| ObjectiveThis study aims to explore the expression level of miR-197 in cartilage tissue of patients with OA,and the effects of miR-197 on the proliferation,migration and secretion of inflammatory factors of chondrocytes,and key genes targeted by miR-197 in chondrocytes.This study aimed to find and explore the pathogenesis and treatment of osteoarthritis to provide research support.Method1.Expression and correlation analysis of miR-197 and EIF4G2 in cartilage of patients with osteoarthritisArticular cartilage was collected from 41 OA patients undergoing total knee arthroplasty,including 25 males and 16 females.In the control group,articular cartilage was collected from 29 knee trauma patients undergoing knee cartilage repair surgery,including 19 men and 10 women.Record and analyze patient medical records.The mRNA expression levels of miR-197 and e IF4G2 in cartilage tissue of 2 groups were compared by RT-qPCR.2.MiR-197 regulates chondrocyte proliferation,migration and inflammatory responseLipofectamin 3000 was used to import exogenous miR-197 mimics,mimics control,miR-197 inhibitor and inhibitor control into the primary cultured chondrocytes of OA patients.3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)method was used to detect the proliferation ability of cells in different groups.Transwell assay was used to evaluate the migration ability of cells in different treatment groups.Enzyme-linked immunosorbent assay(ELISA)was used to detect the expression levels of IL-1β,TNFα and IL-6 in cultured chondrocytes.3.EIF4G2 is a target gene of miR-197 regulating chondrocyte behavior Intertargeting between miR-197 and EIF4G2 was predicted using Target Scan Human 7.2,miRBase,and microRNA databases.Exogenous miR-197 mimics,mimics control,miR-197 inhibitor,inhibitor control pc DNA3.1-EIF4G2 vectors were transfected into the cultured primary chondrocytes with Lipofectamin 3000.The mRNA and protein expression levels of EIF4G2 in miR-197 regulated cells were detected by RT-qPCR and Western Blotting.Dual luciferase reporter assay was used to detect the binding of miR-197 to the UTR region of EIF4G2 mRNA at the 3’ end,thereby inhibiting mRNA expression and translation.MTT was used to detect cell proliferation ability in different groups.Transwell assay was used to evaluate the migration ability of cells in different treatment groups.The expression levels of IL-1β,TNFα and IL-6 in cultured chondrocytes were detected by ELISA.Results1.The general condition of patients with osteoarthritis group and control group was comparedA total of 41 patients with osteoarthritis of the knee(OA group,n=41)who underwent total knee arthroplasty(OA group,n=41)and 29 trauma patients without a history of OA who underwent knee cartilage repair(control group,n=29)were enrolled in this study.Patients in OA group ranged in age from 42-67 years old,with an average age of 60.12 ± 9.20 years,including 25 males and 16 females.The course of disease ranged from 5 to 19 years,with an average course of 7.82±5.18 years.The control group consisted of 29 trauma patients with no history of osteoarthritis who received knee cartilage repair surgery,aged 34-72 years old,with an average age of 58.63 ± 11.03 years old.There were 19 males and 10 females.There was no significant difference in gender and age between OA group and control group(P>0.05).2.Expression of miR-197 and EIF4G2 in articular cartilage in patients with osteoarthritisThe expression of miR-197 in cartilage of OA patients was significantly lower than that of normal cartilage of control group(P<0.001).However,EIF4G2 mRNA expression was significantly increased in cartilage from patients with OA compared to normal cartilage from control group(Fig.1B,P<0.001).Since the expression trend of miR-197 and EIF4G2 mRNA in articulation cartilage was opposite,Spearman’s correlation analysis further analyzed the correlation between miR-197 and EIF4G2 expression,which showed that the expression of miR-197 and EIF4G2 was negatively correlated(Y =-2.796*X + 13.21,r=-0.75,P<0.001).3.MiR-197 promotes the proliferation and migration of chondrocytes and inhibit the expression of inflammatory cytokinesIn chondrocytes cultured in vitro,miR-197 mimics and miR-197 inhibitor transfection significantly regulated the level of miR-197 in chondrocytes.MiR-197 mimics can promote chondrocyte proliferation,migration and inhibit the expression of inflammatory factors(TNFα,IL-1β and IL-6).MiR-197 inhibitor inhibited the proliferation and migration of chondrocytes and the expression of inflammatory factors(TNFα,IL-1β and IL-6).4.MiR-197 regulates proliferation,migration and inflammation by targeting EIF4G2 in chondrocytesThe dual luciferase reporter assay confirmed that miR-197 mimics could significantly inhibit the activity of dual luciferase,while miR-197 inhibitor could significantly promote the activity of dual luciferase.It is suggested that miR-197 can directly bind to the 3 ’non-coding region of EIF4G2 mRNA.Regulation of miR-197 in chondrocytes cultured in vitro can alter the expression levels of EIF4G2 mRNA and protein.Compared with the control vector,EIF4G2 overexpression vector could significantly increase the expression of EIF4G2 mRNA and protein in chondrocytes,inhibit the effect of miR-197 on the proliferation and migration of chondrocytes,and inhibit the inhibition of inflammatory cytokines(TNFα,IL-1β and IL-6)by miR-197.Conclusion:1.The expressions of miR-197 were down-regulated in articular cartilage tissue of osteoarthritis,and EIF4G2 were up-regulated,and the expression levels of the two were negatively correlated.2.Bioinformatics prediction,RT-qPCR,Western blotting and dual luciferase reporter assay confirmed that EIF4G2 was the target gene of miR-197.3.MiR-197 can promote the proliferation and migration of chondrocytes and inhibit the secretion of TNFα,IL-1β and IL-6.4.EIF4G2 is the downstream target gene of miR-197 in regulating the proliferation and migration of chondrocytes and inhibiting the secretion of TNFα,IL-1β and IL-6.Overexpression of EIF4G2 can inhibit the function of miR-197 to a certain extent. |
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