The Biological Effect Of MicroRNA-449a On Osteoarthritis And Mechanism Research

Part I:The research of differential expression of miR-449a between cartilage tissue specimens of osteoarthritis and of control group as well as the correlation between miR-449a and osteoarthritisObjective:In the analysis of the cause of joint abnormalities,osteoarthritis(OA)is the main factor and is also the main cause of disability in the elderly,which has brought huge social and economic burdens worldwide.The main pathological feature of OA is the persistent loss of cartilage due to an imbalance between the anabolic and catabolic activities of the extracellular matrix of the chondrocytes in the articular cartilage.MiR-449a has been reported to play an important role in a variety of diseases,but its role in the pathogenesis of OA has not been fully elucidated.Therefore,this part of the experiment intends to study the changes in the expression level of miR-449a in the OA process firstly.Methods:In terms of cartilage tissue samples,we first used the Kellgren-Lawrence(KL)grading scoring system for knee osteoarthritis to grade the severity of knee osteoarthritis.Human normal cartilage tissue samples were collected from 10 patients with traumatic amputation.Eight intermediate(KL grade III)OA articular cartilage tissue samples and 12 advanced(KL grade IV)OA articular cartilage tissue samples were collected from patients with OA who required total knee arthroplasty.The expression level of miR-449a in cartilage tissue samples was analyzed by qRT-PCR technique,and the changes of miR-449a in the development of OA were analyzed by statistical methods.Results:Compared with normal cartilage tissue,the expression level of miR-449a in OA cartilage tissue samples was significantly up-regulated,and the expression level of miR-449a in the late OA cartilage tissue samples was the highest.Conclusions:The expression level of miR-449a in OA cartilage tissue and normal cartilage tissue is different,and this difference is related to the development of OA,suggesting that miR-449a may play an important role in the pathogenesis of OA.Part ?:The biological role of miR-449a overexpression in the metabolism of extracellular matrix of chondrocytesObjective:The maintenance of normal metabolism of extracellular matrix in articular cartilage is crucial for the normal operation of cartilage function.The progressive degradation of cartilage extracellular matrix is the main pathological feature of the occurrence and development of OA.This part of our experiment is mainly to explore the role of miR-449a in the metabolism of extracellular matrix of chondrocytes.Methods:We used human chondrosarcoma cells(SW1353)instead of human normal chondrocytes to explore the function of miR-449a and we overexpressed and knocked down the miR-449a expression level by using miR-449a mimic and miR-449a inhibitor respectively.The overexpression and inhibition effects of miR-449a mimic and miR-449a inhibitor were firstly detected by qRT-PCR.And then qRT-PCR,western blot and enzyme-linked immunosorbent assay(ELISA)were used to detect type II collagen,aggrecan,matrix metalloproteinase 13(MMP13)and a disintegrin and metalloproteinase with thrombospondin motifs 4(ADAMTS4)in SW1353 cells.Results:qRT-PCR results indicated that miR-449a mimic and miR-449a inhibitor can significantly promote and inhibit the expression of miR-449a respectively.Furthermore,it was confirmed by qRT-PCR,western blot and ELISA that miR-449a overexpression can significantly promote the mRNA and protein expression levels of MMP13 and ADAMTS4,while inhibiting the expression levels of mRNA and protein of type ? collage and aggrecan,but SW1353 cells exhibited the opposite effect when treated with miR-449a inhibitor.Conclusions:These results indicate that in vitro,miR-449a overexpression can promote the catabolism level of extracellular matrix in SW1353 cells,but inhibit the anabolism of extracellular matrix,suggesting that miR-449a can be a target for OA treatment in the future.Part III:The research of miR-449a promoting the degradation of extracellular matrix of chondrocytes by targeting GDF5 geneObjective:It is well known that miRNAs are always activated by inhibiting their specific target genes when they exert a variety of physiological and pathological effects.Therefore,we next intended to verify the downstream specific target genes of miR-449a through a series of experiments,and confirmed the biological role of this target gene in miR-449a-mediated degeneration of chondrocytes' extracellular matrix.Methods:We first predicted the specific target genes of miR-449a by bioinformatics software,and detected the direct targeting relationship between miR-449a and GDF5 mRNA by dual luciferase reporter assay.And the correlation between the expression levels of GDF5 and miR-449 in cartilage tissue samples was detected by qRT-PCR.The role of GDF5 in the promotion of chondrocyte extracellular matrix degradation by miR-449a was verified by miR-449a inhibitor and interfering RNA targeting GDF5(siGDF5).Results:Our dual luciferase reporter assay showed that miR-449a can directly target the 3'-UTR of GDF5,and overexpression of miR-449a or inhibition of miR-449a expression can significantly inhibit or promote GDF5 mRNA and protein expression levels.Furthermore,we found a significant negative correlation between the expression levels of GDF5 and miR-449a in the collected cartilage tissue.These results indicate that miR-449a can directly inhibit GDF5.We furtheruse the siGDF5 to effectively interfere with the expression of GDF5.We observed that inhibition of miR-449a expression promotesextracellular matrixanabolism of SW1353cells and suppresses extracellular matrix catabolism of SW1353cells,and this effect can be reversed by inhibition of GDF5 expression levels.Conclusions:These results suggest that the expression of miR-449a is up-regulated in the progression of OA,and that the progressive degradation of the extracellular matrix of SW1353cells caused by miR-449a overexpression is achieved by direct targetingGDF5.