Cycloastragenol restrains keratinocyte hyperproliferation by promoting autophagy via the miR-145/STC1/Notch1 axis in psoriasis

Abstract Background Psoriasis is characterized by inflammation and hyperproliferation of epidermal keratinocytes. Cycloastragenol (CAG) is an active molecule of Astragalus membranaceus that potentially plays a repressive role in psoriasis. Activated cell autophagy is an effective pathway for alleviating psoriasis progression. Thus, we investigated the role of CAG in the proliferation and autophagy of interleukin (IL)-22-stimulated keratinocytes. Methods A psoriasis model was established by stimulating HaCaT cells with IL-22. Gene or protein expression levels were measured by qRT-PCR or western blot. Autophagy flux was observed with mRFP-GFP-LC3 adenovirus transfection assay under confocal microscopy. Stanniocalcin-1 (STC1) secretion levels were determined using ELISA kits. The apoptosis rate was assessed using flow cytometry. Interactions between miR-145 and STC1 or STC1 and Notch1 were validated by luciferase reporter gene assays, RIP, and Co-IP assays. Results CAG repressed cell proliferation and promoted apoptosis and autophagy in IL-22-stimulated HaCaT cells. Additionally, CAG promoted autophagy by enhancing miR-145. STC1 silencing ameliorated autophagy repression in IL-22-treated HaCaT cells. Moreover, miR-145 negatively regulated STC1, and STC1 was found to activate Notch1. Lastly, STC1 overexpression reversed CAG-promoted autophagy. Conclusion CAG alleviated keratinocyte hyperproliferation through autophagy enhancement via regulating the miR-145/STC1/Notch1 axis in psoriasis.


Introduction
Psoriasis is a chronic inflammatory skin disease that negatively affects patients' physiological and mental health, leading to a substantially decreased quality of life [1].It is induced by excessive inflammation and characterized by the abnormal proliferation and dysfunctional differentiation of epidermal keratinocytes [2].Current psoriasis treatments involve inflammatory agent inhibitors and regulators targeting keratinocytes.But the treatments are restricted by recurrence and adverse effects.Autophagy is a highly conserved lysosomal regulatory system of cell homeostasis that degrades and utilizes misfolded proteins or injured organelles [3].Thus, autophagy activation ameliorates inflammation and keratinocyte hyperproliferation to alleviate psoriasis [4].Enhanced apoptosis also repressed keratinocyte hyperproliferation and disease development [5,6].The specific mechanism of autophagy and apoptosis in keratinocyte proliferation has yet to be fully elucidated.Thus, urgent research focusing on this area is necessary to identify novel drugs or cellular targets to manage this disease.
Cycloastragenol (CAG) is a dominant active molecule in Astragalus membranaceus, which is a traditional medicine in China.CAG exerts repressive effects against viruses, bacteria, tumors, and inflammation in the progression of various diseases [7].Additionally, CAG ameliorated imiquimod-induced psoriasis by inhibiting inflammasome-mediated pyroptosis [8], and experimental evidence indicates that CAG may play a role in repressing psoriasis.However, the precise role of CAG in the excessive inflammation observed in psoriasis remains unclear and requires further investigation.CAG also inhibited lymphocyte proliferation and enhanced myocardial autophagy in cases of moderate cardiac injury [9].Moreover, it induced apoptosis to repress gastric cancer cell proliferation and exerted anticancer effects [10].Based on the ameliorative effects of autophagy and apoptosis in psoriasis, whether CAG can mitigate the inflammation process and hyperproliferation in keratinocytes to restrain psoriasis is unknown.
MicroRNAs (miRNAs) are crucial signaling molecules that can modulate inflammation and proliferation of psoriasis models.For instance, miR-31-5p inhibition ameliorated psoriasis pathogenesis by modulating metabolic processes in the cellular environment and immune cell reactions [11], and miR-149-5p inhibited proliferation and enhanced apoptosis of interleukin (IL)-22-treated keratinocytes [12].miR-145-5p downregulation boosted keratinocyte proliferation and skin inflammation in psoriasis [13], and Astragalus membranaceus treatment decreases tumor cell viability by promoting miR-145 expression [14].Additionally, miR-145 induced cell autophagy to potentially alleviate vascular disease development, such as coronary stenosis [15].Thus, since CAG is a typical active molecule of Astragalus membranaceus, its ability to promote miR-145 expression and regulate the abnormal proliferation and autophagy of epidermal keratinocytes in psoriasis needs to be investigated.
Stanniocalcin-1 (STC1) is a glycoprotein hormone that modulates cell proliferation, death, and stress-coping processes.STC1 upregulated matrix metalloproteinase 1 (MMP1) expression in human immortalized keratinocytes (HaCaT cells), thereby affecting the underlying dermal-epidermal interaction process of psoriasis [16].Plasma MMP1 levels are increased in patients with psoriasis and decline following treatment [17], suggesting that STC1 may play a role in regulating psoriasis pathogenesis.Moreover, the starBase database (http://starbase.sysu.edu.cn/)predicted that STC1 contained potential binding sites for miR-145, but their relationship with keratinocyte hyperproliferation has yet to be explored.
Notch signaling exerts regulatory effects on cellular differentiation, proliferation, and autophagy processes, and Notch1 (a Notch receptor) modulates skin inflammation in the pathogenesis of psoriasis [18].Notch1 inhibition has been reported to promote podocyte autophagy and alleviate inflammatory injury [19].Furthermore, STC1 activated Notch1 signaling by interacting with its extracellular domain in glioblastoma cells [20].However, the interaction between STC1 and Notch1 in the inflammation and autophagy of epidermal keratinocytes in psoriasis remains unreported.
Therefore, we aimed to investigate the role of CAG in the proliferation of IL-22-stimulated HaCaT cells in a psoriasis model.We found that CAG ameliorated the hyperproliferation of IL-22-treated keratinocytes by promoting autophagy.Thus, CAG may be a potential clinical treatment for psoriasis.Furthermore, the miR-145/STC1/Notch1 axis may be a novel molecular target to diagnose and treat psoriasis.

