DR JJ YAN (Orcid ID : 0000-0002-3546-6412)

Accepted Article
Article type : Original Article

Downregulation of miR-145-5p contributes to hyperproliferation of

keratinocytes and skin inflammation in psoriasis

Running head: Functions of miR-145-5p in psoriasis

J. J. Yan1, M. Qiao1, R. H. Li1, X. T. Zhao1, X. Y. Wang2, Q. Sun1△

Affiliations:

1
Department of Dermatology, Qilu Hospital, Shandong University.

2
Department of Dermatology, Qingdao Municipal Hospital (Group).

△Address correspondence to Sun Q: Department of Dermatology, Qilu Hospital, Shandong

University. 44 Wenhuaxi Road, Jinan, Shandong, China. Postal code: 250012, E-mail:
sunqing7226@163.com

Key words: Psoriasis, miR-145-5p, MLK3, chemokine, mouse model

This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/bjd.17256

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 Acknowledgments: This work was supported by the National Natural Science Foundation of China
(No. 8157120310).
Accepted Article
Conflict of Interest: The authors have no conflicts of interest to disclose.

What’s already known about this topic?

1. Psoriasis is a chronic, immune-mediated disease and has a strong genetic component. Aberrant
miRNA expression contributes to psoriasis development and progression.

2. MiR-145-5p has been studied in immune-mediated disease, but not in psoriasis.

What does this study add?

1. MiR-145-5p plays important functions in psoriasis by targeting MLK3.

2. Downregulation of miR-145-5p promotes keratinocytes proliferation and exacerbates skin

inflammation of psoriasis.

Downregulation of miR-145-5p contributes to hyperproliferation of

keratinocytes and skin inflammation in psoriasis

Summary

Background: The extensive involvement of microRNAs (miRNAs) in the pathogenesis of

psoriasis is well documented. However, little is known about the contribution of specific
miRNAs to the prevalence of this disease.

Objectives: Our study aimed to explore the role of miR-145-5p in psoriasis.

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 Methods: MiRNA microarray analysis was performed in 4 patients with psoriasis and 4 controls.
qRT-PCR and fluorescence in situ hybridization (FISH) were used to identify the dysregulated
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miRNAs. Luciferase assays were performed to determine whether miR-145-5p targets Mixed-lineage
kinase 3 (MLK3). Cell counting kit-8 (CCK-8) and Magnetic Luminex® assays were performed to
measure cell proliferation and chemokine secretion. Westernblot analysis was used to investigate
the protein levels of MLK3 and its downstream effectors. Mouse models of psoriasis were
established for in vivo experiments.

Results: MiR-145-5p was down-regulated in psoriasis lesional skin. Luciferase assays showed that
MLK3 is a direct target of miR-145-5p. Overexpression of miR-145-5p in normal human epidermal
keratinocytes (NHEKs) suppressed cell proliferation and secretion of chemokines. In contrast,
silencing miR-145-5p promoted NHEK proliferation and increased chemokine secretion. Silencing
MLK3 abrogated miR-145-5p inhibitor-induced promotion of cell proliferation and chemokine
expression. MiR-145-5p regulates NF-ҡB and STAT3 by targeting MLK3. Delivery of agomiR-145-5p
into the skin decreased epidermal hyperplasia and ameliorated psoriasis-like dermatitis. Delivery of
antagomiR-145-5p led to the opposite effects.

Conclusions: Our findings indicate that miR-145-5p negatively regulates proliferation and chemokine
secretion of NHEKs by targeting MLK3, and downregulation of miR-145-5p contributes to skin
inflammation in psoriasis lesions.

Introduction

Psoriasis is a chronic, immune-mediated disease that manifests in the skin or joints or both and
is characterized by red, scaly skin plaques.1,2 The prevalence rate of psoriasis is 2 to 3%, and it is a
lifelong disease that severely reduces the quality of life of affected individuals.3,4 Psoriasis was
recently proposed to result from a complex interplay among keratinocytes, immune cells, and
inflammatory mediators.5 Psoriasis also has a strong genetic component, and a growing number of
psoriasis susceptibility genes involved in immunity and keratinocyte functions have been
identified.6,7

