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

Zhang,Yang; Yang,Jiujie; Zhou,Xiaoqing; Wang,Nan; Li,Zhi; Zhou,Yubo; Feng,Jianzhou; Shen,Dewei; Zhao,Wei

doi:10.3892/ijmm.2019.4314

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

Authors:
- Yang Zhang
- Jiujie Yang
- Xiaoqing Zhou
- Nan Wang
- Zhi Li
- Yubo Zhou
- Jianzhou Feng
- Dewei Shen
- Wei Zhao
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Affiliations: Spine Division, Department of Orthopedics, Central Hospital Affiliated to Shenyang Medical College, Shenyang, Liaoning 110024, P.R. China
Published online on: August 16, 2019 https://doi.org/10.3892/ijmm.2019.4314
Pages: 1357-1365
Copyright: © Zhang et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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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‑IκΒα 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.

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