Long non-coding RNA 01126 promotes periodontitis pathogenesis of human periodontal ligament cells via miR-518a-5p/HIF-1α/MAPK pathway

Abstract

Background: Periodontitis is a prevalent oral inflammatory disease, which can cause periodontal ligament to a local hypoxia environment. However, the mechanism of hypoxia associated long non-coding RNAs (lncRNAs) involved in periodontitis is still largely unknown.

Methods: Microarray was performed to detect the expression patterns of lncRNAs in 3 pairs of gingival tissues from patients with periodontitis and healthy controls. The expression of lncRNA 01126 (LINC01126), miR-518a-5p and hypoxia-inducible factor-1α (HIF-1α) in periodontal tissues and in human periodontal ligament cells (hPDLCs) under hypoxia was measured by quantitative real-time polymerase chain reaction or western blot. Fluorescence in situ hybridization and cell fraction assay were performed to determine the subcellular localization of LINC01126 and miR-518a-5p. Overexpression or knockdown of LINC01126 or HIF-1α was used to confirm their biological roles in hPDLCs. MTT assays were performed to evaluate hPDLCs proliferation ability. Flow cytometry was used to detect apoptosis. ELISA was used to measure the expression levels of interleukin (IL)-1β, IL-6, IL-8 and TNF-α. Dual-luciferase reporter assays were performed to assess the binding of miR-518a-5p to LINC01126 and HIF-1α. RNA immunoprecipitation assay was used to identify whether LINC01126 and miR-518a-5p were significantly enriched in AGO-containing micro-ribonucleoprotein complexes.

Results: We selected LINC01126, which was the most highly expressed lncRNA, to further verify its functions in periodontitis-induced hypoxia. The expression of LINC01126 was increased in periodontal tissues. In vitro experiment demonstrated that LINC01126 suppressed proliferation, promoted apoptosis and inflammation of hPDLCs under hypoxia via sponging miR-518a-5p. Moreover, we identified HIF-1α acted as a direct target of miR-518a-5p in hPDLCs and LINC01126 promoted periodontitis pathogenesis by regulating the miR-518a-5p/HIF-1α/MAPK pathway.

Conclusion: LINC01126 promotes periodontitis pathogenesis of hPDLCs via miR-518a-5p/HIF-1α/MAPK pathway, providing a possible clue for LINC01126-based periodontal therapeutic approaches.

Keywords: HIF-1α; hypoxia; long non-coding RNA; miR-518a-5p; periodontitis.

Figure 1

Aberrant expression of lncRNAs in periodontitis. (A) Heatmap of the top 50 differentially expressed lncRNAs (|log₂FC| >1.5, P < .05) in 3 pairs of gingival tissues from patients with periodontitis and the healthy controls though lncRNA expression microarray. (B) RNA expressions of the 4 mostly upregulated lncRNAs LINC01126, LINC545726, lncRNA‐KLLP, LINC01314 in periodontal tissues and the controls group (C, D) The top 10 upregulated and downregulated GO functions of the cellular component (CC) domains. (E) GSEA plot of differentially expressed involved in proliferation in periodontal tissues. (F) GSEA plot of genes involved in MAPK signalling pathway in periodontal tissues. Running enrichment score was positive for most differentially expressed genes. (G, H) The top 10 upregulated and downregulated GO functions of the molecular function (MF) domains

Figure 2

LINC01126 was upregulated in hypoxia‐induced hPDLCs. (A) Cell apoptosis ratio under hypoxia or normoxia at 1st day and 3rd day was determined by flow cytometry. (B) Cell proliferative ability at different time points under hypoxia or normoxia was determined by MTT assay. (C‐F) The secretion of inflammatory cytokines including IL‐1β IL‐6, IL‐8 and TNF‐α at different O₂ concentrations after 24 h treatment was determined by ELISA. (G) The expression of LINC01126 at different O₂ concentrations for 48 h was determined by qRT‐PCR. (H) The expression of LINC01126 at different time point under 1% O₂ concentration condition by qRT‐PCR. (I)The protein expression of HIF‐1α after 24h treatment at different O₂ concentrations by Western blot. Histogram shows quantification of the band intensities. Results are presented as mean ± SD (*p < 0.05, **p < 0.01, *** p < 0.001, compared with the control group)

