Study On The Role Of TGFβ/Smad3-mediated MicroRNA-140 In Regulating ADAMTS-5 Expression In Early Cartilage Degradation Of OA

Background/Objective: Transforming growth factor receptor(TGFβ)can affect the metabolic homeostasis and structural integrity of articular cartilage,and microRNA-140 and ADAMTS-5 play a very important role in the metabolism of articular cartilage.This study was to investigate the effect of TGFβ/Smad3 on microRNA-140 regulating the expression of ADAMTS-5 in OA chondrocytes.Methods: Chondrocytes of the knee joint of SD rats(2-3 weeks)were extracted by enzyme digestion in vitro.The basic morphology of chondrocytes and toluidine blue staining of chondrocytes were observed under microscope to identify the chondrocytes.It is induced to become OA chondrocytes by inflammation of IL-1.First of all,mi RNA-140 mimics and microRNA-140 are transfected by plasmid in the OA cartilage primer,and ADAMTS-5inhibitor is used as the positive control group,blank group(standard medium)and empty plasmid group to detect the mRNA expression of ADAMTS-5 by real-time PCR at 24,48 and72 hours,respectively.Meanwhile,the expression of ADAMTS-5 was detected by Western blot at 72 hours to verify the regulation of microRNA-140 on ADAMTS-5.Then,the mRNA expression of microRNA-140 and ADAMTS-5 was detected by Real-time PCR at 24,48 and72 hours by adding Smad3 agonist(recombinant human Smad3 protein),Smad3 inhibitor(SIS3),positive control(ADAMTS-5 inhibitor)and negative control(standard medium)into OA chondral cells.Meanwhile,the expression of ADAMTS-5 was detected by Western blot at72 h to verify the effect of Smad3 on microRNA-140 regulating the expression of ADAMTS-5 in OA chondrocytes.Results: 1.The chondrocytes were irregular polygonal,the cytoplasm was light blue and the nucleus was dark blue.2.Real-time PCR showed that the microRNA-140 mimics group significantly decreased the expression of ADAMTS-5 mRNA in OA chondrocytes compared with the microRNA-140 inhibit group,the positive control group(ADAMTS-5 inhibitor),the blank group(standard medium)and the empty plasmid group(P < 0.05).In addition,the72-hour Western blot showed that the microRNA-140 mimics group significantly decreased the expression of ADAMTS-5 compared with the microRNA-140 inhibit group,the positive control group(ADAMTS-5 inhibitor),the blank group(standard medium)and the empty plasmid group(P < 0.05).3.Real-time PCR showed that the expression of microRNA-140 in OA chondrocytes by Smad3 inhibitor(SIS3)was significantly higher than that by Smad3agonist(recombinant human Smad3 protein),positive control(ADAMTS-5 inhibitor)and negative control(standard medium)at 48 and 72h(P < 0.05),while the expression of ADAMTS-5 mRNA was significantly decreased(P < 0.05).In addition,Smad3 inhibitor(SIS3)significantly decreased the expression of ADAMTS-5 compared with Smad3 agonist(recombinant human Smad3 protein),positive control(ADAMTS-5 inhibitor)and negative control(standard medium)at 72 h Western blot(P < 0.05).Conclusion: TGFβ/Smad3 can affect the regulation of microRNA-140 on ADAMTS-5 in OA chondrocytes.