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.
Supplementary Information
The online version contains supplementary material available at
10.1186/s12967-024-05030-z.
Keywords: Biomarkers, Extracellular vesicles, Keratinocytes, microRNAs,
miR-625-3p, Psoriasis
Background
Psoriasis is a relatively common chronic inflammatory disease
characterized by pathogenic hyperproliferation of keratinocytes and
immune dysregulation [[39]1]. Despite the evolution of disease
treatment, tools for evaluating disease activity are still lacking. The
only currently widely used clinical subjective assessment tools to
evaluate psoriasis are the Psoriasis Area and Severity Index (PASI) and
body surface area (BSA) [[40]2]. Unfortunately, these are subjective
evaluation tools that show relatively high inter-rater variability
[[41]3]. Moreover, the recent coronavirus disease 2019 breakout has
underscored the need for patients to receive clinical services remotely
[[42]4]. Thus, the need for objective markers that reflect the severity
of psoriasis has emerged. Candidate biomarkers including cytokine,
chemokines and adipokines have been studied exhaustively, yet none have
become part of current routine practice. [[43]5]
Extracellular vesicles (EVs) are small structures composed of a
phospholipid bilayer derived from the cell membrane. Specifically, EVs
are classified into exosomes (30–150 nm), micro-vesicles (100–1500 nm),
and apoptotic bodies (500–2000 nm), depending on their size and
biogenesis pathway [[44]6]. If EVs are released into the extracellular
space, they can facilitate communication between cells by transporting
their bioactive contents, which include nucleic acids, proteins, and
various microRNAs (miRNAs), to the recipient cells [[45]6–[46]8]. EV
miRNAs are found in different biofluids, cells, and tissues [[47]7].
These EV miRNAs have demonstrated potential as diagnostic markers of
various inflammatory diseases including psoriasis. [[48]9–[49]11]
Given the noticeable scarcity of studies focusing on objective markers
for psoriasis severity, particularly those utilizing EV miRNAs, our
objective was to identify a reliable biomarker of psoriasis disease
activity using plasma-derived EV miRNAs. We analyzed the levels of EV
miRNAs in both the plasma and skin of patients and identified
keratinocytes as the origin of differentially expressed miRNA (DE
miRNA) in the EVs. Furthermore, we showed that the keratinocyte-derived
miRNA promotes keratinocyte proliferation, a hallmark of psoriasis.
Methods
Study subjects, acquisition of biospecimens, and study flow
This study included patients diagnosed with psoriasis between May 2016
and March 2021, and patients diagnosed with Behçet’s disease (BD)
between April 2016 and May 2020. The diagnosis of psoriasis was made
based on clinical characteristics and histological features on skin
biopsy. None of the patients with psoriasis received systemic treatment
or phototherapy at the time of sample collection. PASI and BSA were
also evaluated on the day of sampling. Patients with BD met the
International Criteria for BD [[50]12] and the Japanese Criteria for BD
[[51]13]. BD patients were chosen as a control group for our research,
given their shared autoinflammatory traits and T-helper 1 and T-helper
17 dominant immune response [[52]1, [53]13]. Plasma samples were
collected from 63 patients with psoriasis (8 patients for screening, 42
for validation, and 20 for pre- and post-treatment comparisons; some
patients participated in screening, validation, and pre-and
post-treatment comparisons) and 12 patients with BD.
The blood samples for obtaining the patient's plasma were collected in
an EDTA-containing tube and immediately centrifuged at 2000 × g for
10 min at 4 °C to remove cellular components. The plasma samples were
divided into aliquots in microcentrifuge tubes and stored at −80 ℃ in a
deep freezer until used for experimental analysis. Tissue samples from
psoriatic lesions were obtained using a 3-mm disposable biopsy punch,
and snap-frozen tissue samples from patients were stored in a nitrogen
tank at the Ajou University Hospital Human Biobank until analysis.
