Knockdown of miR-222 inhibits inflammation and the apoptosis of LPS-stimulated human intervertebral disc nucleus pulposus cells.

Abstract

It has been demonstrated that miR-222 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 miR-222 in IVD tissues. Nucleus pulposus (NP) cells were treated with lipopolysaccharide (LPS) to simulate IVD degeneration. The expression level of miR-222 was detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) in cells and tissues. Cell apoptosis was analyzed by flow cytometry. Additionally, western blot analysis was used to determine the levels of Toll-like receptor 4 (TLR4), Iκβ-alpha (IκBα) and p65. Interleukin (IL)-1β, tumor necrosis factor-α (TNF-α) and IL-6 protein expression levels were determined by enzyme-linked immunosorbent assay (ELISA). The target gene of miR-222 was determined by TargetScan7.2 and dual luciferase reporter gene analysis. Western blot analysis and RT-qPCR were used to determine the mRNA and protein levels of tissue inhibitor of metalloproteinase 3 (TIMP3). The mRNA expression level of miR-222 was found to be increased in IVD tissues and in LPS-stimulated cells, and its expression was positively associated with the clinical MRI grade. In vitro, apoptosis was promoted/inhibited by miR-222 mimics/inhibitors. Transfection with miR-222 mimics/inhibitors significantly increased/decreased the production of TNF-α, IL-1β and IL-6 and suppressed/enhanced collagen II and aggrecan expression. The protein levels of TLR4, p-IκBα and p-p65 were upregulated/downregulated by transfection with the mimics/inhibitors. In addition, it was demonstrated that TIMP3 was a direct target gene of miR-222, and was negatively regulated by miR-222 in NP cells. The silencing of TIMP3 reversed the inhibitory effects of miR-222 inhibitor on cell apoptosis, which was induced by LPS. Thus, on the whole, the findings of this study demonstrate that miR-222 functions as a promoter of IVD development, partly via the regulation of TIMP3.