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 MiRNAs are 18-22 nucleotide, single-stranded, noncoding small RNA molecules that modulate
protein expression by inhibiting mRNA translation and stability.8,9 Recently, miRNA dysregulation in
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the pathogenesis of psoriasis has attracted attention, and an increasing number of specific miRNAs
have been associated with the pathogenesis of psoriasis by regulating target genes.5,10,11

In this study, we performed a miRNA microarray and found that miR-145-5p was
downregulated in psoriasis. Recent studies suggest that miR-145-5p suppresses inflammation in
several inflammatory diseases.12-16 The potential regulatory mechanisms of miR-145-5p have been
investigated, and miR-145-5p was shown to regulate inflammatory diseases by modulating various
signalling pathways, such as the AKT/GSK pathway,15 the MAPK signalling pathway,16 and the mTOR
and NF-κB signalling pathway.17

However, the role of miR-145-5p in the pathogenesis of psoriasis remains unclear. It was
reported that MLK3 is a member of the serine/threonine kinase family and preferentially activates
JNK kinase. This kinase was also found to be involved in the transcriptional activity of NF-ҡB.
Furthermore, we predicted that MLK3 was a potential target gene of miR-145-5p. Therefore, we
investigated the role of miR-145-5p and MLK3 in psoriasis.

Materials and methods

Patients and tissue samples

Ten specimens were obtained from psoriasis vulgaris patients (five males, five females, aged
23-42 years) who had received no systemic treatments or phototherapy or externally used drugs for
at least 3 months before skin biopsies were collected from Qilu Hospital of Shandong University. Ten
normal skin biopsies were obtained from healthy volunteers (five males, five females, aged 25-40
years). We obtained biopsies by surgical operations, and the size of the skin biopsies was 1×0.5 cm.
The study was approved by the Ethics Committee of Shandong University, China, and all subjects
provided written informed consent.

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 MiRNA microarray
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Total RNA extracted from patients with psoriasis (n=4) and healthy volunteers (n=4) was analysed
using the Exiqon miRCURY LNA microRNA array, 7th generation (miRBase v18, condensed Probe_ID
version, Exiqon, Vedbaek, Denmark).

Cell isolation and culture

The skin specimens of young children’s foreskins were obtained from Qilu Hospital. The specimens
were digested with dispase II (Sigma, lot# BCBR9297V) at 4°C overnight to dissect the epidermis
from the dermis. Then, the epidermal specimens were digested with a 0.25% trypsin-0.01% EDTA
mixture (37°C, 10-15 min) to obtain single cell suspensions. Cells were grown and maintained in
keratinocyte medium (ScienCell, CA, USA) at 37°C in a humidified atmosphere of 5% CO2.

Cell transfection

Cells were transfected with miR-145-5p mimic (50 nM) or miR-145-5p mimic ctrl (50 nM);
miR-145-5p inhibitor (100 nM) or miR-548a-3p inhibitor ctrl (100 nM) (RiboBio, Guangzhou, China);
or siMLK3/ctrl (60 nM) (GenePharma, Shanghai, China) with Lipofectamine 2000 (Invitrogen,
Carlsbad, CA, USA) following the manufacturer’s instructions. Cell viability was determined by CCK-8
assays 48 h later (Fig. S1).

qRT-PCR

Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad,CA, USA). The miRNA expression
was detected according to the manual of the All-in-One miRNA qRT-PCR Detection System
(GeneCopoeia, Guangzhou, China). The primer sequences for miRNA and mRNA are listed in Tables
S1 and S2. GAPDH and 5.8S rRNA were used as internal controls for mRNA and miRNA, respectively.

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 FISH analysis of miR-145-5p expression in human and mouse tissues
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Diluted double CY3-labeled miR-145-5p probe (8 ng/ul, 5'-CY3-AGGGA TTCCT GGGAA AACTG

GAC-CY3-3'，Servicebio, Wuhan, China) was used for hybridization. DAPI (Servicebio, G1012) was
used for nuclear staining.

CCK-8 proliferation assay

Cell proliferation rates were measured at different time intervals using CCK-8 assays (Beyotime,
Shanghai, China) following the manual. Optical density (OD) values at 450 nm were measured using
a Synergy H1 Microplate Reader (BioTek, USA).

