MicroRNA-26a/b have protective roles in oral lichen planus

Abstract

Oral lichen planus (OLP) is a kind of oral epithelial disorder featured with keratinocyte apoptosis and inflammatory reaction. The pathogenesis of OLP remains an enigma. Herein, we showed that the levels of miR-26a/b were robustly down-regulated in oral mucosal biopsies, serum and saliva in OLP patients compared with healthy control. Moreover, we found the binding sites of vitamin D receptor (VDR) in the promoter regions of miR-26a/b genes and proved that the induction of miR-26a/b was VDR dependent. The reduction of miR-26a/b expression was also detected in the oral epithelium of vitamin D deficient or VDR knockout mice. miR-26a/b inhibitors enhanced apoptosis and Type 1T helper (Th1) cells-related cytokines production in oral keratinocytes, whereas miR-26a/b mimics were protective. Mechanistically, we analyzed miRNA target genes and confirmed that miR-26a/b blocked apoptosis by directly targeting Protein Kinase C δ (PKCδ) which promotes cellular apoptotic processes. Meanwhile, miR-26a/b suppressed Th1-related cytokines secretion through targeting cluster of the differentiation 38 (CD38). In accordant with miR-26a/b decreases, PKCδ and CD38 levels were highly elevated in OLP patients' samples. Taken together, our present investigations suggest that vitamin D/VDR-induced miR-26a/b take protective functions in OLP via both inhibiting apoptosis and impeding inflammatory response in oral keratinocytes.

Fig. 1. miR-26a/b are decreased in OLP patients.

a miR-26a/b levels in human oral epithelial samples determined by real-time PCR. b Correlation of fold change between TNFα and miR-26a/b in oral epitheliums from OLP patients (r = −0.8118, P = 3.59 × 10⁻⁶, Spearman’s correlation test for miR-26a; r = -0.8383, P = 2.36 × 10^-6, Spearman’s correlation test for miR-26b). c miR-26a/b levels in serum of participants measured by real-time PCR. d Correlation between TNFα concentrations and miR-26a/b levels in serum of OLP patients (r = −0.6488, P = 5.18 × 10^-8, Spearman’s correlation test for miR-26a; r = −0.7227, P = 5.19 × 10^-8, Spearman’s correlation test for miR-26b). e qPCR analysis of OLP saliva showing 40% decreases of miR-26a/b versus healthy controls. f Correlation between TNFα status and miR-26a/b expression in saliva derived from OLP patients (r = −0.6128, P = 1.57 × 10⁻⁷, Spearman’s correlation test for miR-26a; r = −0.7525, P = 1.63 × 10⁻⁷, Spearman’s correlation test for miR-26b). **P < 0.01, ***P < 0.001 vs. corresponding healthy controls; n = 14 each group.

Fig. 2. VDR induces miR-26a/b by binding with VDRE in HOKs.

a Schematic illustration of VDR binding sites in the promoter regions of miR-26a/b genes. b ChIP analysis showing the increases of miR-26a/b levels after 36-hour VDR plasmids transfection in HOKs, bar indicates log₂ fold change, n = 3. qPCR analysis of miR26-a/b expression in HOKs challenged by activated CD4⁺ T cells (c) or LPS (d) with or without VDR plasmids, n = 3. Differential expression of miR-26a/b in oral keratinocytes of VDRKO (e), paricalcitol-treated (f), or vitamin D-deficient (g) mice, n = 5. Paricalcitol is an analog of vitamin D. h Correlation of fold change in OLP biopsies between VDR and miR-26a/b (r = 0.85081, P = 0.00036, Spearman’s correlation test for miR-26a; r = 0.79941, P = 0.02852, Spearman’s correlation test for miR-26b), n = 14. i Correlation between 25(OH)D concentrations and miR-26a/b levels in serum from OLP patient (r = 0.44655, P = 2.86 × 10⁻¹¹, Spearman’s correlation test for miR-26a; r = 0.58412, P = 2.87 × 10^-11, Spearman’s correlation test for miR-26b), n = 14. *P < 0.05, **P < 0.01, ***P < 0.001 vs. corresponding control. Ctrl control, VDRKO VDR knockout, Pari paricalcitol, VD-D vitamin D deficiency.