Tissue acquisition and cell culture
Skin tissue samples (4-mm punch biopsies) were collected from the psoriatic plaque of psoriasis patients (n = 11) or from the skin of healthy volunteers (n = 7) under local anesthesia with 2% lignocaine at Changsha Hospital of Traditional Chinese Medicine, Hunan, China.All the psoriasis patients' disease severity belonged to the moderate stage (PASI 10-20).The participants did not receive any skin-treatment drug for at least 1 month before skin tissue collection.All procedures were conducted in accordance with the approved guidelines provided by the Institutional Review Committee of the above hospital (study approval number: 2023051).
HEKn cells (primary neonatal human epithelial keratinocytes) were acquired from Thermo Fisher Scientific (Waltham, MA, USA) and cultured in medium supplemented with human keratinocyte growth supplement and 1% penicillin/streptomycin (P/S).HaCaT cells (human immortalized keratinocytes) obtained from Procell Life Science & Technology (Wuhan, China) were maintained in minimal essential medium (Procell Life Science & Technology) supplemented with 15% fetal bovine serum (FBS; Gibco BRL, Indianapolis, IN, USA) and 1% P/S.HEK-293T cells (Procell Life Science & Technology) were cultured in a humidified incubator at 37 °C in an atmosphere of 5% CO 2 in Dulbecco modified Eagle medium (DMEM; Gibco BRL) supplemented with 10% FBS and 1% P/S.Cytokine mixture (C-mix; 20 ng/mL of IL-17, IL-22, interferon [IFN] γ, and tumor necrosis factor [TNF] α, respectively) was added to the keratinocyte culture to mimic an in vitro psoriasis-like state.For stimulation with recombinant IL-22 or C-mix, HaCaT cells or HEKn cells were starved in serum-free DMEM for 24 h before treatment with IL-22 (100 ng/mL) or C-mix, respectively, for a further 24 h or not (control treatment), according to the relevant experimental protocol [21,22].HaCaT cells were stimulated with CAG (0, 0.01, 0.05, 0.1, 1, and 5 μM) for 24 h, and the final experimental dose of CAG was determined according to the HaCaT cell viabilities.To inhibit the autophagy flux, cells were pretreated for 1 h with 1 mM 3-methyladenine (3-MA; MedChemExpress, Monmouth Junction, NJ, USA).

Cell transfection
For the overexpression or inhibition of miR-145, targeted mimics or inhibitors (used at a concentration of 40 pmol/mL) and the associated negative control (NC) were purchased from Genechem (Shanghai, China).Small interfering (si) RNAs targeting STC1 and the associated NC were also acquired from Genechem.To overexpress STC1, full-length STC1 cDNA was subcloned into a pcDNA3.1 vector (Invitrogen, Carlsbad, CA, USA) to construct a pcDNA3.1-STC1vector.Then, the constructed vector was transfected into HaCaT cells for 48 h using Lipofectamine 2000 Transfection Reagent (#11668027, Invitrogen), as per the manufacturer's instructions.