Using plasma samples from eight patients (five patients with PASI ≥ 10
and three with PASI < 5), we performed next-generation sequencing (NGS)
of miRNAs to screen DE miRNAs. Forty patients were further enrolled to
validate the screened DE miRNAs (two patients participated in both
screening and validation). We performed real-time quantitative reverse
transcription-polymerase chain reaction (qRT-PCR) to validate the DE
miRNAs identified by screening DE miRNAs using NGS. Skin samples were
obtained from 18 patients who consented to additional tissue collection
at the time of the biopsy. The tissue samples were homogenized in
QIAzol lysis Reagnet (Qiagen, Hilden, Germamy) using TissueLyser II
(Qiagen, Hilden, Germamy). Total RNA was extracted using the
TRIzol-based extraction method.
Isolation of EVs, RNA extraction, and NGS of miRNAs
After thawing the plasma, it was centrifuged for 10 min at 300 × g and
4 ℃, followed by centrifugation at 10,000 × g for 30 min and 4 ℃ for
30 min. From the supernatants, EVs were extracted using the miRCURY
exosome isolation kit (Qiagen, Hilden, Germany) and total miRNA was
extracted from the exosomes using a miRNA isolation kit (miRNeasy
Serum/Plasma Kit; Qiagen, Hilden, Germany) according to the
manufacturer's protocols. The total RNA quantity and quality were
assessed by spectrophotometry (NanodropTM ND-1000, Thermo Fisher
Scientific, Copenhagen, Denmark). miRNA sequencing was performed by
Theragen Bio Co. Ltd. (Suwon, South Korea). Please see “Supplementary
Materials and Methods” for a detailed description of the process.
Size exclusion chromatography and ultracentrifugation
As described previously, EVs from the selected plasma samples of
psoriasis patients were isolated using mini-size exclusion
chromatography (mini-SEC) [[54]14]. Briefly, plasma was centrifuged for
10 min at 300 × g and 4 ℃, followed by centrifugation at 10,000 × g for
30 min, 4 ℃ for 30 min. The supernatant obtained after the second
centrifugation was loaded on the Sepharose CL-2B (Sigma Aldrich, St.
Louis, MO, USA) column. The flow-through was collected in 6 fractions
of 1 ml each. The fractions containing EVs (fractions #4, #5, and #6)
were pooled and transferred to a new ultracentrifuge tube and then
ultracentrifuged at 100,000 × g for 70 min using an SW32 Ti rotor
(Beckman Coulter, California, USA) to pellet EVs. The pellets of EVs
were resuspended in TRIzol reagent (Thermo Fisher Scientific,
Copenhagen, Denmark) for RNA isolation in phosphate-buffered saline
(PBS) for transmission electron microscopy and nanoparticle tracking
analysis. Additional details regarding experimental materials and
methods can be found in the “Supplementary Materials and Methods”
section.
Statistical analysis
All data are presented as mean ± standard deviation. Statistical
analyses were performed using GraphPad Prism 9.3.1 (GraphPad Software
Inc., La Jolla, CA, USA) and the R statistical program 4.2.1 (R
Foundation for Statistical Research, Vienna, Austria). When comparing
two groups, we used an unpaired Student's t-test, applying Welch's
correction when an F-test indicated differing variances between the
sample groups. A paired t-test was used to compare values of miRNA
levels between pre-treatment and post-treatment samples. Spearman's
correlation test was used to assess the relationship between the
validated miRNAs and their association with PASI and BSA scores. For
multiple comparisons, we employed an unpaired one-way analysis of
variance (ANOVA). Statistical significance was established at P < 0.05.