Luciferase reporter assay

The recombinant pmirGLO-MLK3-MUT and pmirGLO-MLK3-WT plasmids were purchased from

RiboBio (Guangzhou, China). Cells were co-transfected with miR-145-5p mimic or negative control
(50 nM) and pmirGLO-MLK3-WT or pmirGLO-MLK3-MUT (250 ng/well) with Lipofectamine 2000
(Invitrogen, Carlsbad, CA, USA). The luciferase value was detected using the Luc-Pair Duo-Luciferase
Assay Kit 2.0 (GeneCopoeia, Guangzhou, China) after 48 h.

Chemokine measurement using Magnetic Luminex® Assays

After 24 h of transfection as mentioned above, cells were stimulated by IL-17A (100 ng/ml), and
culture supernatants were collected after 12 h. Chemokines were measured using the Magnetic
Luminex® Assay multiplex kit (R&D Systems, MN, USA). Median fluorescence intensity was
determined using the Luminex® 200 analyser platform.

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 Western blotting
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Western blotting was performed as described in our previous experiments.18,19 The following
primary antibodies were used in this study: rabbit mAb MLK3 (1:1000, CST, #2817), NF-ҡB
p65(1:1000, CST, #8242), p- NF-ҡB p-p65 (1:1000, #3033, CST), p-STAT3 (1:2000, CST, #9145), GAPDH
(1:1000, Abcam), IL-17 (2 µg/ml, Abcam, ab79056), CCL2 (1:2000, Abcam, ab25124), CCL20 (0.2
µg/ml, Abcam, ab9829) and mouse mAb STAT3 (1:1000, CST, #9139).

Immunohistochemistry(IHC)

IHC was performed as previous described. 18,19 The primary antibodies, including Ki67 (1:500,
Servicebio, GB13030-2), CD3 (1:100, Servicebio, GB13014), IL-17A (1:1000, Servicebio, GB13111) and
MLK3 (1:1000, Santa Cruz), were used in this study.

IMQ-induced mouse models of psoriasis

IMQ (5% Aldara; 3 M Pharmaceuticals) mouse models of psoriasis were induced as reported
previously.20 Female C57BL/6J (n = 50) mice were purchased at 8 weeks of age from the animal
centre of Shandong University. The mice were divided into ten groups: the Ctrl + vehicle/IMQ group,
agomiR-145-5p + vehicle/IMQ group, agomiR Ctrl + vehicle/IMQ group, antagomiR-145-5p +
vehicle/IMQ group and antagomiR Ctrl + vehicle/IMQ group. Clinical scores were assessed by 3
independent researchers, who assigned scores of 0 to 4 (0, none; 1, mild; 2, moderate; 3, severe;
and 4, very severe) for erythema, scaling, and thickness. All animal experiments were approved by
the Ethics Committee of Shandong University.

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 In vivo miR-145-5p interference
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For overexpression or suppression of miR-145-5p expression in vivo, 1 nmol (diluted in 20 µl
phosphate buffered saline) of agomiR-145-5p/ctrl or antagomiR-145-5p/ctrl (RiboBio, Guangzhou,
China) was injected intradermally into the shaved back skin of each mouse every day for 3 days. The
skin samples were collected and stored at -80°C.

Statistical analysis

All data are summarized and presented as the mean ± standard deviation (SD) from at least 3
separate experiments. The differences between independent groups were analysed using Student's t
test with SPSS 17.0 analysis software (SPSS, Chicago, IL, USA) and GraphPad Prism 7.00 (La Jolla, CA,
USA). P < 0.05 was considered significant.

Results

MiRNA microarray validation and target prediction

Several studies have shown that aberrant miRNA expression contributes to psoriasis development
and progression. However, miRNA microarray analysis comparing psoriasis skin tissues and normal
controls is relatively rare. To identify new miRNAs associated with psoriasis and enrich the miRNA
expression profiles in psoriasis, we performed a miRNA microarray. According to our miRNA
microarray results of psoriasis samples, we found that 116 miRNAs were differentially expressed
(fold change ≥ 2.0, P <0.05) (Fig. 1a; Table S3). The top ten up- and downregulated miRNAs are listed
in Table S4. We selected eight of the most strongly downregulated miRNAs and several previously
described miRNAs (e.g., miR-21, miR-31, miR-146a) to validate using qRT-PCR (n = 10) (Fig. 1b) and
chose miR-145-5p for further study. We predicted its target genes using TargetScan, miRmap,
miRanda and miRTarbase and then focused on 39 genes with high possibility association with
miR-145-5p (Fig. 1c; Table S5). We performed pathway analysis using mirPath v.3 and found that

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 miR-145-5p was closely related to the MAPK signalling pathway (Table S6). According to these
results, we selected MLK3 as a chief candidate gene of miR-145-5p and explored their potential
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functions in psoriasis.

