miR-183-3p suppresses proliferation and migration of keratinocyte in psoriasis by inhibiting GAB1

Background MicroRNAs (miRNAs) target genes involved in the hyperproliferation of keratinocytes or immune dysfunction of psoriasis. This study prospectively determined the involvement of miR-183-3p in the pathogenesis of psoriasis. Methods Differentially expressed miR-183-3p between psoriatic lesional and non-lesional skin were determined by quantitative RT-PCR and in situ hybridization (ISH). CCK8 and wound healing assays were performed to assess cell viability and migration of human keratinocyte cell line (HaCaT). The target of miR-183-3p was validated by luciferase activity assay. Results Lower miR-183-3p expression was observed in psoriatic lesional skin compared to psoriatic non-lesional skin. MiR-183-3p over-expression inhibited the viability and migration of HaCaT cells, while inhibition of miR-183-3p promoted the viability and migration of HaCaT cells. Moreover, miR-183-3p could bind to the 3′ UTR of GAB1 (growth factor receptor binding 2-associated binding protein 1) and decrease the mRNA and protein expression of GAB1 in HaCaT cells. In addition, higher GAB1 expression was observed in psoriatic lesional skin than psoriatic non-lesional skin. Conclusion MiR-183-3p exhibited inhibition property in the proliferation and migration of HaCaT cells via down-regulation of GAB1, suggesting the potential therapeutic strategy for psoriasis.


Background
Psoriasis is a common chronic inflammatory disease, with increasing incidence and a prevalence between 1 and 3% worldwide [1]. The etiology and pathogenesis of psoriasis is complicated and still unclear. Currently, it is believed that the pathogenesis of psoriasis mainly involves immune, genetic, psychological and environmental factors [2]. The main pathological changes of psoriasis are keratinocyte dysplasia, keratinosis, neovascularization and inflammatory cell infiltration [3]. Meanwhile, hyperproliferation and abnormal migration of keratinocytes, responsible for psoriasis lesional microenvironment, are critical features of psoriasis [4]. Considering the fact that psoriasis is easy to relapse and difficult to cure, exploringnew therapeutical targets involved in the proliferation and migration of keratinocytes is beneficial for the cure of psoriasis.
MiRNAs (microRNAs), with a length of about 18-25 nucleotides, are a class of non-coding single-stranded small RNA molecules that are highly conserved in evolution and ubiquitous in plants and animals [5]. MiRNAs account for 1-5% of the entire human genome and regulate the expression of 30% of protein-coding genes in human [5]. MiRNAs could bind to the 3'UTR of a specific target gene mRNA through base complementary pairing to degrade its target mRNA or inhibit its translation, thereby negatively regulate protein expression at post-transcriptional levels [5]. Moreover, miRNAs have the abilities to regulate cell proliferation and immune response, are critically implicated in the pathogenesis of immunological disorders, including psoriasis [6]. For example, Sonkoly et al. found that miR-203 could inhibit the expression of suppressor of cytokine signaling-3, which was involved in the inflammatory response of keratinocyte [7]. MiR-125b reduced the expression of fibroblast growth factor receptor 2 to regulate the proliferation and differentiation of keratinocytes [8]. MiR-4516 could reduce the proliferatory and migratory abilities of keratinocytes toameliorate psoriasis [9].
MiR-183-3p was found to be associated with chronic systolic heart failure [10] and lung adenocarcinoma [11]. More recently, miR-183-5p was identified as a mediator of inflammatory disease and involved in the chronic constriction injury-induced neuropathic pain [12],. Besides, down-regulation of miR-183-3p was also discovered in psoriatic skin [13]. However, the role and mechanism of miR-183-3p in psoriasis remains elusive. Therefore, this study aimed to examine the functional role of miR-183-3p in the proliferation and migration of keratinocytes, and identified the underlying mechanism, thus providing more potential therapeutic strategy for psoriasis.

MiR-183-3p was down-regulated in psoriatic lesional skin
To evaluate the expression level of miR-183-3p in psoriatic patients, 41 paired psoriatic lesional (LS) or nonlesional (Con) skin biopsies were collected and qRT-PCR was peformed. Result showed a significant reduction of miR-183-3p in LS compared to Con (Fig. 1a). Moreover, ISH showed that higher expression of miR-183-3p in Con group was found in basal and suprabasal cell layers than that in LS group (Fig. 1b). In general, miR-183-3p was down-regulated in psoriatic lesional skin.