Fig. 3. miR-26a/b target PKCδ to inhibit apoptosis in oral keratinocytes.

a–d Western blot showing changes of cleaved caspase 9, cleaved PARP, and cleaved caspase 3 levels in HOKs added with miR-26a/b mimics (a) or inhibitors (b). c-d Western blot analysis of cleaved caspase 9, cleaved PARP, and cleaved caspase 3 expression in mouse oral keratinocytes. C57BL/6 mice were subject to miR-26a/b mimics (c) or inhibitors (d) by tail vein injection. e Putative miR-26a/b binding site in the 3′UTR of PKCδ mRNA. f Schematic illustration of binding site location in the 3′UTR of hPKCδ cDNA. g Luciferase reporter assay showing miR-26a/b target binding site in the 3′UTR of hPKCδ in HOKs which were transfected with Luc-PKCδ-3′UTR or pRL-TK (control) plasmids. Luciferase activity was quantified after 24 h. h HOKs were co-transfected with Luc-PKCδ-3′UTR or Luc-PKCδ-3′UTR-Mut and miR-26a/b inhibitors as indicated. i–j PKCδ expression and phosphorylation in HOKs with miR-26a/b mimics or inhibitors were measured by real-time PCR (i) or western blot (j). Western blot showing Bax translocation, cyt c release, and downstream apoptotic factors expression in HOKs during miR-26a/b mimics (k) or inhibitors (l) treatment with or without PKCδ plasmids or siRNA transfection as indicated. *P < 0.05, **P < 0.01, ***P < 0.001 vs. corresponding control; n = 3 for assays in vitro and n = 5 for assays in vivo. Ctrl control, Mito mitochondria, Cyto cytoplasm, cyt c cytochrome c, mi mimic, In inhibitor, Mut mutation.

Fig. 4. miR-26a/b regulate cytokines and target CD38.

a Heat map showing alterations of cytokines and corresponding receptors expression in HOKs with activated CD4⁺ T cells or LPS stimulation by real-time PCR. b Real-time PCR analysis of relative IFNγ status in activated CD4⁺ T cells or LPS-stimulated HOKs with 36-h miR-26a/b pre-treatment. c Putative miR-26a/b binding site in the 3′UTR of CD38 mRNA. d Schematic showing binding site location in the 3′UTR of hCD38 cDNA. e Luciferase reporter assay showing miR-26a/b target binding site in the 3′UTR of hCD38 in HOKs. f–g Luciferase activity quantification in HOKs. Cells were co-transfected with Luc-CD38-3′UTR or Luc-CD38-3′UTR-Mut and miR-26a/b inhibitors as shown. *P < 0.05, **P < 0.01, ***P < 0.001 vs. corresponding control; n = 3. Ctrl control, In inhibitor, Mut mutation.

Fig. 5. miR-26a/b suppress cytokines via targeting CD38.

a IFNγ, TNFα, IL-2, and IL-12 levels detected by real-time PCR in HOKs after CD38 plasmids transfection. (b) qPCR showing IFNγ, TNFα, IL-2 and IL-12 levels in activated CD4⁺ T cells or LPS-stimulated HOKs, following CD38 inhibitor treatment. Real-time PCR analysis of IFNγ, TNFα, IL-2 and IL-12 levels in HOKs during miR-26a (c) or miR-26b (d) mimic treatment with or without CD3 plasmids transfection as indicated. Real-time PCR analysis of IFNγ, TNFα, IL-2, and IL-12 levels in HOKs during miR-26a (e) or miR-26b (f) inhibitor treatment with or without siRNA transfection. *P < 0.05, **P < 0.01, ***P < 0.001 vs. corresponding control; ^#P < 0.05, ^##P < 0.01 vs miR-26a/b mi or In group; n = 3. Ctrl control, mi mimic, In inhibitor.

Fig. 6. Apoptosis and inflammatory response are enhanced in oral keratinocytes derived from OLP biopsies.

a Western blot analysis of oral epithelial cells from OLP patients (I) or healthy individuals (NI) with a set of antibodies as indicated. b TUNEL staining of biopsies harvested from OLP patients or healthy control. c Average TUNEL positive-oral keratinocytes per microscopic field (magnification, ×400), 20 random fields were counted for each group. d Violin plot showing CD38 mRNA expression in healthy or OLP oral epitheliums determined by real-time PCR. e Real-time PCR analysis of IFNγ, IL-2 and IL-12 mRNA levels in human biopsies. n = 14 each group, NI non-inflammation, I inflammation.

Fig. 7

Schematic illustration recapitulates biological process and function of miR-26a/b in oral keratinocytes.