MLK3 expression levels in psoriatic lesional skin and healthy skin were detected by qRT-PCR
(n=10) and IHC analyses. As shown in Figure 1d, e, MLK3 expression was significantly upregulated in
psoriatic skins compared with that of the normal control.

FISH was used to detect the expression and distribution of miR-145-5p. As shown in Figure 1f,
we found that miR-145-5p expression levels were lower in the psoriasis lesional skin than in the
control skin.

MiR-145-5p targets MLK3

Luciferase assays were performed to determine whether miR-145-5p functioned by targeting MLK3.
Prediction of miR-145-5p targeting of MLK3 was performed by TargetScan (Fig. 2a). We mutated two
seed sites for miR-145-5p to form a mutant (MUT) 3'-UTR of MLK3 (Figs 2b, S2). Co-transfection with
the miR-145-5p mimic and pmirGLO-MLK3-WT led to a significant 32% reduction in reporter gene
activity compared to that of the control (P < 0.05). However, reporter gene activity showed no
significant change with pmirGLO-MLK3-MUT and the miR-145-5p mimic (Fig. 2c). These results
indicated that MLK3 is a direct target gene of miR-145-5p.

Proliferative effects of miR-145-5p and MLK3 on NHEKs

To study the effects of miR-145-5p on NHEK proliferation, we transfected miR-145-5p mimic/ctrl or

miR-145-5p inhibitor/ctrl into NHEKs through liposomes. As shown in Figure 3a, b, miR-145-5p
mimic/ inhibitor were successfully transfected and functional in the cells. CCK-8 proliferation assays

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 indicated that the miR-145-5p mimic suppressed the proliferation of NHEKs compared with the
negative control cells (Fig. 3c). However, the miR-145-5p inhibitor promoted the proliferation of
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NHEKs (Fig. 3d).

NHEKs were transfected with siMLK3 or siMLK3 ctrl to detect the potential effect of MLK3 on
cell proliferation. The qRT-PCR results showed that MLK3 was successfully inhibited by siMLK3 (Fig.
3b). The results of the CCK-8 proliferation assays demonstrated that silencing MLK3 suppressed
NHEK proliferation (Fig. 3e).

Effects of miR-145-5p and MLK3 on chemokine secretion after IL-17A stimulation

IL-17 is involved in the regulation of proinflammatory chemokines and plays a central role in the
pathophysiology of immune-mediated diseases such as psoriasis.21 Therefore, in this study, we
investigated the role of miR-145-5p and MLK3 in the IL-17 signalling pathway.

NHEKs were transfected as mentioned above, and treated with IL-17A to assess its effect on
chemokine secretion. Though the IL-17 signalling pathway (KEGG database), we found eight
chemokines, CCL2, CCL7, CCL20, CXCL1, CXCL2, CXCL5, CXCL8 and CXCL-10, produced by NHEKs after
IL-17A stimulation. Then, we performed Magnetic Luminex® Assays to dissect the role of miR-145-5p
and MLK3 in the NHEK response to IL-17A. The results indicated that both the miR-145-5p mimic and
siMLK3 inhibited chemokine secretion after IL-17A treatment (Fig. 4a, b). Conversely, the
miR-145-5p inhibitor promoted the secretion of chemokines (Fig. 4a).

The effects of miR-145-5p on cell proliferation and chemokine secretion are mediated by

MLK3

To analyse the role of MLK3 in the anti-proliferative and anti-inflammatory effects of miR-145-5p in
NHEKs, we manipulated the expression of MLK3 using MLK3 siRNA. NHEKs were divided into four

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 groups: the miR-145-5p inhibitor group, the inhibitor ctrl group, the miR-145-5p inhibitor+siMLK3
group (co-transfected with miR-145-5p inhibitor and siMLK3) and the miR-145-5p inhibitor
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ctrl+siMLK3 group (co-transfected with miR-145-5p inhibitor ctrl and siMLK3). Then, the cells were
treated with 100 ng/mL IL-17A for 12 h. The NHEK proliferative rates and expression levels of
chemokines were measured by CCK-8 assays and Magnetic Luminex® Assays, respectively. As shown
in Figure 5a, we found that the enhanced proliferative effect of the miR-145-5p inhibitor was
significantly abrogated by siMLK3. As shown in Figure 5b, the miR-145-5p inhibitor-mediated
promotion of chemokine secretion in NHEKs was significantly abrogated by siMLK3. These results
indicated that the effects of miR-145-5p on cell proliferation and chemokine secretion were
mediated by MLK3.