GAB1 was up-regulated in psoriatic lesional skin
Considering the negative corrlation between miR-183-3p and GAB1, the expression of GAB1 in psoriatic lesional skin was then determined. Results fromqRT-PCR ( Fig. 4a) and immunohistochemistry (Fig. 4b) showed that GAB1 was elevated in LS group compared to Con group. Moreover, the correlation analysis indicated a significantly negative correlation (p = 0.0033) between miR-183-3p and GAB1 in psoriatic patients (Fig. 4c).

Discussion
Increasing evidence has revealed that miRNAs were abnormally expressed in psoriasis, and regulate immune dysfunction and keratinocytes proliferation in psoriasis [6]. Results from this study revealed the downregulation of miR-183-3p in psoriatic lesional skin, which were consistent with a previous report [13], indicating its regulatory role in the progression of psoriasis.
Bcl-2 interacting protein 3 like [10] or high-mobility group nucleosome binding domain 5 [24] were validated as binding targets of miR-183-3p and involved in chronic systolic heart failure or prostate cancer, respectively. The direct binding target involved in miR-183-3pmediated psoriasis remains elusive. This is the first study identifing GAB1 as a binding target of miR-183-3p during psoriasis. GAB1 functions as a docking protein to promote the proliferation and differentiation of epidermis [25]. Moreover, GAB1 was found to be involved in the migration of keratinocyte, as well as wound healing, suggesting its potential role in the pathogenesis of psoriasis [26]. Recently, GAB1 was shown to be aberrantly expressed in multiple sclerosis and psoriasis [27]. Results from this study showed that GAB1 was up-regulated in psoriatic lesional skin, and suggested a significantly negative correlation with miR-183-3p. In addition, miR-183-3p could repress the mRNA and protein expression of GAB1, suggesting that miR-183-3p could suppress the proliferation and migration of keratinocyte in psoriasis by inhibiting GAB1. Ras [25] and phosphoinositide 3-kinase pathway [26] were closely associated with GAB1-mediated epidermal differentiation and cell proliferation/migration in cultured skin keratinocytes, respectively. The underlying mechanism related to miR-183-3p/GAB1 axismediated psoriasis needs further investigations.

Conclusion
In conclusion, miR-183-3p effectively suppressed the viability and migration of keratinocytes through downregulation of GAB1. These results suggested the beneficial effect of miR-183-3p knockdown during psoriasis.

Patients and specimens
Forty-one paired lesional or non-lesional skin biopsies (5 mm) were collected from patients who diagnosed as psoriasis at the Affiliated Hospital of North Sichuan Medical College. The protocol was approved by the Ethics Committee of Affiliated Hospital of North Sichuan Medical College, and all patients signed written informed consent.

In situ hybridization
Formalin-fixed and paraffin-embedded sections (5 μm in thickness) of the biopsy specimens were firstly incubated with acetylation solution for 10 min, and then incubated with permeabilization buffer for 30 min. After prehybridizing at 50°C for 1 h, the sections were hybridized with digoxygenin-labeled miRCURY locked nucleic acid probes (Exiqon, Vedbaek, Denmark) overnight. After incubation with alkaline phophatase-conjugated sheep antidigoxigenin Fab fragments (1:3000, Abcam, Cambridge, MA, USA) for 1 h, the sections were visualized under inverted microscope (Nikon Eclipse Ti, Tokyo, Japan).

Immunohistochemistry
Formalin-fixed and paraffin-embedded sections (0.5 μm in thickness) of the biopsy specimens were incubated with rabbit antihuman GAB1 antibody (1:500, Abcam) overnight. After incubation with horse radish peroxidase-goat anti-rabbit secondary antibody, the sections were visualized under inverted microscope.

Wound healing
HaCaT cells (1 × 10 4 cells/well) were seeded and then transfected with miR-183-3p mimics/inhibitor (20 nM) or their NC. After transfection for 48 h, each well was scratched and then cultured for another 24 h. Wound area was photographed and calculated.

qRT-PCR
RNAs or miRNAs were isolated from psoriatic lesional or non-lesional skin and HaCaT cells via Trizol (Thermo Fisher). Total RNAs or miRNAs were then reversetranscribed into cDNAs via High Capacity Reverse Transcription System Kit (Takara, Dalian, China) or Micro-RNA Reverse Transcription Synthesis Kit (Thermo Fisher). qRT-PCR was conducted with SYBR Green Master (Roche, Mannheim, Germany) on Applied Biosystems 7500 Real-time PCR Systems (Thermo Fisher). GAPDH or U6 was used as endogenous control with the following primer sequences (Table 1).

Statistical analysis
Data were shown as mean ± SEM and analyzed by Graphpad Prism 6. The statistical analyses were determined with one-way analysis of variance at significance of * p < 0.05, ** p < 0.01 or *** p < 0.001. The correlation between GAB1 and miR-183-3p expression was determined by Spearman's correlation coefficients.