Spine Division, Department of Orthopedics, Central Hospital Affiliated to Shenyang Medical College, Shenyang, Liaoning 110024, P.R. China.
Int J Mol Med. 2019 Oct;44(4):1357-1365. doi: 10.3892/ijmm.2019.4314. Epub 2019 Aug 16.
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.
已经证实，miR-222 在人类椎间盘（IVD）退变组织中上调；然而，其潜在机制尚不清楚。在本研究中，我们旨在阐明 miR-222 在 IVD 组织中的作用机制。用脂多糖（LPS）处理髓核（NP）细胞以模拟 IVD 退变。通过逆转录-定量聚合酶链反应（RT-qPCR）检测细胞和组织中 miR-222 的表达水平。通过流式细胞术分析细胞凋亡。此外，通过 Western blot 分析测定 Toll 样受体 4（TLR4）、Iκβ-α（IκBα）和 p65 的水平。通过酶联免疫吸附测定（ELISA）测定白细胞介素（IL）-1β、肿瘤坏死因子-α（TNF-α）和 IL-6 蛋白表达水平。通过 TargetScan7.2 和双荧光素酶报告基因分析确定 miR-222 的靶基因。Western blot 分析和 RT-qPCR 用于测定组织金属蛋白酶抑制剂 3（TIMP3）的 mRNA 和蛋白水平。发现 miR-222 在 IVD 组织和 LPS 刺激的细胞中的表达增加，并且其表达与临床 MRI 分级呈正相关。在体外，miR-222 模拟物/抑制剂促进/抑制细胞凋亡。转染 miR-222 模拟物/抑制剂显著增加/减少 TNF-α、IL-1β 和 IL-6 的产生，并抑制/增强胶原 II 和聚集蛋白聚糖的表达。转染 miR-222 模拟物/抑制剂上调/下调 TLR4、p-IκΒα 和 p-p65 的蛋白水平。此外，证实 TIMP3 是 miR-222 的直接靶基因，在 NP 细胞中受 miR-222 负调控。沉默 TIMP3 逆转了 miR-222 抑制剂对 LPS 诱导的 NP 细胞凋亡的抑制作用。因此，总的来说，本研究的结果表明，miR-222 作为 IVD 发育的促进剂发挥作用，部分通过调节 TIMP3。
