Keratinocyte-derived circulating microRNAs in extracellular vesicles: a novel biomarker of psoriasis severity and potential therapeutic target

Abstract

Background: Psoriasis is a chronic inflammatory disorder characterized by pathogenic hyperproliferation of keratinocytes and immune dysregulation. Currently, objective evaluation tools reflecting the severity of psoriasis are insufficient. MicroRNAs in extracellular vesicles (EV miRNAs) have been shown to be potential biomarkers for various inflammatory diseases. Our objective was to investigate the possibility of plasma-derived EV miRNAs as a marker for the psoriasis disease severity.

Methods: EVs were extracted from the plasma of 63 patients with psoriasis and 12 with Behçet's disease. We performed next-generation sequencing of the plasma-derived EV miRNAs from the psoriasis patients. Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to validate the level of EV miRNA expression. In situ hybridization was used to discern the anatomical location of miRNAs. qRT-PCR, western blotting, and cell counting kits (CCKs) were used to investigate IGF-1 signaling in cells transfected with miRNA mimics.

Results: We identified 19 differentially expressed EV miRNAs and validated the top three up-and down-regulated EV miRNAs. Among these, miR-625-3p was significantly increased in patients with severe psoriasis in both plasma and skin and most accurately distinguished moderate-to-severe psoriasis from mild-to-moderate psoriasis. It was produced and secreted by keratinocytes upon stimulation. We also observed a significant intensification of IGF-1 signalling and increased cell numbers in the miR-625-3p mimic transfected cells.

Conclusions: We propose keratinocyte-derived EV miR-625-3p as a novel and reliable biomarker for estimating the severity of psoriasis. This biomarker could objectively evaluate the severity of psoriasis in the clinical setting and might serve as a potential therapeutic target. Trial registration None.

Keywords: Biomarkers; Extracellular vesicles; Keratinocytes; Psoriasis; miR-625-3p; microRNAs.

Fig. 1

Differentially expressed (DE) miRNAs in extracellular vesicles (EV miRNAs) identified from blood and skin of patients with severe and mild psoriasis. A Experimental scheme for acquisition and analysis of EV miRNA (Screening phase shown in blue arrows and validation phase shown in red arrows). B DE miRNAs on volcano plot (Selected EV miRNAs shown in bold). Blue and red colored dots indicate miRNAs with |log2 fold change|≥ 2 and P ≤ 0.05, and grey colored dots represent miRNAs with no significant changes. C and D qRT-PCR results of selected C downregulated and D upregulated miRNAs in the plasma of psoriasis patients. E qRT-PCR results of upregulated miRNAs in skin of psoriasis patients. For comparisons of differences between groups C-E, ordinary one-way ANOVA was used. (F) The skin miR-625-3p level plotted against PASI and BSA. EV miRNAs; miRNAs in extracellular vesicles. The significance of the correlation was assessed by the Spearman's rank correlation test. *P < 0.05 and **P < 0.01

Fig. 2

EV miR-625-3p is highly associated with PASI and BSA, most accurately differentiates mild and moderate-to-severe psoriasis, is psoriasis-specific, and declines significantly when successfully treated. A and B Plasma miR-625-3p, miR-4488 and miR-342-3p level plotted against A PASI and B BSA. The significance of the correlation was tested using the Spearman's rank correlation test. C Receiver Operating Characteristic (ROC) curves and associated AUC value of each upregulated EV miRNAs. A higher area under the ROC curve (AUC) indicates superior model discrimination, ranging from 0 (none) to 1 (perfect). D Experimental scheme for analysis of EV miRNA level in BD patients and before-and-after treatment. E qRT-PCR results of plasma levels of miR-625-3p and miR-4488 according to disease activity of patients with BD. The difference between groups was assessed using ordinary one-way ANOVA. F Expression level of EV miRNAs from plasma of patients before (Pre) and after (Post) treatment. Paired t-test was used to compare the two groups. *P < 0.05

Fig. 3

Psoriatic basal keratinocytes as a source of EV miR-625-3p. A and B The expression of miR-625-3p in the skin detected using ISH in A low power field (× 100), and B high-power field (× 400). C Expression levels of miR-625-3p or miR-4488 measured by qRT-PCR after stimulation with 50 ng/ml IL-12 and/or 50 ng/ml IL-23 for 24 h in collected HaCaT or Jurkat cells. D The expression levels of miR-625-3p or miR-4488 in EVs from the cell medium detected by qRT-PCR after stimulation with 50 ng/ml of IL-12 and IL-23 for 24 h. Unpaired Student's t-test was used to compare the two groups. Data are representative of two independent experiments and values are expressed in means ± SEM. *P < 0.05 and **P < 0.01

Fig. 4

IGF-1 signalling in keratinocytes and its association to miR-625-3p. A Prediction of target gene and gene function using three miRNA databases (miRDB/TargetScan/miRTarBase). The overlapping predicted target genes from each database were subjected to GO term and KEGG pathway enrichment analysis. B The expression efficiency of miR-625-3p mimic validated using qRT-PCR analysis by HaCaT cells transfection with negative control mimics or miR-625-3p mimics. HaCaT cells were cultured for 48 h after transfection. C qRT- PCR results of insulin growth factor -1 receptor (IGF1R) and IGF-binding proteins gene expression. D IGFBP1 and IGFBP3 level quantified by western blotting 48 h after transfection. E Putative miR-625-3p binding sites in the 3’-UTR of human IGFBP3 mRNA. F Western blot analysis of the phospho-Akt protein level. G qRT-PCR results of Ki67 gene expression. H Cell viability of HaCaT cells transfected with negative control or miR-625-3p-mimic cultured for 24, 48, and 72 h determined using CCK analysis. Data are representative of two independent experiments and values are expressed in means ± SEM. Horizontal lines above bars indicate statistical comparisons with statistical differences between categories. Statistical analyses were performed by the unpaired Student's t-test (B–D, F and G) and 2-way ANOVA (H). *P < 0.05, **P < 0.01, ****P < 0.001 and ****P < 0.0001