Exploration of miR-145-5p and MLK3 effects on the NF-ҡB and STAT signalling pathway

Previous published observations showing that MLK3 functions by regulating downstream proteins,
including NF-ҡB and STATs, prompted us to investigate their relationship with miR-145-5p.22-24 To
study the effects of miR-145-5p on MLK3 protein expression, we delivered the miR-145-5p
mimic/ctrl or miR-145-5p inhibitor/ctrl into NHEKs through liposomes. The MLK3 levels in NHEKs
were determined by Western blotting 48 h after transfection. As shown in Figure 6a, MLK3 protein
expression in the miR-145-5p mimic group was strongly decreased (P < 0.01). However, in the
miR-145-5p inhibitor group, MLK3 protein expression significantly increased compared with that in
the ctrl groups (P < 0.01).

We performed Western blotting to detect the interference efficiency of siMLK3, as shown in

Figure 6b. MLK3 was significantly inhibited by siMLK3. To study the effects of MLK3 on NF-ҡB,
p-NF-ҡB, STAT3 and p-STAT3 protein expression, we delivered siMLK3 ctrl or siMLK3 into NHEKs
through liposomes. The NF-ҡB, p-NF-ҡB, STAT3 and p-STAT3 protein expression in the siMLK3 ctrl
and siMLK3 groups were determined by Western blotting 48 h after transfection. As shown in Figure
6c, the expression of NF-ҡB and STAT3 showed no significant difference (P > 0.05). However, the
p-NF-ҡB and p-STAT3 expression levels were strongly decreased compared with those in the ctrl

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 groups. As expected, miR-145-5p knocked down the expression of MLK3, whereas interfered with
MLK3 knockdown of the p-NF-ҡB and p-STAT3 expression levels. These results suggest that
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miR-145-5p likely regulates the NF-ҡB and STAT signalling pathways by targeting MLK3 in psoriasis.

MiR-145-5p suppresses epidermal hyperplasia and inflammatory cell infiltration in the

epidermis of mouse models

IMQ-induced skin inflammation is a well-characterized mouse model for psoriasis. To characterize

the effects of miR-145-5p on epidermal proliferation and skin inflammation in psoriasis, we
intradermally injected agomiR-145-5p/ctrl and antagomiR-145-5p/Ctrl into the shaved back skin of
the mice for 3 consecutive days. Then, mice were topically treated with IMQ as mentioned above.
The results of FISH and qRT-PCR showed that miR-145-5p was overexpressed in the agomiR-145-5p
+vehicle/IMQ groups and inhibited in the antagomiR-145-5p +vehicle/IMQ groups mouse skins (Figs
7a, S3). As shown in Figure 7b-i, using the IHC, qRT-PCR and Western blot analyses, we found that
the expression levels of MLK3, CCL2, CCL20 and IL-17 were upregulated in the skin of IMQ-induced
mouse models and negatively related to miR-145-5p. On day 5, vehicle-treated mice in different
groups did not show apparent signs of skin inflammation (Figs 8a, S4); mice of the agomiR-145-5p +
vehicle/IMQ groups had significantly decreased skin thickness and severity scores compared with
agomiR-145-5p Ctrl + vehicle/IMQ group mice (Fig. 8b); in contrast, antagomiR-145-5p + vehicle/IMQ
group mice had increased skin thickness and more obvious skin inflammation than the
antagomiR-145-5p Ctrl + vehicle/IMQ group mice (Fig. 8b). Histologic analysis showed IMQ-induced
epidermal thickening, which was further suppressed by agomiR-145-5p and enhanced by
antagomiR-145-5p. IHC analyses showed increased epidermal hyperplasia in the antagomiR-145-5p +
vehicle/IMQ groups based on a significantly increased number of Ki67+ epidermal cells (Fig. 8c). IHC
analyses for the T cell and Th17 markers CD3 and IL-17A also showed that IMQ increased the
number of CD3+ and IL-17A+ cells in the skin, and a significantly higher increase was observed in the
antagomiR-145-5p + vehicle/IMQ group. In contrast, the levels of CD3+ and IL-17A+ cells in the skins
of mice were significantly decreased in the agomiR-145-5p + vehicle/IMQ group (Fig. 8C). In
conclusion, local delivery of agomiR-145-5p alleviated epidermal hyperplasia and psoriasiform skin
inflammation; delivery of antagomiR-145-5p led to increased epidermal thickness and exacerbated
skin inflammation (Fig. 9).

