Circ_0061012 contributes to IL-22-induced proliferation, migration and invasion in keratinocytes through miR-194-5p/GAB1 axis in psoriasis

Abstract Psoriasis is a chronic inflammation-associated skin disorder featured by excessive proliferation and abnormal differentiation of keratinocytes. Here, we intended to investigate the role of circular RNA 0061012 (circ_0061012) in psoriasis progression. The expression of circ_0061012, SLMO2-ATP5E readthrough (SLMO2-ATP5E) messenger RNA (mRNA), microRNA-194-5p (miR-194-5p) and GRB2 associated binding protein 1 (GAB1) mRNA was determined by quantitative real-time polymerase chain reaction (qRT-PCR). Cell proliferation and metastasis were analyzed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and transwell assays. Western blot assay was used to measure the protein levels of Ki67, matrix metallopeptidase 9 (MMP9) and GAB1. Dual-luciferase reporter assay and RNA immune co-precipitation (RIP) assay were used to verify the interaction between miR-194-5p and circ_0061012 or GAB1. Circ_0061012 abundance was significantly enhanced in lesional skin samples from psoriasis patients than that in normal skin specimens from healthy volunteers. Interleukin-22 (IL-22) treatment increased the expression of circ_0061012 in a dose-dependent manner. Circ_0061012 silencing alleviated IL-22-induced promoting effects in the proliferation, migration and invasion of HaCaT cells. Circ_0061012 interacted with miR-194-5p, and miR-194-5p knockdown counteracted circ_0061012 silencing-mediated influences in IL-22-induced HaCaT cells. GAB1 was a target of miR-194-5p in HaCaT cells, and miR-194-5p hampered proliferation and metastasis which were induced by IL-22 partly through targeting GAB1. Circ_0061012 elevated the expression of GAB1 through sponging miR-194-5p in HaCaT cells. Circ_0061012 accelerated IL-22-induced proliferation and metastasis in HaCaT cells through enhancing GAB1 expression via sponging miR-194-5p in psoriasis.


Introduction
Psoriasis is an autoimmune skin disorder characterized by hyper-proliferation, aberrant motility and differentiation of keratinocytes and the infiltration of inflammatory cells [1][2][3]. Immune, genetic and environmental factors are all responsible for the pathogenesis of psoriasis [4]. Pro-inflammatory cytokines secreted by immune cells promote the proliferation of keratinocytes [5].  belongs to IL-10 superfamily [6], and the messenger RNA (mRNA) level of IL-22 was positively correlated with the severity of psoriasis [7,8]. Here, we established psoriasis cell model through stimulating keratinocytes HaCaT with IL-22.
Compared with linear RNAs, circular RNAs (circRNAs) are stably distributed due to their covalently closed loop structure [9]. CircRNAs exert crucial functions in aging, tissue development, cancers and diseases [10]. Circular RNA 0061012 (Circ 0061012) was highly expressed in the lesional skin samples compared with healthy skin samples [11]. Nevertheless, the precise role of circ 0061012 in psoriasis progression remains to be discovered.
MicroRNAs (miRNAs) were found to modulate the development of psoriasis [12,13]. Numerous miRNAs were abnormally expressed in the lesional tissues of psoriasis patients [14,15]. Yu et al. proved that miR-194 overexpression hampered the proliferation while accelerated the differentiation of keratinocytes through GRHL2 in psoriasis [16]. Nevertheless, the exact role of miR-194-5p in psoriasis is not fully addressed.
GRB2 associated binding protein 1 (GAB1) acts as the docking protein to facilitate the growth and differentiation of epithelium [17]. GAB1 was observed to regulate the migration ability of keratinocytes, implying GAB1 might exert essential role in psoriasis progression [18]. Liu et al. claimed that miR-183-3p restrained the growth and migration abilities of HaCaT cells via inhibiting GAB1 in psoriasis [19]. We discovered the potential mechanism behind the role of GAB1 in psoriasis.
In the present study, the expression pattern and biological role of circ 0061012 in psoriasis were explored. Subsequently, the downstream targets of circ 0061012 were predicted by bioinformatics software to further illustrate the working mechanism of circ 0061012 in psoriasis progression.

Clinical samples
Twenty-seven psoriasis patients along with twenty-seven healthy volunteers were enrolled in Hubei Provincial Hospital of Traditional Chinese Medicine. Four-millimeter punch biopsies were collected from psoriatic lesional skin tissues of psoriasis patients. Punch biopsies collected from the healthy skin tissues were utilized as control group. All cases had signed informed consents. The protocols were approved by the Ethic Committee of Hubei Provincial Hospital of Traditional Chinese Medicine.

