MicroRNA-200c Attenuates Periodontitis by Modulating Proinflammatory and Osteoclastogenic Mediators

Abstract

This study tested whether microRNA (miR)-200c can attenuate the inflammation and alveolar bone resorption in periodontitis by using an in vitro and a rat model. Polyethylenimine (PEI) was used to facilitate the transfection of plasmid DNA encoding miR-200c into primary human gingival fibroblasts (HGFs) and gingival tissues of rats. We first analyzed how proinflammatory and osteoclastogenic mediators in HGFs with overexpression of miR-200c responded to Porphyromonas gingivalis lipopolysaccharide (LPS-PG) challenge in vitro. We observed that overexpression of miR-200c significantly reduced interleukin (IL)-6 and 8 and repressed interferon-related developmental regulator-1 (IFRD1) in HGFs. miR-200c also downregulated p65 and p50. In a rat model of periodontitis induced by an LPS injection at the gingival sulcus of the second maxillary molar (M2), we analyzed how the mediators in rat gingiva and alveolar bone resorption responded to miR-200c treatment by a local injection of PEI-plasmid miR-200 nanoplexes. We observed that the local injection of miR-200c significantly upregulated miR-200c expression in gingiva and reduced IL-6, IL-8, IFRD1, and the ratio of receptor activator of nuclear factor kappa-B ligand/osteoprotegerin. Using micro-computed tomography analysis and histomorphometry, we further confirmed that local treatment with miR-200c effectively protected alveolar bone resorption in the rat model of periodontitis by reducing the distance between the cemento-enamel junction and the alveolar bone crest and the inter-radicular space in the upper maxilla at M2. These findings imply that miR-200c may serve as a unique means to prevent periodontitis and associated bone loss.

Keywords: alveolar bone loss; microRNA; periodontitis; polyethylenimine; proinflammatory.

FIG. 1.

Cell viability, proliferation, and miR-200c expression in HGF cells after transfection with PEI-miR-200c nanoplex. (A) Fold change of miR-200c expression in HGF cells evaluated by quantitative polymerase chain reaction. (B, C) Microphotographs of HGFs with Live/Dead staining (B) and the percentage of viable cells (C) 3 days after transfection with PEI-miR-200c. Bars = 100 μm. (D) MTT assay [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)] results for the HGF 4 days after different treatments; *P < 0.05 versus EV (10 μg); performed in replicate. EV, empty vector; IL, interleukin; HGF, human gingival fibroblast; PEI, polyethylenimine. Color images are available online.

FIG. 2.

miR-200c reduces protein level of proinflammatory and osteoclastogenic mediators after LPS stimulation. (A–E) Relative fold change of transcripts of IL-6 (A), IL-8 (B), p50 (C), p65 (D), and IFRD1 (E) in HGFs with different transfections 24 h after stimulation with 1 μg/mL of LPS-PG. *P < 0.05 versus HGFs untreated with LPS; ^#P < 0.05 versus LPS-EV; performed in replicate. (F) Targetscan.org predicted the possible binding site of miR-200c-p3 and the 3′UTR of IFRD1 at the position 1,120–1,126. IFRD1, interferon-related developmental regulator-1; LPS-PG, Porphyromonas gingivalis lipopolysaccharide.

FIG. 3.

miR-200c reduces proinflammatory and osteoclastogenic mediators after LPS stimulation. Protein concentration of IL-6 (A), IL-8 (B) in the supernatant; the protein concentration of p65 (C) and IFRD1 (D) in the lysis of HGFs with different transfections 24 h after stimulation with 1 μg/mL of LPS-PG. *P < 0.05 versus LPS untreated; performed in replicate.

FIG. 4.

miR-200c reduces both inflammatory and osteoclastogenic mediators in vivo. (A) Fold change of miR-200c in gingival tissue of rats 4 or 7 days after receiving PEI-miR-200c at 1 or 10 μg and controls. (B–D) Relative fold change of transcripts of IL-6 (B), IL-8 (C), IFRD1 (D), and the ratio of RANKL/OPG (E) at gingival tissues of rats 1 week after receiving different treatments under LPS-PG challenge at 10 μg. *P < 0.05 versus untreated; ^#P < 0.05 versus EV; n = 3 for each treatment.

FIG. 5.

miR-200c protects LPS-induced ABL in rat periodontitis. Rats were injected bi-weekly with 10 μg LPS and treated weekly with one injection of PBS, PEI facilitating EV (1 and 10 μg), miR-200c (1 and 10 μg) up to 4 weeks. (A) Three-dimensional reconstructed micro-computed tomography images of maxillary alveolar bone on the palatal sides of rats 4 weeks after receiving an LPS injection with and without miR-200c. Red dot lines indicate the ABC and CEJ at maxillary second molars. Scale bars: 1 mm. (B, C) The outcome of the ABL (B) and the inter-radicular space (C) at the M2 after 4 weeks. (D, E) Structural parameters of alveolar bone 4 weeks after different treatment, including (Tb. Th) (D), and the percentages of BV/TV, BS/BV, and the total alveolar bone porosity (E). *P < 0.05 versus untreated; ^#P < 0.05 versus LPS-PBS; n = 8 for each treatment. ABC, alveolar bone crest; ABL, alveolar bone loss; BS, bone surface; BV, bone volume; CEJ, cemento-enamel junction; M2, second maxillary molar; PBS, phosphate-buffered saline; Tb. Th, trabecular thickness; TV, tissue volume. Color images are available online.

FIG. 6.

Histological analysis of alveolar bone and periodontal ligament adjacent to the M2 of 4 weeks LPS-treated rats. (A) and (B) Representative micrographs of sham and periodontitis rats treated with PBS, PEI facilitating EV (1 and 10 μg), and miR-200c (1 and 10 μg) with hematoxylin and eosin staining (A) and Masson's trichrome staining (B). Magnification = 4. (C) Ratio of the remaining ACB to the RL adjacent to the M2. The ratio of the ACB to the RL was calculated by using the formula (ABC-RA)/(CEJ-RA), where ABC-RA is the distance from ABC to RA and CEJ-RA is the distance from CEJ to RA. *P < 0.05 vs untreated; ^#P < 0.05 versus LPS-PBS; n = 3 for each treatment. ACB, alveolar crest bone; RA, root apex; RL, root length. Color images are available online.

FIG. 7.

Potential mechanism(s) of anti-inflammatory/osteoclastogenic function of miR-200c to attenuate periodontitis.