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 Discussion
Accepted Article
MiRNAs are important gene regulatory molecules that target multiple mRNAs and regulate
numerous cellular processes, and miRNA dysregulation frequently occurs in many types of
inflammatory diseases, including psoriasis.25,26 MiRNA microarrays, which are widely used as an
efficient method for massive miRNA analysis, are commonly used in the detection of miRNA
expression profiles. In this study, our miRNA microarray results showed that miR-145-5p was one of
the most significantly downregulated miRNAs in psoriasis lesions. Of those dysregulated miRNAs,
miR-145-5p was especially interesting because it was proved to be closely related to immune
mediated diseases. However whether miR-145-5p contributes to the pathogenesis of psoriasis
remains unknown.

Psoriasis is a chronic inflammatory disease. It is now widely accepted that IL-23/IL-17A axis
plays a central role in the development of psoriasis. Several studies have shown that miR-145-5p
inhibits cell proliferation and immune response.15,27 We identified a protective association of
miR-145-5p with psoriasis and downregulation of miR-145-5p contributes to keratinocytes
proliferation and skin inflammation. Subsequent functional studies revealed that miR-145-5p
inhibited the proliferation of NHEKs and changed the chemokine expression profiles of NHEKs after
stimulation with IL-17A. We further confirmed that MLK3 is a downstream target gene of
miR-145-5p.

In this study, using CCK-8 proliferation assays, we confirmed that miR-145-5p negatively
regulates the proliferation of NHEKs. To investigate the function of miR-145-5p in the IL-17 signalling
pathway, we manipulated the expression levels of miR-145-5p in NHEKs and then stimulated them
using IL-17A. In the miR-145-5p inhibitor group, the chemokine expression levels were significantly
increased. These results indicated that downregulation of miR-145-5p increased keratinocytes
proliferation and promoted IL-17-mediated inflammation.

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 We detected that MLK3 was expressed at a higher level in psoriasis tissues. MLK3 plays
important roles not only in regulation of inflammatory mediator secretion by controlling the
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transcription activity of JNK and NF-ҡB28-31 but also in the mitogen regulation of B-Raf and ERK.32-34
Silencing MLK3 in NHEKs significantly attenuated the cell proliferation and chemokine secretion
promotion of the miR-145-5p inhibitor. These results suggested that the anti-proliferative and
anti-inflammatory roles of miR-145-5p were mediated by MLK3.

We found that the expression levels of IL-17, CCL2 and CCL20 were significantly upregulated in
the skin of IMQ-induced mouse models compared with that of the ctrl group. Through in vivo
miR-145-5p interference, we found that overexpression of miR-145-5p in the epidermis suppressed
epidermal hyperplasia and inflammatory cell infiltration. Conversely, silencing of miR-145-5p in the
epidermis led to exacerbated pathology of skin inflammation, increased epidermal hyperplasia and
CD3+ and IL-17A+ cell infiltration. These results indicated that miR-145-5p might play a protective role
in psoriasis.

In conclusion, we demonstrated that miR-145-5p could suppress the proliferation and

chemokine secretion of NHEKs by regulating the expression of MLK3. Furthermore, we identified a
protective role for miR-145-5p in psoriasis by regulating downstream proteins, including STAT3 and
NF-ҡB. To our knowledge, this is the first study to investigate the function of miR-145-5p and MLK3
in psoriasis, and we identified miR-145-5p as a potential therapeutic target for psoriasis in the clinic.