Cell viability assay
Cell viability was determined using Cell Counting Kit-8 (CCK-8; C0041, Beyotime Biotech, Beijing, China).Briefly, HaCaT cells or HEKn cells (2 × 10 3 cells/well) were seeded in 96-well plates and cultured for 24 h.Following the relevant experimental treatment, CCK-8 solution (10 μL in 90 μL of phosphate-buffered saline [PBS]) was added to each well, and the plate was incubated at 37 °C for 2 h, as per the manufacturer's instructions.Then, the absorbance value at 450 nm was measured using a SpectraMax iD3/iD5 microplate reader (Molecular Devices, San Jose, CA, USA).

Enzyme-linked immunosorbent assay (ELISA)
The STC1 concentration in the HaCaT cell culture supernatant was determined using a Human Stanniocalcin 1/STC Kit (STC1) ELISA kit (ab213829, Abcam, Cambridge, UK).The assay was performed according to the manufacturer's instructions, and the absorbance at 450 nm was read on a SpectraMax iD3/iD5 microplate reader (Molecular Devices).

Autophagic flux assays
To observe the autophagic flux, HaCaT cells or HEKn cells were transfected with ad-mRFP-GFP-LC3 (HB-AP210 0001, Hanbio Biotechnology, Shanghai, China) for 24 h at a multiplicity of infection of 20, as per previous research [23].Following relevant treatment, the fluorescent signals were captured under confocal microscopy (Leica TCS SP5, Leica Microsystems, Wetzlar, Germany).GFP and mRFP intensity were manually detected under fluorescent puncta.

Flow cytometry analysis
Apoptosis was determined using an annexin V-FITC/PI Apoptosis Kit (40302ES50, Yeasen Biotechnology, Shanghai, China) for dual-staining of the cells and subsequent detection on a NovoCyte Advanteon flow cytometer system (Agilent Technologies, Santa Clara, CA, USA).As per the kit's protocol, the cells were resuspended in 100 μL of 1 × binding buffer and then darkly double-stained with annexin V-FITC (5 μL) and propidium iodide (PI) (5 μL) for 15 min.Next, 1 × binding buffer (400 μL) was added to the mix to detect apoptotic cells.Cells staining positive for annexin V-FITC and negative for PI indicated early apoptosis, and cells staining positive for both annexin V-FITC and PI indicated late apoptosis.

Dual-luciferase reporter gene assay
Potential binding sites of miR-145 on the 3′ untranslated region (UTR) of STC1 wild type (STC1-WT) and its mutated site (STC1-MUT) were designed and cloned into the firefly luciferase gene in pmirGLO vectors (Promega, Madison, WI, USA).Then, HEK-293T cells that had been cultured for 12 h were co-transfected with the STC1-WT/STC1-MUT reporter gene plasmid and miR-145 mimics or NC.After 48 h of incubation, a Dual-Luciferase Reporter Assay System (Promega) was used to examine luciferase activity.

Co-immunoprecipitation (Co-IP) analysis
The collected HaCaT cells were incubated in lysis buffer from Pierce HA-Tag Co-IP Kit (#26180, Thermo Fisher Scientific) for 1 h.Following centrifugation (12,000 rpm, 10 min), the cell lysate supernatant was incubated overnight at 4 °C with anti-HA tag (1:1000, ab9110, Abcam) or anti-IgG (1:1000, ab172730, Abcam).Then, Protein A/G magnetic beads were added to the rinsed samples and incubated for a further 4 h.Next, the beads were washed with PBS (three times), followed by IP lysis buffer (two times).Finally, the precipitated proteins were eluted and identified using western blot analysis.

RNA immunoprecipitation (RIP) assays
RIP analysis was performed using a Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, Burlington, MA, USA).Briefly, cells were immersed in RIP buffer, and the extraction of cells was performed in interaction with magnetic beads that were bound with antibodies, specifically anti-IgG (1:500, ab172730, Abcam) or Argonaute-2 (AGO2; ab32381, Abcam).Subsequently, the determination of miR-145 and STC1 enrichment in the immunoprecipitated complexes was accomplished through qRT-PCR analysis.
Statistical analysis SPSS 23.0 statistical software (IBM Corp., Armonk, NY, USA) was used for all statistical analyses.Comparisons between two groups were analyzed using Student t-test, and comparisons between multiple groups were analyzed using one-way ANOVA followed by Tukey test.Data were presented as means ± standard deviation (SD).Results were considered statistically significant if p < 0.05.

