MiR-125a-3p alleviates hyperproliferation of keratinocytes and psoriasis-like inflammation by targeting TLR4/NF-κB pathway

Abstract

Introduction: Psoriasis is a chronic auto-inflammatory dermatosis characterized by hyperproliferation of keratinocytes. Emerging evidence has validated the dysregulated expression of microRNAs (miRNAs/miRs) in psoriasis patients.

Aim: To probe into the role and precise mechanism of miR-125a-3p in HaCaT cells and imiquimod (IMQ)-stimulated psoriasis-like mice.

Material and methods: In M5-treated HaCaT cells and IMQ-stimulated psoriasis-like mice, real-time quantitative polymerase chain reaction and western blot analysis were performed for detecting gene expression. Hematoxylin and eosin staining was used to evaluate pathological morphology of IMQ-induced psoriasis skin. The proliferation of keratinocytes was assessed using Cell Counting Kit-8 assay and Ki67 positive staining. The combination between miR-125a-3p and Toll-like receptor 4 (TLR4) was confirmed by luciferase reporter assay.

Results: Our study showed reduced miR-125a-3p expression in psoriasis patients, psoriasis-like inflammatory cell models, and IMQ-generated psoriasis-like mouse models. MiR-125a-3p repressed the activity of keratinocytes in vitro by suppressing cell proliferation, inhibiting the production of psoriasis-related genes and inflammatory genes, and inactivating the NF-κB and interleukin (IL)-1β pathways. Notably, the psoriasis-like inflammation was repressed by intradermal injection of agomiR-125a-3p in psoriatic mouse models in vivo. Mechanically, miR-125a-3p targeted and negatively regulated TLR4. Furthermore, the elevated expression of TLR4 reversed the influences of miR-125a-3p mimics on HaCaT cells.

Conclusions: Upregulation of miR-125a-3p protects keratinocytes against hyperproliferation and inflammatory damage by inhibiting TLR4, suggesting that the miR-125a-3p/TLR4 axis might become a novel target for the prevention of psoriasis.

Keywords: Toll-like receptor 4; hyperproliferation; inflammation; miR-125a-3p; psoriasis.

Figure 1

Downregulation of miR-125a-3p in psoriasis patients and M5-treated keratinocytes. A – MiR-125a-3p expression in skin specimens from healthy donors (n = 35) and psoriasis patients (n = 35) by RT-qPCR analysis. B – Correlation of PASI score with miR-125a-3p expression in psoriatic biopsies of 35 psoriasis patients. C–J – Expression of psoriasis-related genes (S100A7, S100A8, and S100A9) and inflammatory genes (CXCL1, CXCL2, IL-1β, CCL2, and CCL20) in HaCaT cells by RT-qPCR. K – MiR-125a-3p expression in HaCaT cells with or without M5 treatment by RT-qPCR. Experimental data are expressed as the mean ± SD. **p < 0.01, ***p < 0.001

Figure 2

Effects of miR-125a-3p in vitro. A – Transfection efficiency of miR-125a-3p mimics or miR-125a-3p inhibitors in HaCaT cells. B – CCK-8 assay for assessing the proliferation of HaCaT cells transfected with miR-125a-3p mimics or miR-125a-3p inhibitors. C–J – The mRNA expression of psoriasis-related genes and inflammatory genes in HaCaT cells. K – The protein levels of p-NF-κB p65 and IL-1β in transfected HaCaT cells with or without M5 treatment by western blot analysis. L–S – The mRNA expression of psoriasis-related genes and inflammatory genes in HaCaT cells. T – The protein levels of p-NF-κB p65 and IL-1β in HaCaT cells. Experimental data are expressed as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001

Figure 3

AgomiR-125a-3p attenuates IMQ-induced psoriasis in mouse models. A – Treatment regimens of agomiR-125a-3p in IMQ-induced psoriasis mice (n = 15/group). B – MiR-125a-3p expression in the groups of Vehicle, IMQ + agomiR-NC, and IMQ + agomiR-125a-3p. C – PASI score of mouse models in the indicated groups was recorded daily during the experimental periods. D – H&E staining for the histological feature of the back skin tissues. Scale bar: 100 μm. Ki67 staining for the proliferated cells. Scale bar: 50 μm. E, F – Skin epidermal thickness and the percentage of Ki67 staining were quantified. G – Protein expression of p-NF-κB p65 and IL-1β in each group. H–N – The expression of S100A7, S100A8, S100A9, CXCL1, CXCL2, IL-1β, CCL2, and CCL20 genes in back skin tissues from mouse models. N = 15/group. Experimental data are expressed as the mean ± SD. **p < 0.01, ***p < 0.001

Figure 4

MiR-125a-3p targets TLR4. A – Predicated consequential pairing of TLR4 and miR-125a-3p shown by TargetScan database. B – Constructed TLR4-Mut and the predicated binding site between TLR4 and miR-125a-3p. C – Luciferase reporter assay for validating the combination between miR-125a-3p and TLR4. D, E – The protein expression of TLR4 in the transfected- or treated-HaCaT cells. F – TLR4 mRNA expression in skin tissues from healthy donors (n = 35) and psoriasis patients (n = 35) by RT-qPCR analysis. G – Protein expression of TLR4 in mice. H, I – Correlation of TLR4 protein positivity and PASI scores in psoriasis patients (n = 35) or IMQ-stimulated psoriasis mice (n = 35). J, K – Correlation of TLR4 protein positivity and miR-125a-3p in psoriasis patients (n = 35) or IMQ-stimulated psoriasis mice (n = 35). Experimental data are expressed as the mean ± SD. ***p < 0.001

Figure 5

Overexpressed TLR4 reverses the influences of miR-125a-3p on HaCaT cells. A – Overexpression efficiency of TLR4 in HaCaT cells by RT-qPCR. B – Cell proliferation in the miR-NC, miR-125a-3p, and miR-125a-3p + TLR4 groups by CCK-8 assay. C–J – Gene mRNA expression in the indicated groups. K – Protein levels of p-NF-κB p65 and IL-1β in the indicated groups. Experimental data are expressed as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001