Results
Hsa-miR-625-3p expression is significantly increased in both the plasma and
skin of patients with psoriasis and is associated with PASI and BSA
The baseline demographics of patients with psoriasis are summarised in
Table [55]1. The EV miRNAs extracted from 8 patients (clinical
characteristics described in Table [56]1 and characteristics of
isolated EVs shown in Additional file [57]1: Figure S1) underwent NGS
to identify candidate miRNAs (Fig. [58]1A, blue arrows). Out of the 805
miRNAs detected, we identified 19 DE miRNAs, including 9 upregulated
and 10 downregulated miRNAs (Fig. [59]1B and Additional file [60]1:
Table S1). We carefully chose three DE miRNA candidates based on their
fold change and p-value: miR-625-3p, miR-4488, and miR-342-3p from the
upregulated miRNAs, and miR-5698, miR-1255b-5p, and miR323a-5p from the
downregulated miRNAs. To validate possible miRNAs for use as
biomarkers, we performed qRT-PCR of the selected miRNAs with a larger
group of patients (Fig. [61]1A, red arrows), classified by their PASI
score (PASI < 5, 5 ≤ PASI < 10, and PASI ≥ 10, with each group
consisting of 14 patients). Although the selected downregulated miRNAs
did not display significant expression differences between the groups
(Fig. [62]1C), all the chosen upregulated miRNAs exhibited significant
increases between the PASI < 5 and the PASI ≥ 10 groups. Furthermore,
both miR-4488 and miR-625-3p showed significant differences between the
PASI < 5 and 5 ≤ PASI < 10 groups (Fig. [63]1D). In particular,
miR-625-3p showed a significant difference between the PASI < 5 and
5 ≤ PASI < 10 groups, as well as between the PASI < 5 and PASI ≥ 10
groups using EV miRNAs isolated from the skin (Fig. [64]1E). miR-625-3p
was the only EV miRNA from the skin with a strong association
(ρ ≥ 0.60) [[65]24] between both PASI and BSA (Fig. [66]1F), whereas
other miRNAs demonstrated limited associations. (miR-4488 and PASI:
Spearman’s ρ = 0.5862, p = 0.0236; miR-342-3p and PASI: Spearman’s
ρ = 0.3718, p = 0.1719; Additional file [67]1: Figure S2).
Table 1.
Demographic and clinical characteristics of the patients with psoriasis
Patient numbers 3 5 p-value 14 14 14 p-value 10 10 p-value
Characteristics Screening Validation Treatment response
PASI < 5 10 ≥ PASI 0 < PASI < 5 5
[MATH: ≤ :MATH]
PASI < 10 10 ≥ PASI Poor responders* Good responders*
Age (mean ± SD, yr) 36.3 ± 5.6 52.6 ± 15.4 0.1124 36.3 ± 9.3
40.1 ± 13.4 37.1 ± 11.6 0.6837 39.8 ± 15.8 41.7 ± 15.5 0.7890
Sex (M:F) 2:1 4:1 7:7 10:4 8:6 7:3 7:3
BMI (mean ± SD, kg/m^[68]2 or lb/ inches
[MATH: × :MATH]
703) 24.2 ± 1.1 25.5 ± 1.0 0.2032 25.5 ± 4.2 24.1 ± 3.3 26.4 ± 5.1
0.3755 26.5 ± 3.2 25.4 ± 2.4 0.3888
Initial PASI (mean ± SD) 3.9 ± 0.5 13.6 ± 1.9 0.0002 3.5 ± 1.0
6.5 ± 1.0 18.9 ± 6.0 < 0.0001 10.8 ± 5.1 13.1 ± 5.7 0.3251
Initial BSA (mean ± SD, %) 4.7 ± 2.1 23.4 ± 4.0 0.0003 6.1 ± 4.2
9.4 ± 5.3 31.9 ± 12.3 < 0.0001 15.7 ± 16.1 20.6 ± 11.5 0.4457
Disease duration (yr) 6.3 ± 3.3 12.8 ± 8.9 0.2486 10.5 ± 8.8 10.0 ± 8.3
12.5 ± 9.8 0.7575 10.8 ± 5.9 13.8 ± 5.2 0.2430
Psoriasis onset age(mean ± SD, yr) 30.0 ± 2.4 39.8 ± 11.8 0.1749
25.8 ± 7.3 30.1 ± 11.8 24.7 ± 13.8 0.4484 29.0 ± 16.3 27.9 ± 16.3
0.8816
Joint involvement (%) 0 0 > 0.9999 0 0 14.2 0.1225 0 0 > 0.9999
Nail involvement (%) 66.7 80 > 0.9999 21.4 14.2 42.9 0.2017 50 40
> 0.9999
[69]Open in a new tab
SD, standard deviation; BMI, body mass index; PASI, Psoriasis Area and
Severity Index; BSA, body surface area. All participants in the study
were of Asian ethnicity. 'Good responders' were defined as patients who
achieved a PASI 50 response, while 'poor responders' were those who did
not achieve a PASI 50 response following treatment. To assess the
differences between the groups, Welch’s t-test was employed for the
screening phase and for the evaluation of treatment response. One-way
ANOVA was utilized in the validation phase. For comparisons of joint
and nail involvement, Fisher’s exact test was used for the screening
phase and for evaluating treatment response, while the Chi-square test
was applied in the validation phase
Fig. 1.
