and circ₀001103 overexpression vectors were transfected into chondrocytes and then treated with the medium containing 10 ng/mL IL-1? for 24 h.The chondrocytes were divided into control group(IL-1?+pCD5-ciR group)and overexpression group(IL-1?+circ₀001103 group).(3)The expression of circ₀001103 in chondrocytes induced by IL-1? was detected by qRT-PCR.The activity of chondrocytes was evaluated by MTT assay.Cell proliferation was examined by EdU assay.Cell apoptosis rate was assessed by flow cytometry.The gene and protein expression levels of IL-8,IL-6,TNF-?,COL2A1,ADAMTS4,pro-apoptotic gene BAX and anti-apoptotic gene BCL2 were detected by qRT-PCR and Western blot respectively.Results:(1)The expression of circ₀001103 was down-regulated in IL-1?induced chondrocytes(P<0.05);(2)Compared with the control group,IL-1? treatment reduced chondrocyte viability,proliferation ability,COL2A1(type ? collagen encoding gene)expression(P<0.05),and increased inflammatory factors IL-8,IL-6,TNF-?,matrix degradation enzyme ADAMTS4 expression(P<0.05).In addition,apoptosis rate was increased,with up-regulation of pro-apoptotic gene BAX and down-regulation of anti-apoptotic gene BCL2(P<0.05).(3)Compared with the control group(IL-1?+pCD5-ciR),the chondrocyte viability,proliferation and the expression of COL2A1 were up-regulated in circ₀001103 overexpression group(IL-1?+circ₀001103 group)(P<0.05).The expression of IL-8,IL-6,TNF-? and ADAMTS4 were decreased(P<0.05).In addition,apoptosis rate was decreased in circ₀001103 overexpression group(P<0.05).The expression of proapoptotic gene BAX was decreased while the expression of anti-apoptotic gene BCL2 was increased in circ₀001103 overexpression group(P<0.05).Conclusion:Overexpression of circ₀001103 can enhance chondrocyte activity and promote cell proliferation in IL-1?-induced OA cell model,and inhibit cell apoptosis,inflammation and cartilage matrix degradation metabolism.Part III:circ₀001103 inhibits the mechanism of OA process bytargeting the miR-375/SIRT1 axisObjective:To explore the possible mechanism of circ₀001103 in regulating IL1?-induced chondrocyte proliferation,apoptosis,inflammation and cartilage degeneration.Methods:(1)Screening the downstream targets of circ₀001103:The targeting relationship between circ₀001103 and miR-375 and the targeting relationship between miR-375 and the downstream gene SIRT1 were verified by dual luciferase reporter assay and RNA binding protein immunoprecipitation(RIP)assay.(2)qRT-PCR and Western blot were used to detect the expression of miR-375 and SIRT1 in normal and OA articular cartilage and IL-1? induced chondrocytes respectively.The correlation between miR-375 and circ₀001103 expression and the correlation between miR-375 and SIRT1 expression in OA articular cartilage tissues was analyzed by Pearson method.(3)Experimental grouping:circ₀001103 and miR-con,circ₀001103 overexpression vector and miR-375 mimics,in-miR-con,in-miR-375,in-miR-375 and si-con,in-miR-375 and si-SIRT1 was transfected into chondrocytes and treated with IL-1? at a concentration of 10 ng/mL for 24 h,which were marked as circ₀001103+miR-con group,circ₀001103+miR-375 group,IL-1?+pCD5-ciR group,IL-1?+circ₀001103 group,IL-1?+circ₀001103+miR-con group,IL1?+circ₀001103+miR-375 group.The MTT assay was used to detect cell viability.EdU test was used to evaluate cell proliferation ability.Flow cytometry was used to assess the apoptosis rate.qRT-PCR and Western blot were used to detect the expression of IL-8,IL-6,TNF-?,COL2A1,ADAMTS4,BAX,and BCL2.Results:(1)Circ₀001103 could target miR-375,and miR-375 could target SIRT1.Compared with normal cartilage tissue,the expression of miR-375 in osteoarthritis cartilage tissue was increased(P<0.05),while the expression of SIRT1 was decreased(P<0.05).(2)Circ₀001103 was negatively correlated with miR-375(r=-0.4691,P=0.089),miR-375 was negatively correlated with SIRT1(r=-0.522,P=0.0031).Compared with the control group,the expression of miR-375 in chondrocytes induced by IL-1? in the IL-1? group was increased(P<0.05),while the expression of SIRT1 was decreased(P<0.05).circ₀001103 could negatively regulate the expression of miR-375 and positively regulate the expression of SIRT1.(3)Compared with IL-1?+circ₀001103+miR-con group,the cell viability of IL1?+circ₀001103+miR-375 group was decreased(P<0.05),the rate of EdU positive cells was decreased(P<0.05),and the expression of COL2A1 and BCL2 were decreased(P<0.05),while the expression of IL-8,IL-6,TNF-?,ADAMTS4,BAX were increased(P<0.05),and the rate of cell apoptosis was increased(P<0.05).Compared with IL-1?+in-miR-con group,the cell viability of IL-1?+in-miR-375 group was increased(P<0.05),the rate of EdU positive cells was increased(P<0.05),and the expression of COL2A1 and BCL2 were increased(P<0.05),while the expression of IL-8,IL-6,TNF-?,ADAMTS4,and BAX were decreased(P<0.05),and the rate of apoptosis was decreased(P<0.05).(4)Compared with IL-1?+in-miR-375+si-con group,the cell viability of IL1?+in-miR-375+si-SIRT1 group was decreased(P<0.05),and the rate of EdU positive cells was decreased(P<0.05),the expression of COL2A1 and BCL2 were decreased(P<0.05),while the expression of IL-8,IL-6,TNF-?,ADAMTS4,and BAX were increased(P<0.05),and the rate of apoptosis was increased(P<0.05).Conclusion:The overexpression of circ₀001103 could up-regulate the expression of SIRT1 by targeting the expression of miR-375 to enhance the viability of chondrocytes induced by IL-1? and promote cell proliferation,and could inhibit cell inflammation and cartilage degradation,as well as inhibit cell apoptosis.