MicroRNA-200c Represses IL-6, IL-8, and CCL-5 Expression and Enhances Osteogenic Differentiation

Abstract

MicroRNAs (miRs) regulate inflammation and BMP antagonists, thus they have potential uses as therapeutic reagents. However, the molecular function of miR-200c in modulating proinflammatory and bone metabolic mediators and osteogenic differentiation is not known. After miR-200c was transduced into a human embryonic palatal mesenchyme (HEPM) (a cell line of preosteoblasts), using lentiviral vectors, the resulting miR-200c overexpression increased osteogenic differentiation biomarkers, including osteocalcin (OCN) transcripts and calcium content. miR-200c expression also down-regulated interleukin (IL)-6, IL-8, and chemokine (C-C motif) ligand (CCL)-5 under lipopolysaccharide (LPS) stimulation and increased osteoprotegerin (OPG) in these cells. miR-200c directly regulates the expression of IL-6, IL-8 and CCL-5 transcripts by binding to their 3'UTRs. A plasmid-based miR-200c inhibitor effectively reduces their binding activities. Additionally, miR-200c delivered using polyethylenimine (PEI) nanoparticles effectively inhibits IL-6, IL-8 and CCL-5 in primary human periodontal ligament fibroblasts and increases the biomarkers of osteogenic differentiation in human bone marrow mesenchymal stem cells (MSCs), including calcium content, ALP, and Runx2. These data demonstrate that miR-200c represses IL-6, IL-8 and CCL-5 and improves osteogenic differentiation. miR-200c may potentially be used as an effective means to prevent periodontitis-associated bone loss by arresting inflammation and osteoclastogenesis and enhancing bone regeneration.

Fig 1. miR-200c overexpression in HEPM cells and the effects on their proliferation.

A: Microphotographs of HEPM cells and the cells with miR-200c or scrambled miRs under phase-contrast. Bar = 10μm. B: Fold change of miR-200c expression in non-treated HEPM cells and the cells with miR-200c and scrambled miRs. C: The doubling time of non-treated HEPM cells and the cells in the context of miR infection. **: p<0.01.

Fig 2. miR-200c modulates proinflammatory mediators in human preosteoblasts.

A and B: the transcripts of IL-6 (A) and IL-8 (B) in non-treated HEPM cells and the cells with miR-200c or scrambled miRs cultured in DMEM supplemented with LPS at 0, 1, 5 and 10 μg/mL after 24 hours; *:p<0.05 vs non-treated; C: the amounts of IL-8 secreted by HEPM cells with miR-200c or scrambled miRs cultured in DMEM supplemented with or without LPS at different time points; *: p<0.05 vs cells with scrambled miRs; D and E: the amounts of IL-6 (D) and CCL-5 (E) secreted by HEPM cells with miR-200c or scrambled miRs cultured in DMEM supplemented with or without LPS after 24 hrs; F: the amounts of OPG secreted by HEPM cells with different miRs cultured in DMEM supplemented with or without LPS after 32 hours. *: p<0.05.

Fig 3. miR-200c increases osteogenic biomarkers in human preosteoblasts.

A and B: the amounts of the transcript of OCN (A) and calcium content (B) in non-treated HEPM cells and the cells with miR-200c or scrambled miRs cultured in DMEM supplemented β-glycerophosphate and ascorbic acid after 1 and 2 weeks, respectively. *: p<0.05.

Fig 4. Intracellular delivery of miR-200c using PEI nanoparticles to human primary periodontal ligament fibroblasts and bone marrow MSCs.

A: TEM image of PEI-miR-200c nanoplexes. B and C: Fold change of the transcript of miR-200c in non-treated human periodontal ligament fibroblasts (B) and bone marrow MSCs (C) and the cells transfected with empty vector (EV) (10μg/per well) and miR-200c (1, 5, 10μg/per well).

Fig 5. miR-200c delivered using PEI nanoparticles inhibits IL-6, IL-8, and CCL-5 in primary human periodontal ligament fibroblasts.

A-C: The transcripts of IL-6 (A), IL-8 (B), and CCL-5 (C) in the cells with miR-200c or empty vector cultured in DMEM supplemented with LPS after 24 hours; D and E: the amounts of IL-6 (D), IL-8 (E), and CCL-5 (F) secreted by the cells with miR-200c or empty vector cultured in DMEM supplemented with LPS after 12 and 32 hrs, respectively. *: p<0.05 vs empty vector with the same amount.

Fig 6. miR-200c directly targets the 3’UTR of IL-6, IL-8, and CCL-5.

A: The sequence and miR-200c binding region located in the 3’UTR and mutated 3’UTR of IL-6, IL-8, and CCL-5. B-D: Normalized luciferase activities of the 3’ UTR IL-6, IL-8, and CCL-5-luciferase reporters and their 3’UTR-mutated-luciferase reporters treated with empty vector or miR-200c. *: p<0.05.

Fig 7. PMIS-200c reduces binding activity of miR-200c to the 3’UTR of IL-6, IL-8, and CCL-5 and the function of miR-200c.

A-C: Normalized luciferase activities of the 3’ UTR IL-6, IL-8, and CCL-5-luciferase reporters and their 3’UTR-mutated-luciferase reporters co-treated with miR-200c and PMIS-EV or PMIS-200c at different ratios of concentration. D-F: the transcripts of IL-6 (D), IL-8 (E), and CCL-5 (F) in the cells co-treated with miR-200c and PMIS-EV or PMIS-200c cultured in DMEM supplemented with LPS after 24 hours; *: p<0.05.

Fig 8. Enhancement of osteogenic differentiation of human bone marrow MSCs with overexpression of miR-200c using PEI nanoparticles.

A: Images of ALP and von-Kossa staining in MSCs overexpressing miR-200c, one and two weeks after treatment with osteogenic medium. B and C: the transcripts of ALP (B) and Runx2 (C) in MSCs overexpressing miR-200c, one week after treatment with osteogenic medium. D and E: Quantitative measurement of ALP levels (D) and calcium content (E) in MSCs overexpressing miR-200c, one and two week after treatment with osteogenic medium. Each measurement was made in triplicate. *: p<0.05.