[70]Fig. 1
[71]Open in a new tab
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
EV miR-625-3p most accurately differentiates mild and moderate-to-severe
psoriasis, is psoriasis-specific, and reflects treatment response.
We also confirmed a significant association between EV miR-625-3p and
PASI (Fig. [72]2A). All selected miRNAs showed a significant
correlation (p < 0.0001) between their expression levels and PASI, but
miR-625-3p showed an exceptionally high level of correlation. A similar
result was observed in its correlation with BSA; it had the highest
correlation coefficient value compared to other miRNAs (Fig. [73]2B).
In the receiver operative characteristic (ROC) curve, the area under
the curve (AUC) value was also the highest for miR-625-3p, with an
exceptionally high value of 0.9515, thereby confirming its diagnostic
value as a biomarker for differentiating mild-to-moderate psoriasis
(PASI < 10) from moderate-to-severe psoriasis (PASI ≥ 10)
(Fig. [74]2C). Based on these results, we narrowed the potential
biomarkers to miR-625-3p and miR-4488. To exclude the possibility that
the miRNAs are markers of inflammation, which is not
psoriasis-specific, we analyzed the EV miRNAs of BD patients
(Fig. [75]2D, blue arrows). When comparing patients with active BD and
those with inactive BD, albeit statistically insignificant
(p = 0.0593), we observed an increasing tendency in miR-4488 in
symptomatic BD patients (Fig. [76]2E). The results suggest that
miR-4488, in contrast to miR-625-3p, is likely not specific to
psoriasis. To confirm whether the EV miRNA levels depicted a treatment
response, we collected blood samples from 10 patients each pre- and
post-treatment (Fig. [77]2D, red arrows). The clinical characteristics
of each group are shown in Table [78]1. Good responders, defined as
patients who achieved a 50% reduction in PASI score (PASI50) following
treatment, exhibited a significant reduction in levels of miR-625-3p.
In contrast, the poor responders (patients who did not achieve a PASI50
after treatment) showed no significant change. No significant changes
were observed in miR-4488 for both groups. (Fig. [79]2F). Taken
together, miR-625-3p was the only biomarker that precisely represented
psoriasis activity.
Fig. 2.
[80]Fig. 2
[81]Open in a new tab
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
EV miR-625-3p originates from activated keratinocytes
To confirm that miR-625-3p from EVs, rather than complexed circulating
miR-625-3p, serves as a novel biomarker of psoriasis severity, we
performed mini-size exclusion chromatography (which is also known to
isolate EVs effectively) [[82]25] and compared the relative expression
of miR-625-3p for each method (Additional file [83]1: Figure S3). The
expression results revealed a significantly strong correlation (Pearson
correlation coefficient = 0.8501), indicating that EV miR-625-3p
accurately reflected psoriasis severity. Our results also.
Since EV miR-625-3p reflected the treatment response and was highly
correlated with PASI, we hypothesized that EV miR-625-3p originated
from lesional psoriatic skin. To discern the anatomical location of
miR-625-3p present in the skin, we performed in situ hybridization
(ISH) on the paraffin-embedded skin tissue of patients. Interestingly,
miR-625-3p was detected in basal keratinocytes but not in infiltrating
immune cells (Fig. [84]3A). In a high-power field, miR-625-3p was
mainly observed in basal keratinocytes' cytoplasm and extracellular
matrix (Fig. [85]3B), in contrast to miR-4488, which exhibited diffuse
epidermal staining. We therefore hypothesized that miR-625-3p
originates from psoriatic basal keratinocytes. To mimic psoriatic
conditions, we stimulated HaCaT cells (human keratinocyte cell line)
and Jurkat cells (human T lymphocyte cell line) with IL-12 and IL-23
(both 50 ng/mL), which showed a significant increase in miR-625-3p
expression in keratinocytes, not T cells (Fig. [86]3C). EV miR-625-3p
was also increased in the supernatants of stimulated keratinocytes,
whereas miR-4488 was not (Fig. [87]3D). These results suggest that
activated keratinocytes are capable of miR-625-3p production and
secretion.