Figure 3

LINC01126 promoted hypoxia‐induced apoptosis, inflammation, suppressed proliferation in hPDLCs. (A) The expression of LINC01126 after transfection with pCDH‐01126, sh‐01126 and the corresponding controls. (B‐D) Cell proliferative ability after overexpression or knockdown of LINC01126 under hypoxia or normoxia. (E) ELISA showing the secretion of inflammatory cytokines including IL‐1β IL‐6, IL‐8 and TNF‐α at hypoxic environment (1% O₂) after 24 h treatment was determined by ELISA. (F‐G) Cell apoptosis after overexpression or knockdown of LINC01126 under hypoxia or normoxia. Results are presented as mean ± SD (*P < .05, **P < .01, compared with the control group)

Figure 4

LINC01126 serves as a sponge for miR‐518. (A) Venn diagrams showing the number of potential miRNAs targeting LINC01126 (including miR‐518a‐5p). The potential miRNAs were predicted by four databases: LncBase, miRcode, Starbase, StarBaseV2. (B) Fluorescence in situ hybridization results demonstrated the subcellular location of INC01126 and miR‐518. Nuclei were stained by DAPI (blue), the scale bar = 50 μm. (C) The predicted binding of miR‐518a‐5p with LINC01126 3′‐UTR. The sequence of wild‐type (WT) and mutant (Mut) LINC01126 target sites were shown. (D) RNA immunoprecipitation assay of endogenous AGO2 binding to RNA in hPDLCs. IgG was used as the negative control. The expression of LINC01126 and miR‐518a‐5p was analysed by qRT‐PCR and the results were normalized relative to the input control. (E) Dual‐luciferase reporter assay validating the interaction between miR‐518a‐5p and LINC01126. (F) The expression of miR‐518a‐5p after transfection with pCDH‐01126, sh‐01126 and the corresponding controls. (G) The expression of miR‐518a‐5p after transfection with miR‐518a‐5p mimic, miR‐518a‐5p‐inhibitor and the corresponding controls. Results are presented as mean ± SD (**P < .01, compared with the control group)

Figure 5

HIF‐1α is a target of LINC01126/ miR‐518a‐5p. (A) Relative expression of LINC01126 and in nuclear and cytoplasmic fractions of hPDLCs. (B) Venn diagrams showing the number of potential genes targeting miR‐518a‐5p (including HIF‐1α gene). The potential genes were predicted by three databases: TargetScan, miRWalk and miRTar. (C) The predicted binding of miR‐154‐5p with HIF‐1α 3ʹ UTR. (D) Dual‐luciferase reporter assay validating the interaction between miR‐518a‐5p and LINC01126. (E) The mRNA expression of HIF‐1α after transfection with miR‐518a‐5p mimic, miR‐518a‐5p inhibitor and the corresponding controls. (F) The mRNA expression of HIF‐1α after transfection with pCDH‐01126, LINC01126‐miR‐518a‐5p, sh‐01126, sh‐01126‐miR‐518a‐5p and the corresponding controls. (G) The protein expression level after transfection with pCDH‐01126, sh‐01126 and the corresponding controls. (H‐I) The protein expression of HIF‐1α after transfection with pCDH‐01126, sh‐01126 and the corresponding controls under different O₂ condition. Histograms show quantification of the band intensities. Results are presented as mean ± SD (**P < .01, compared with the control group)

Figure 6

HIF‐1α promotes hypoxia‐induced apoptosis and suppressed proliferation in hPDLCs. (A, B) The mRNA and protein expression of HIF‐1α after transfection with transfection with sh‐LINC01126, pCDH‐HIF‐1α, sh‐HIF‐1α and the corresponding controls. Histogram shows quantification of the band intensities. (C‐E) Cell proliferative ability after overexpression or knockdown of HIF‐1αunder different O₂ concentrations was determined by MTT assay. (F) Cell apoptosis ratio after overexpression or knockdown of HIF‐1α under different O₂ concentrations. (G‐H) The expression of HIF‐1α mRNA after knockdown of LINC01126 under hypoxia (1% O₂, 5% O₂). Results are presented as mean ± SD (*P < .05, **P < .01, compared with the control group)

Figure 7

LINC01126/miR‐518/HIF‐1α aggravates periodontitis pathogenesis via activating MAPK pathway. (A‐C) The protein expression of p38, JNK1/2, ERK1/2 after transfection with pCDH‐HIF‐1α, sh‐HIF‐1α and the corresponding controls under different O₂ concentrations. Histogram shows quantification of the band intensities. (D‐F) The protein expression of p38, JNK1/2, ERK1/2 after transfection with pCDH‐01126, sh‐HIF‐1α/ pCDH‐01126 and the corresponding controls under different O₂ concentrations. Histogram shows quantification of the band intensities. Results are presented as mean ± SD (*P < .05, **P < .01, compared with the control group)

Figure 8

Schematic model showing the regulation periodontitis pathogenesis by of LINC01126