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‑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.

Figure 1

LPS-induced nucleus pulposus cell apoptosis is inhibited by miR-222 inhibitor. (A) miR-222 level in human intervertebral disc tissues was detected by reverse transcription-quantitative polymerase chain reaction. (B) miR-222 level in lipopolysaccharide-stimulated nucleus pulposus cells was detected using reverse transcription-quantitative polymerase chain reaction. (C) Transfection efficiency of miR-222 mock, mimics and inhibitor was determined by reverse transcription-quantitative polymerase chain reaction. (D) Nucleus pulposus cell apoptosis was analyzed by flow cytometry. ^*P<0.05 and ^**P<0.01 vs. control; ^#P<0.05 and ^##P<0.01 vs. cells stimulated with LPS only. LPS, lipopolysaccharide.

Figure 2

Effects of miR-222 on TNF-α, IL-1β and IL-6, collagen II and aggrecan expression levels in LPS-treated nucleus pulposus cells. (A) TNF-α, (B) IL-1β and (C) IL-6 expression levels in nucleus pulposus cells treated with LPS were detected by ELISA. The expression levels of (D) collagen II and (E) aggrecan in nucleus pulposus cells treated with LPS were detected using reverse transcription-quantitative polymerase chain reaction ^*P<0.05 and ^**P<0.01 vs. control, ^#P<0.05 and ^##P<0.01 vs. cells stimulated with LPS only. TNF-α, tumor necrosis factor- α; IL, interleukin; LPS, lipopolysaccharide.

Figure 3

Levels of TLR4, p-IκBα and p-p65 were suppressed by miR-222 inhibitor. (A) Protein levels of TLR4, p-IκBα and p-p65 were determined by western blot analysis. The relative levels of proteins described in (B, C and D) were normalized to ^**P<0.01 vs. control; ^#P<0.05 vs. cells stimulated with LPS only. TLR4, Toll-like receptor 4; LPS, lipopolysaccharide.

Figure 4

miR-222 targets TIMP3. (A) The 3′-UTR of TIMP3 was predicted as a miR-222 target using TargetScan 7.2. (B) Luciferase activity of a reporter containing a wild-type (WT) TIMP3 3′ UTR or a mutant (MUT) TIMP3 3′ UTR is shown. (C) The expression of TIMP3 was detected using reverse transcription-quantitative polymerase chain reaction. (D) The protein level of TIMP3 was determined by western blot analysis. The relative level of TIMP3 described in (E) was normalized to ^**P<0.01 vs. control; ^#P<0.05 and ^##P<0.01 vs. cells stimulated with LPS only. TIMP3, tissue inhibitor of metalloproteinase 3; LPS, lipopolysaccharide.

Figure 5

Overexpression of TIMP3 reverses the apoptosis of LPS-stimulated nucleus pulposus cells induced by miR-222 inhibitor. (A) TIMP3 mRNA levels was determined by reverse transcription-quantitative polymerase chain reaction. (B) TIMP3 protein levels were determined by western blot analysis. (C) The relative TIMP3 protein level is presented. (D) Cell apoptosis was analyzed by flow cytometry. ^**P<0.01 vs. control; ^##P<0.01 vs. cells stimulated with LPS only; ^&&P<0.01 vs. inhibitor + siNC.