Fig. 3.
[88]Fig. 3
[89]Open in a new tab
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
miR-625-3p induces keratinocyte proliferation via IGF-1/Akt signalling
interference
The specific target of miR-625-3p in psoriasis has not yet been
identified. Using multiple miRNA databases (Additional file [90]1:
Figure S4), we investigated a possible role in psoriasis pathogenesis,
which revealed its possible association with insulin-like growth factor
(IGF), particularly with insulin-like growth factor binding
(Fig. [91]4A). Thus, using a miR-625-3p mimic (625-3p mimic), which was
successfully transfected to keratinocytes (Fig. [92]4B), we analyzed
the expression levels of insulin-like growth factor binding protein
(IGFBP) associated genes and proteins. There was a significant increase
in IGF1R and decreases in IGFBP2 and IGFBP3 mRNA expression levels
(Fig. [93]4C). Interestingly, we observed that the protein expression
of IGFBP3, and not IGFBP1, was significantly decreased following
treatment with the 625-3p mimic (Fig. [94]4D). Using bioinformatic
prediction, we verified the potential miR-625-3p binding site in the
human IGFBP3 mRNA (Fig. [95]4E). These findings suggested the
possibility of augmented IGF-1 signalling, which was confirmed by a
significant increase in phosphorylated Akt (Fig. [96]4F). Given that
enhanced IGF-1signalling is associated with cell proliferation
[[97]26], we evaluated the expression of Ki-67 mRNA, and noted a
significant elevation (Fig. [98]4G). We also observed a significant
increase in cell numbers within the miR-625-3p mimic transfected cell
population (Fig. [99]4H).
Fig. 4.
[100]Fig. 4
[101]Open in a new tab
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
Discussion
More than 250 DE miRNAs have been identified in the skin or blood of
psoriasis patients [[102]8, [103]9]. miRNAs are small single-stranded
non-coding RNA molecules vulnerable to external stimuli and
degradation. However, plasma-derived EV miRNAs are likely more stable,
as their encasement within a phospholipid bilayer protects them from
degradation in the bloodstream [[104]5, [105]27]. Thus, we focused on
plasma-derived EV miRNAs identified as stable markers of multiple
diseases [[106]28, [107]29], as candidate markers of psoriasis
severity.
EV microRNAs have been extensively investigated as potential biomarkers
in various diseases, such as autoimmune diseases, cancer, and
neurodegenerative diseases [[108]30–[109]32]. They can reflect the
secreting cells' characteristics and provide early disease detection
opportunities. We identified a single miRNA, miR-625-3p, as a biomarker
to determine psoriasis disease activity. miR-625-3p levels were
increased in EVs as well as in the skin of patients with psoriasis.
While miR-625-3p has been studied previously in cancers such as oral
squamous cell carcinoma and malignant melanoma [[110]33–[111]35],
studies on EV miR-625-3p are limited. Previous reports showed a
decrease in miR-625-3p levels in psoriatic skin compared to
non-psoriatic skin, in contrast to our results [[112]36, [113]37]. This
discrepancy might have resulted from the differences in patient
characteristics and experimental settings, such as the lack of EV
extraction and separation of the epidermis from the dermis [[114]38].
Further evaluation with more patients in a unified experimental setting
is necessary for the universal use of our disease severity marker.
To our knowledge, this is the first study to localize the site of
miR-625-3p expression in skin. Further, we showed that cytokine-treated
KCs could excrete EV miR-625-3p, confirming the origin of the EV miRNA.