IL-22 stimulation
When cell confluence reached approximately 80%, HaCaT cells were pre-treated in serum-free medium for 24 h, and these cells were exposed to increased doses of IL-22 (Sangon Biotech) for an additional 24 h.

Subcellular fractionation location and RNase R digestion
Cytoplasmic/nuclear RNA isolation was implemented with the PARIS™ Kit Protein and RNA Isolation system (Thermo Fisher Scientific, Waltham, MA, U.S.A.). Cell supernatant was utilized to measure the levels of cytoplasmic RNAs, and nuclear pellet was collected to determine the levels of nuclear RNAs. U6 small nuclear RNA and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were utilized as nuclear endogenous control and cytoplasmic endogenous control, respectively. RNA levels were determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR). The distribution ratio of cytoplasm to nucleus was analyzed through dividing the expression of circ 0061012 in cytoplasmic fraction by the expression of circ 0061012 in nuclear fraction.
Total RNA was digested with 3 U/μg RNase R (Epicentre Technologies, Madison, WI, U.S.A.) for 40 min at room temperature. RNA levels were measured via qRT-PCR.

qRT-PCR
To measure the enrichment of circ 0061012, SLMO2-ATP5E readthrough (SLMO2-ATP5E) mRNA and GAB1 mRNA, moloney murine leukemia virus reverse transcriptase (Takara, Dalian, China) was used to synthesize complementary DNA (cDNA). The reverse transcription of miR-194-5p was conducted using the TaqMan microRNA reverse transcription kit (Applied Biosystems, Carlsbad, CA, U.S.A.). SYBR Green Master Mix Kit (Bio-Rad, Hercules, CA, U.S.A.) and primers (shown in Table 1) were used for amplification reaction. The RNA expression was analyzed using the 2 − C t method. GAPDH served as the internal control for circ 0061012, SLMO2-ATP5E mRNA and GAB1 mRNA, while miR-194-5p enrichment was normalized to U6.

MTT assay
HaCaT cells were seeded into 96-well plates to settle down. The next day, at appropriate time points, 20 μl

Dual-luciferase reporter assay
Bioinformatics software (StarBase) was used to predict the targets of circ 0061012 and miR-194-5p. The fragment of circ 0061012 or the 3 untranslated region (3 UTR) of GAB1, including the binding sites with miR-194-5p, along with these fragments with mutant miR-194-5p-binding sites, were cloned into pmirGLO (Promega, Madison, WI, U.S.A.). HaCaT cells were seeded into 24-well plates. The next day, these cells were co-transfected with luciferase reporter plasmids and miR-194-5p or miR-NC. After 48 h of transfection, luciferase activity was determined with the Dual-Luciferase Reporter Assay System (Promega).

RNA immune co-precipitation assay
HaCaT cells were lysed and cell lysates were incubated with protein-A Sepharose beads (Bio-Rad) which pre-coated with Argonaute-2 (Ago2) antibody (ab32381; Abcam) or Immunoglobulin G (IgG) antibody for 3 h. The complexes were enriched by protein-A Sepharose beads. After isolating with TRIzol reagent (Thermo Fisher Scientific), qRT-PCR was implemented for the determination of RNA expression.

Statistical analysis
Statistical analysis of three independent experiments with at least three technical repetitions was conducted using GraphPad Prism 7.0 (GraphPad, La Jolla, CA, U.S.A.), and the statistical data were expressed as mean + − standard deviation. Differences were evaluated by Student's t-test (two groups) or one-way analysis of variance (ANOVA) followed by Tukey's test (multiple groups). Correlation analysis was conducted using Spearman's correlation analysis test. P-value less than 0.05 was considered as statistically significant.

