Research On Effects And Mechanism Of MiRNA-146a In Mechanic Injury Human Chondrocytes

miRNA-146a is one of the first identified miRNAs expressed differentially in os-teoarthritis (OA) cartilage.We found the expression of miRNA-146a was up-regulated inmechanical injury chondrocytes by using gene microarray.However, the role it plays inchondrocytes mechanic injury pathogenesis is not clear. The aim of this study is to identifya molecular target of miRNA-146a, thereby elucidating its function in chondrocytes dur-ing mechanical injury pathogenesis.The results of the miRNA microarray and gene microarray demonstrated that the ex-pression of miRNA-146a was up-regulated in mechanical injury chondrocytes. Mean-while, the bioinformatics analysis showed that SMAD4is a potential target gene of miR-NA-146a. Combined with the gene microarray report that the expression levels of SMAD4was down-regulated.We put forward the hypothesis that SMAD4mediates the injury ef-fects of miRNA-146a in mechanic injury chondrocytes.Objective:On the basis of this hypothesis, studies were carry out to demonstrate the role ofmiRNA-146a in chondrocytes mechanic injury pathogenesis and to investigate its mole- cular mechanism; to prove SMAD4is a direct target of miRNA-146a, and SMAD4me-diates the injury effects of miRNA-146a in mechanic injury chondrocytes.We hope thiscan provide some suggestion for the clinical treatment of Acute injury arthritis.Methods:1. Application MTT method to determine the cell grgen type,through the separation ofowth curve by the MTT,a blue-colla chondrocytes method, II aniline immunohis-tochemical stains to find differences of the original generations to cultivate, humannormal chondrocyteand and OA chondrocyteand2. To develop a set of cell pressure loading unit with proprietary intellectual propertyrights so as to provide a necessary tool for the research of cellular mechanics.We uti-lized the pressure loading machine to establish the mechanic injury cell model, whichto the largest extent simulated the the high energy mechanic injury.3. To screen the differentially expressed genes and miRNAs between normal chondro-cytes and mechanic injury chondrocytes.And to validate the differential expression ofmiRNA-146a in normal Chondrocytes and mechanic injury Chondrocytes by usingRealtime RT-PCR, and To validate the differential expression of SMAD4and VEGFby using Realtime RT-PCR and Western Blot analysis.4. To investigate the function of miRNA-146a, we transfected human normal Primarychondrocytes chemically synthesized double-stranded oligonucleotides (miRNA-146amimic) that mimic the endogenous mature miRNA-146a function and modified anti-sense oligonucleotides that inhibit miRNA-146a function (miRNA-146a inhibitor) orscramble oligonucleotides. The cells transfected with nothing (blank) were used ascontrol.5. To investigate the function of miRNA-146a affected cell survival, apoptosis, and cellcycle by using miRNA-146a mimics or inhibitor. and To validate the differential ex-pression of SMAD4and VEGF by using Realtime RT-PCR and Western Blot analysis.6. To investigate the function of miRNA-146a affected cell survival, apoptosis, and cellcycle by using miRNA-146a mimics or inhibitor in mechanic injury chondrocytes 7. To validate SMAD4is a direct target of miRNA-146a by using the dual luciferase re-porter gene system.Results:1. According to appraisal result of chondrocyte phenotypes and biological activity, en-sure the primary culture of the cells into active cells in good condition, to proceed tothe next step of the experiment.2. Firstly, chondrocytes were compressed by1Mpa to5, or10Mpa for10,30,60min.According to the result of MTT, we took the10Mpa for60min group as iMpact in-jury cell’s models.3. miRNA-146a is the first of miRNAs expressed differentially in in mechanic injurychondrocytes. And the bioinformatics analysis showed that SMAD4is a potential tar-get gene of miRNA-146a. Combined with the gene microarray report that the expres-sion levels of SMAD4was down-regulated.4. the expression of miRNA-146a was changed by the chemically synthesizeddouble-stranded oligonucleotides in human normal Primary chondrocytes5. In human normal Primary chondrocytes, the SMAD4expression was inhibited (pro-moted) after up-regulating (down-regulating) the miRNA-146a levels.And theVEGFexpression was inhibited (promoted) after down-regulating (up-regulating) the miR-NA-146a levels.6. Expression of miRNA-146a led to an increase of apoptosis rate in mechanic injurychondrocytes.7. SMAD4is a direct target of miRNA-146aConclusion:1. Pressure responsive miRNA-146a is overexpressed in an experimentally induced me-chanic injury model, accoMpanied byupregulation of VEGF and downregulation ofSmad4in vivo.2. SMAD4is a direct target of miRNA-146a3. miRNA-146a upregulation of VEGF is mediated by SMAD and can induce the apop- tosis in chondrocytes4. miRNA-146a may contribute to OA pathogenesis by increasing VEGF levels and byiMpairing the TGF-β signaling pathway through targeted inhibition of Smad4in car-tilage.