Considering the proximity of the basal keratinocytes, where EV
miR-625-3p is likely secreted, with dermal blood vessels in psoriasis,
it seems plausible that these keratinocyte-derived EVs are secreted
into the dilated blood vessels and are detectable in circulation.
Recent studies showed that psoriatic keratinocyte-derived EVs can
stimulate neutrophils to produce inflammatory cytokines, proliferate
resting T cells, and provide immune-stimulatory abilities
[[115]38–[116]40]. We present a novel association between IGF-1
signalling and EV miR-625-3p expression in our study. IGF signalling is
known for its role in keratinocyte proliferation [[117]26, [118]41].
Furthermore, IGFBP3 has been suggested as a factor contributing to
epidermal hyper-proliferation in psoriasis [[119]42]. Collectively,
miR-625-3p may contribute to the pathogenesis of psoriasis through
interference with IGF-1 signalling.
Despite the exceptionally high likelihood of disease severity
differentiation (based on our AUC value), our study has a few
limitations. First, it was conducted in a single center, leading to a
relatively small sample size of only one ethnicity. Also, the gender
distribution and age range of included patients could potentially be
affected by participation bias. Future research needs to confirm the
validation of EV miR-625-3p in a larger population with various races
and different clinical environments. Second, EV miRNA profiling among
samples can be affected by biological and technical variations,
including the EV extraction kit’s selectivity, which can lead to
inconsistencies between specimens. Third, our study lacked sufficient
longitudinal data to reveal the dynamic changes in EV miR-625-3p
expression. Future research is imperative to monitor these changes,
particularly in the context of long-term therapeutic interventions.
Lastly, while we presented a putative target of miR-625-3p, we have not
yet elucidated the exact in vivo mechanism of its regulation in
psoriasis pathogenesis. We are currently investigating the specific
target(s) and their mechanisms in the disease pathogenesis.
Conclusions
EV miR-625-3p, originating from psoriatic keratinocytes, reflects both
the severity of psoriasis and the response to treatment. As there is a
current and urgent need for an objective biomarker detectable in the
blood that accurately reflects psoriasis disease status, we propose EV
miR-625-3p as a potentially useful biomarker for assessing disease
activity. This biomarker also holds the potential for being a novel
target for psoriasis therapy.
Supplementary Information
[120]12967_2024_5030_MOESM1_ESM.docx^ (445.8KB, docx)
Additional file 1: Figure S1. Isolation of plasma EVs by
ultracentrifugation (A) NTA demonstrating the size distribution of EVs
diluted samples of plasma-derived EVs using ultracentrifugation. (B)
Representative TEM image of plasma-derived EVs showing a membrane
structure composed of a lipid bilayer (Bar = 200 nm). NTA; nanoparticle
tracking analysis; EV; extracellular vesicle; TEM; transmission
electron microscope. Figure S2. miR-4488 and miR-342-3p from the
psoriatic lesional skin show a weak-to-moderate association with PASI
and BSA scores. Skin miR-4488 and miR-342-3p levels plotted against
PASI and BSA. The significance of the correlation was tested using
Spearman's rank correlation test. *P < 0.05. PASI, Psoriasis Area and
Severity Index; BSA, body surface area. Figure S3. EV miR-625-3p
correlates across different isolation methods (A) Representative TEM
image of plasma-derived EVs using mini-size exclusion chromatography
(Bar = 200 nm). (B) Relative expression levels of EV miR-625-3p
isolated using miRCURY exosome Kit showing a strong positive
correlation with relative expression of EV miR-625-3p isolated after
mini-size exclusion chromatography. Result shown represent combined
data of two experiments. The significance of the correlation was tested
using the Pearson's correlation test. ****P < 0.0001. Figure S4. Venn
diagram showing number of predicted gene-targets for miR-625-3p using
three different algorithms (miRDB/TargetScan/miRTarBase). The top 100
overlapping genes (6 genes overlapping all three, 94 genes overlapping
any two) were chosen for target prediction. Table S1. DE microRNA
candidates* identified from next-generation sequencing (NGS). Table S2.
miRNA and mRNAPrimers Used for RT-qPCR.
Acknowledgements