Acknowledgements

This study was supported by The National Natural Science Foundation of China (No. 8157120310).
We would like to acknowledge the helpful comments on this paper received from our reviewers.

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 34 Chadee DN, Kyriakis JM. MLK3 is required for mitogen activation of B-Raf, ERK and
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Supporting Information

Fig. S1. Cell viability of was determined by the CCK-8 assays.

Fig. S2. Plasmid vector structure (a) and sequencing results (b).

Fig. S3. MiR-145-5p expression was analysed with qRT-PCR.

Fig. S4. Function analysis of miR-145-5p in vehicle-treated mice. (a) Macroscopic images and HE
staining of skin sections obtained from mice of different groups on day 5 (n=5). (b) Severity scores
(n=5). (c) Quantification of epidermal thickness (n=5). (d) IHC for MLK3, Ki67, CD3 and IL-17A (n=5).
(e) MLK3 protein expression levels shown by the mean optical density (MOD) (n=5). (f-h)
Quantification of Ki67+, CD3+ and IL-17A+ cells in the epidermis (n=5). (i-l) The mRNA expression
levels of MLK3, CCL2, CCL20 and IL-17A in the skin tissues. (m-n) The expression of MLK3, CCL2 ,
CCL20 and IL-17.

Table S1. MiRNA primers used for qRT-PCR.

Table S2. mRNA primers used for qRT-PCR.

Table S3. All differentially expressed miRNAs.

Table S4. Top 10 up-/down-regulated differentially expressed miRNAs.

Table S5. The targets intersection of miR-145-5p.

Table S6. Pathways related to miR-145-5p analysis using mirPath v.3.

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 Figure Legends
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Fig. 1. Microarray result validation and target prediction of miR-145-5p. (a) A total of 116 miRNAs
were differentially expressed (fold change ≥ 2.0, P <0.05). (b) Eight of the most strongly
downregulated and three previous described miRNAs were validated using qRT-PCR (n = 10). (c)
Target prediction of miR-145-5p was performed using TargetScan, miRmap, miRanda and
miRTarbase. (d) MLK3 (mRNA) expression was detected using qRT-PCR (n=10). (e) MLK3 protein
expression levels in different groups shown by the mean optical density (MOD) (n=10). (f) The
expression of miR-145-5p was detected using FISH; miR-145-5p (red) and nuclei (blue). The results
are based on three independent experiments. The expression levels of miRNA and MLK3 were
calculated using the 2-ΔΔCt method, and the results are plotted as relative fold changes. Scale bars =
50 μm. Data in (b, d, e) are expressed as the mean value ± standard deviation. Comparisons between
two groups were performed with Student’s t test. *indicates P < 0.05, ** indicates P < 0.01, ***
indicates P < 0.001.

Fig. 2. MLK3 is a direct target gene of miR-145-5p. (a) The potential miR-145-5p seed regions at the
3'UTR of MLK3 mRNA predicted by TargetScan. (b) The vector sequence needs to be constructed
(double mutant). (c) Cells were co-transfected with pmirGLO-MLK3-WT+miR-145-5p mimic/ctrl or
co-transfected with pmirGLO-MLK3-MUT+miR-145-5p mimic/ctrl; luciferase activity was normalized
by the ratio of Renilla luciferase signals and firefly signals. The significance of luciferase activity was
analysed using Student’s t test; * indicates comparison with ctrl, P < 0.05.

Fig. 3. Effect of miR-145-5p and siMLK3 on NHEK proliferation in vitro. (a) MiR-145-5p expression
levels were detected by qRT-PCR 24 h after transfection with miR-145-5p mimic/ctrl or inhibitor/ctrl.
(b) MLK3 (mRNA) expression levels were detected by qRT-PCR 24 h after transfection with
miR-145-5p mimic/ctrl, inhibitor/ctrl or siMLK3/ctrl. (c-e) Proliferation assays of NHEKs were
performed after transfection with miR-145-5p mimic/ctrl, miR-145-5p inhibitor/ctrl or siMLK3/ctrl at
0, 24, 48 and 72 h by CCK-8 assays. Optical density (OD) values at 450 nm of each point were
calculated from 3 independent experiments and are presented as the mean ± SD. (* indicates P <
0.05, ** indicates P < 0.01, *** indicates P < 0.001).

