[No authors listed]
It has been demonstrated that miRâ222 is upregulated in human intervertebral disc (IVD) degeneration tissues; however, the underlying mechanisms remain unclear. In this study, we aimed to elucidate the mechanisms of action of miRâ222 in IVD tissues. Nucleus pulposus (NP) cells were treated with lipopolysaccharide (LPS) to simulate IVD degeneration. The expression level of miRâ222 was detected by reverse transcriptionâquantitative polymerase chain reaction (RTâqPCR) in cells and tissues. Cell apoptosis was analyzed by flow cytometry. Additionally, western blot analysis was used to determine the levels of Tollâlike receptor 4 (TLR4), Iκβâalpha (IκBα) and p65. Interleukin (IL)â1β, tumor necrosis factorâα (TNFâα) and ILâ6 protein expression levels were determined by enzymeâlinked immunosorbent assay (ELISA). The target gene of miRâ222 was determined by TargetScan7.2 and dual luciferase reporter gene analysis. Western blot analysis and RTâqPCR were used to determine the mRNA and protein levels of tissue inhibitor of metalloproteinase 3 (TIMP3). The mRNA expression level of miRâ222 was found to be increased in IVD tissues and in LPSâstimulated cells, and its expression was positively associated with the clinical MRI grade. In vitro, apoptosis was promoted/inhibited by miRâ222 mimics/inhibitors. Transfection with miRâ222 mimics/inhibitors significantly increased/decreased the production of TNFâα, ILâ1β and ILâ6 and suppressed/enhanced collagen II and aggrecan expression. The protein levels of TLR4, pâIκÎα and pâp65 were upregulated/downregulated by transfection with the mimics/inhibitors. In addition, it was demonstrated that TIMP3 was a direct target gene of miRâ222, and was negatively regulated by miRâ222 in NP cells. The silencing of TIMP3 reversed the inhibitory effects of miRâ222 inhibitor on cell apoptosis, which was induced by LPS. Thus, on the whole, the findings of this study demonstrate that miRâ222 functions as a promoter of IVD development, partly via the regulation of TIMP3.
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