Circ 0061012 is highly expressed in the lesional skin of psoriasis patients and IL-22-induced HaCaT cells
The up-regulated expression of circ 0061012 was observed in the lesional skin tissues of psoriasis patients (n=27) relative to healthy skin tissues of normal volunteers (n=27) ( Figure 1A). Meanwhile, circ 0061012 expression was significantly enhanced in HaCaT cells upon IL-22 treatment in a dose-dependent manner as shown in Figure 1B. Prior to investigate the contribution of circ 0061012 in psoriasis progression, we explored its subcellular distribution in HaCaT cells. U6 acted as nuclear marker while GAPDH was used as cytoplasmic marker. As shown in Figure 1C, circ 0061012 was mainly distributed in the cytoplasm of HaCaT cells. RNA samples isolated from HaCaT cells were divided into two equal parts, and these two parts were treated with RNase R nor not. The expression of circ 0061012 remained unaffected while the level of its linear form was notably reduced after RNase R digestion ( Figure 1D). Overall, circ 0061012 was highly expressed in psoriasis patients and it was mainly distributed in the cytoplasmic fraction of HaCaT cells.

IL-22 promotes cell proliferation, migration and invasion of HaCaT cells through up-regulating circ 0061012
To

Circ 0061012 interacts with miR-194-5p in HaCaT cells
StarBase database was used to predict the targets of circ 0061012, and miR-194-5p was predicted as a possible target of circ 0061012. The complementary sequence between circ 0061012 and miR-194-5p was listed in Figure 3A. To validate the hypothesis that miR-194-5p interacted with circ 0061012, we conducted dual-luciferase reporter assay and RNA immune co-precipitation (RIP) assay. MiR-194-5p accumulation resulted in a significant reduction in luciferase activity in WT-circ 0061012 group compared with miR-NC group ( Figure 3B), suggested that miR-194-5p was a target of circ 0061012 in HaCaT cells. Meanwhile, compared with miR-NC and MUT-circ 0061012 group, luciferase activity was unaffected in miR-194-5p and MUT-circ 0061012 group ( Figure 3B), suggested that circ 0061012 interacted with miR-194-5p via the complementary sites shown in Figure 3A. Compared with IgG group, both miR-194-5p and circ 0061012 were pulled-down in Ago2 group ( Figure 3C), demonstrating the interaction between miR-194-5p and circ 0061012. There was a notable down-regulation in miR-194-5p expression in psoriasis lesional tissues relative to healthy skin tissues ( Figure 3D). MiR-194-5p expression was negatively correlated with the level of circ 0061012 ( Figure 3E). With the increased dose of IL-22, miR-194-5p expression was gradually decreased in HaCaT cells ( Figure  3F). IL-22-induced up-regulation in circ 0061012 expression was further enhanced with the addition of circ 0061012 overexpression plasmid in HaCaT cells ( Figure 3G). IL-22 stimulation decreased the abundance of miR-194-5p, and its expression was partly recovered in IL-22 and si-circ 0061012 group ( Figure 3H). Besides, IL-22-induced down-regulation in miR-194-5p expression was further potentiated with the addition of circ 0061012 in HaCaT cells ( Figure 3H). These findings suggested that miR-194-5p was a target of circ 0061012, and miR-194-5p was negatively regulated by circ 0061012 in HaCaT cells.

MiR-194-5p interacts with the 3 UTR of GAB1 in HaCaT cells
GAB1 was predicted as a target of miR-194-5p by StarBase software ( Figure 5A). HaCaT cells were co-transfected with miR-NC or miR-194-5p and WT-GAB1 3 UTR or MUT-GAB1 3 UTR. Luciferase activity was prominently reduced in miR-194-5p and WT-GAB1 3 UTR group rather than miR-194-5p and MUT-GAB1 3 UTR group ( Figure  5B), suggesting the interaction between miR-194-5p and GAB1 in HaCaT cells. Meanwhile, miR-194-5p and GAB1 were both enriched in Ago2 group relative to IgG group ( Figure 5C), which further verified the target interaction between miR-194-5p and GAB1 in HaCaT cells. GAB1 mRNA level was prominently enhanced in skin tissues of psoriasis patients when compared with healthy volunteers ( Figure 5D). The results of Western blot assay revealed that GAB1 protein level was gradually up-regulated with the increased concentration of IL-22 in HaCaT cells ( Figure  5E). There was a negative correlation between the expression of miR-194-5p and GAB1 ( Figure 5F)