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 Fig. 4. Effects of miR-145-5p and MLK3 on chemokine secretion after IL-17A stimulation. (a)
Magnetic Luminex® Assays of the secretion of CCL2, CCL7, CCL20, CXCL1, CXCL2, CXCL5, CXCL8 and
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CXCL-10 in the miR-145-5p overexpression/inhibition or ctrl group; (b) or siMLK3/ctrl group after
treatment with IL-17A at 100 ng/mL for 12 h. Data in (a, b) are presented as the mean value ±
standard deviation. * P<0.05; ** P<0.01; *** P< 0.001 vs. miR-145-5p mimic ctrl or inhibitor ctrl
group.

Fig. 5. The effects of miR-145-5p on cell proliferation and chemokine secretion are mediated by
MLK3. (a) Proliferation assays of NHEKs were performed after transfection with the miR-145-5p
inhibitor/ctrl or co-transfection with siMLK3 at 0, 24, 48 and 72 h by CCK-8 assays. (b) Magnetic
Luminex® Assays of the secretion of CCL2, CCL7, CCL20, CXCL1, CXCL2, CXCL5, CXCL8 and CXCL-10 in
the miR-145-5p inhibitor/ctrl or miR-145-5p inhibitor/ctrl +siMLK3 group after treatment with IL-17A
at 100 ng/mL for 12 h. The results are based on three independent experiments. Comparisons
between two groups (miR-145-5p inhibitor group vs miR-145-5p inhibitor+siMLK3 group and
miR-145-5p inhibitor ctrl group vs miR-145-5p inhibitor ctrl+MLK3 group) were performed with
Student’s t test. * P<0.05; **P<0.01; *** P< 0.001.

Fig. 6. MiR-145-5p negatively regulated MLK3 protein expression and affects the NF-ҡB and STAT
signalling pathways. (a) NHEKs were transfected with miR-145-5p mimic/ctrl or inhibitor/ctrl, and
then, MLK3 expression levels were evaluated by Western blotting 48 h later. (b-c) The siCtrl or siMLK
was delivered into NHEKs through liposomes. The MLK3, NF-ҡB, p-NF-ҡB, STAT3 and p-STAT3 protein
expression levels in the siCtrl and siMLK3 groups were determined by Western blotting 48 h after
transfection. GAPDH was used as a control. The band intensity values were analysed using ImageJ.
Experiments were performed independently three times. (* indicates P < 0.05, ** indicates P < 0.01,
*** indicates P< 0.001).

Fig. 7. In vivo miR-145-5p interference. (a) The miR-145-5p expression levels in the skin tissues of
different groups were detected using FISH, miR-145-5p (red) and nuclei (blue). (b) IHC for MLK3 in
the epidermis (n=5). (c) MLK3 protein expression levels in different groups shown by the mean

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 optical density (MOD) (n=5). (d-g) The mRNA expression levels of MLK3, CCL2, CCL20 and IL-17A in
the skin tissues of different groups. (h) The expression of MLK3, CCL2, CCL20 and IL-17 in
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IMQ/vehicle-treated mouse skin tissues was detected using Western blot analysis. (i) In IMQ-induced
mouse models, effects of agomiR-145-5p and antagomiR-145-5p on its target gene MLK3 and related
chemokines were detected by Western blot. Scale bars = 50 μm. *indicates P < 0.05, ** indicates P <
0.01, *** indicates P < 0.001.

Fig.8. MiR-145-5p alleviates epidermal hyperplasia and psoriasiform skin inflammation. (a)
Macroscopic images of back skin and HE staining of skin sections obtained from mice of different
groups on day 5 (n=5). (b) Quantification of epidermal thickness of HE-stained back skin sections
(n=5) (left); cumulative scores of erythema, scaling, and skin thickness of different groups on day 5
(n=5) (right). (c) IHC for Ki67, CD3 and IL-17A in the epidermis (n=5) (up); quantification of Ki67+,
CD3+ and IL-17A+ cells (down) in the epidermis (n=5). Scale bars = 50 μm. *indicates P < 0.05, **
indicates P < 0.01, *** indicates P < 0.001.

Fig.9. A hypothetic model for the contribution of miR-145-5p to the pathogenesis of psoriasis.
Downregulation of miR-145-5p in the back skin of IMQ-induced mouse models led to increased
epidermal thickness and exacerbated skin inflammation.

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