Knockdown of miR-222 inhibits inflammation and the apoptosis of LPS-stimulated human intervertebral disc nucleus pulposus cells.
Int J Mol Med
; 44(4): 1357-1365, 2019 Oct.
Article
em En
| MEDLINE
| ID: mdl-31432092
ABSTRACT
It has been demonstrated that miR222 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 miR222 in IVD tissues. Nucleus pulposus (NP) cells were treated with lipopolysaccharide (LPS) to simulate IVD degeneration. The expression level of miR222 was detected by reverse transcriptionquantitative polymerase chain reaction (RTqPCR) in cells and tissues. Cell apoptosis was analyzed by flow cytometry. Additionally, western blot analysis was used to determine the levels of Tolllike receptor 4 (TLR4), Iκßalpha (IκBα) and p65. Interleukin (IL)1ß, tumor necrosis factorα (TNFα) and IL6 protein expression levels were determined by enzymelinked immunosorbent assay (ELISA). The target gene of miR222 was determined by TargetScan7.2 and dual luciferase reporter gene analysis. Western blot analysis and RTqPCR were used to determine the mRNA and protein levels of tissue inhibitor of metalloproteinase 3 (TIMP3). The mRNA expression level of miR222 was found to be increased in IVD tissues and in LPSstimulated cells, and its expression was positively associated with the clinical MRI grade. In vitro, apoptosis was promoted/inhibited by miR222 mimics/inhibitors. Transfection with miR222 mimics/inhibitors significantly increased/decreased the production of TNFα, IL1ß and IL6 and suppressed/enhanced collagen II and aggrecan expression. The protein levels of TLR4, pIκΒα and pp65 were upregulated/downregulated by transfection with the mimics/inhibitors. In addition, it was demonstrated that TIMP3 was a direct target gene of miR222, and was negatively regulated by miR222 in NP cells. The silencing of TIMP3 reversed the inhibitory effects of miR222 inhibitor on cell apoptosis, which was induced by LPS. Thus, on the whole, the findings of this study demonstrate that miR222 functions as a promoter of IVD development, partly via the regulation of TIMP3.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Lipopolissacarídeos
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Apoptose
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MicroRNAs
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Núcleo Pulposo
Tipo de estudo:
Etiology_studies
Limite:
Adult
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Aged
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Female
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Humans
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Male
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Middle aged
Idioma:
En
Ano de publicação:
2019
Tipo de documento:
Article