Circ 0061012 up-regulates the level of GAB1 through sponging miR-194-5p in IL-22-induced HaCaT cells
Given the negative regulatory relationship between miR-194-5p and circ 0061012 or GAB1, we subsequently aimed to investigate the modulatory relationship between circ 0061012 and GAB1. HaCaT cells were transfected with si-NC, si-circ 0061012, si-circ 0061012 + anti-miR-NC or si-circ 0061012 + anti-miR-194-5p prior to IL-22 stimulation. The mRNA and protein abundance of GAB1 was enhanced with IL-22 treatment, and this effect was attenuated with the silencing of circ 0061012 ( Figure 7A,B). Circ 0061012 silencing-mediated reduction in the mRNA and protein levels of GAB1 was counteracted by the addition of anti-miR-194-5p ( Figure 7A,B), suggested that circ 0061012 enhanced the abundance of GAB1 mRNA and protein through sponging miR-194-5p in HaCaT cells upon IL-22 treatment. As shown in Figure 7C, we concluded that circ 0061012 elevated the expression of Ki67 and MMP9 to promote psoriasis progression through targeting miR-194-5p/GAB1 axis.

Discussion
We explored the biological contribution of circ 0061012 in psoriasis progression. Circ 0061012 expression was notably enhanced in the lesional samples relative to healthy skin specimens. Loss-of-function experiments demonstrated that IL-22 accelerated the proliferation and metastasis of HaCaT cells partly through up-regulating circ 0061012. Subsequently, circ 0061012/miR-194-5p/GAB1 signal axis was identified to be involved in the pathogenesis of psoriasis. Our study discovered for the first time the crucial role of circ 0061012 in the proliferation and metastasis of keratinocytes.
CircRNAs modulate the progression of human diseases. For instance, circ-BANP accelerated the development of lung cancer through targeting miR-503/LARP1 axis [20]. Circ 0000950 accelerated neuron apoptosis, inhibited the outgrowth of neurite and enhanced the levels of inflammation-related cytokines via miR-103/PTGS2 axis in Alzheimer's disease [21]. A large number of circRNAs were dysregulated in psoriasis specimens compared with healthy skin specimens, and circ 0061012 was one of the circRNAs that were up-regulated in psoriasis [11]. Consistent with former article [11], we found that circ 0061012 expression was elevated in psoriasis lesional specimens compared with healthy specimens. Also, circ 0061012 level was increased in IL-22-induced HaCaT cells in a dose-dependent manner. IL-22 stimulation promoted cell proliferation, migration and invasion of HaCaT cells, and these effects were largely attenuated by the silencing of circ 0061012, demonstrated that IL-22 promoted psoriasis progression partly through enhancing circ 0061012 expression.
MiRNAs are involved in the regulation of psoriasis progression [12,13]. Xu et al. found that miR-155 contributed to the proliferation and suppressed cell apoptosis through regulating PTEN signal pathway in psoriasis [22]. Tang et al. demonstrated that the down-regulation of miR-187 accelerated the proliferation of keratinocytes in psoriasis [23]. MiR-194-5p was predicted to be a binding partner of circ 0061012 via StarBase database, and this interaction was confirmed by dual-luciferase reporter assay and RIP assay. MiR-194-5p restrained the malignant behaviors of many cancer cells [24][25][26]. Wang et al. claimed that miR-194-5p suppressed the motility of bladder cancer cells through E2F3 [24]. MiR-194 was reported to suppress cell proliferation and induce cell differentiation of keratinocytes via GRHL2 [16]. MiR-194-5p expression was declined in psoriasis specimens relative to healthy skin specimens. Furthermore, IL-22 treatment dose-dependently decreased the level of miR-194-5p in HaCaT cells. Compensation experiments revealed that circ 0061012 silencing restrained IL-22-induced proliferation and metastasis in HaCaT cells through targeting miR-194-5p.
The interaction between miR-194-5p and GAB1 was verified by dual-luciferase reporter assay and RIP assay. GAB1 facilitated the inflammation and fibrosis in systemic sclerosis [27]. GAB1 was abnormally expressed in sclerosis and psoriasis [28]. GAB1 contributed to the proliferation and differentiation of epidermal cells [17]. GAB1 expression was aberrantly up-regulated in lesional skin tissues of psoriasis patients and IL-22-induced HaCaT cells compared with that in healthy skin tissues and un-treated HaCaT cells. Rescue experiments suggested that miR-194-5p restrained proliferation and motility of HaCaT cells upon IL-22 treatment through targeting GAB1. Circ 0061012 acted as the molecular sponge of miR-194-5p to elevate the expression of GAB1 in HaCaT cells.
In conclusion, circ 0061012 contributed to IL-22-induced proliferation and motility of HaCaT cells through targeting miR-194-5p/GAB1 axis, providing a novel insight into developing new therapeutic targets for psoriasis patients.

Data Availability
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.