CAG attenuated the IL-22-induced autophagy inhibition in HaCaT cells
To investigate the modulatory mechanisms of psoriasis, we established a psoriasis cellular model using HaCaT cells.To explore the precise role of CAG in IL-22-induced psoriasis, the cells were pretreated with CAG (0, 0.01, 0.05, 0.1, 1, and 5 μM) for 24 h.CAG treatment markedly reduced the cell viability in a dose-dependent manner when the concentration was <0.1 μM (Figure 1A).The minimum CAG concentration inhibiting HaCaT cell proliferation was 0.1 μM, and we used this concentration for subsequent experiments.A mixture of cytokines (C-mix) is usually selected as an inducer to establish cellular models of psoriasis [24].HaCaT cells and HEKn cells that had received CAG pretreatment were treated with the autophagy flux inhibitor 3-MA for 1 h.Then, the HaCaT cells and HEKn cells were further treated with IL-22 and C-mix, respectively.We observed that CAG treatment repressed the IL-22-or C-mix-induced increased cell viability, whereas 3-MA treatment reversed the trend of CAG (Figure 1B and Supplementary Figure 1A).Additionally, IL-22 or C-mix stimulation significantly inhibited LC3 II/LC3 I expression, promoted p62 expression, and inhibited the autophagy flux, while CAG treatment enhanced LC3 II/LC3 I expression, repressed p62 expression, and induced the autophagy flux in HaCaT cells or HEKn cells treated with IL-22 or C-mix, respectively.However, 3-MA overturned the effects of CAG (Figure 1C and D, Supplementary Figure 1B and C).Moreover, CAG reversed the IL-22 or C-mix-induced apoptosis reduction, while 3-MA treatment abolished the modulation trend of CAG in HaCaT cells and HEKn cells (Figure 1E and Supplementary Figure 1D).Furthermore, CAG ameliorated the IL-22-or C-mix-induced increased IL-1β and TNF-α mRNA levels (Figure 1F).Overall, these data indicated that IL-22 or C-mix stimulation inhibited autophagy and apoptosis and promoted cell proliferation, while CAG alleviated the autophagy inhibition induced by IL-22 or C-mix to regulate apoptosis and abnormal cell proliferation in the psoriasis model.

IL-22 or C-mix significantly decreased miR-145 expression in
HaCaT cells or HEKn cells, respectively, whereas CAG treatment markedly enhanced miR-145 expression (Figure 2A and Supplementary Figure 1E).Further, two groups of HaCaT cells were transfected with either miR-145 inhibitor or inhibitor NC and treated with CAG and IL-22.We observed that miR-145 inhibition significantly reversed the enhanced expression of miR-145 by CAG treatment (Figure 2B).CAG repressed the cell viability that was increased by IL-22, whereas miR-145 inhibition abolished this effect of CAG (Figure 2C).CAG also reversed the decreased expression of LC3 II/LC3 I, the increased expression of p62, and the inhibited autophagy flux induced by IL-22, whereas miR-145 inhibition reversed the CAG-induced effects (Figure 2D and E).Further, CAG alleviated the IL-22-induced inhibition of apoptosis, and miR-145 silencing reversed the promotion of apoptosis by CAG in IL-22-stimulated HaCaT cells (Figure 2F).Collectively, our findings demonstrated that CAG promoted cell autophagy by enhancing miR-145 expression to induce apoptosis and repress excessive cell proliferation in IL-22 treated HaCaT cells.

Silenced STC1 ameliorated autophagy repression in IL-22-treated HaCaT cells
We investigated the STC1 mRNA levels in the skin tissues of psoriasis patients and healthy volunteers (Nomal).The STC1 mRNA levels in psoriasis patients were significantly higher compared with the healthy volunteers (Figure 3A).Additionally, IL-22 treatment significantly increased STC1 mRNA and STC1 protein levels in HaCaT cells (Figure 3A and B).Thereafter, HaCaT cells were transfected with si-STC1 and si-NC and treated with IL-22.The level of STC1 protein expression in the IL-22 + si-STC1 group was markedly lower than that of the IL-22-alone group or the IL-22 + si-NC group (Figure 3C), suggesting that the transfection efficacy was ideal.Moreover, STC1 silencing reversed the IL-22-enhanced cell viability (Figure 3D).The repression of STC1 promoted LC3 II/LC3 I expression, reduced p62 expression, and enhanced autophagy flux in IL-22-treated HaCaT cells, alleviating the IL-22induced autophagy inhibition (Figure 3E and F).Additionally, STC1 silencing overturned the IL-22-repressed apoptosis of HaCaT cells (Figure 3G).In summary, silenced STC1 alleviated autophagy inhibition, promoted cell apoptosis, and repressed cell proliferation in IL-22-stimulated cellular model of psoriasis.

MiR-145 bound with STC1 and downregulated its expression
The starBase database predicted potential binding sites for miR-145 on STC1 (Figure 4A).Further, the relevant luciferase activities were decreased by the transfection of miR-145 mimics in the STC1-WT groups, whereas the luciferase activities in the STC1-MUT groups were not significantly altered (Figure 4B).RIP analysis also validated the immunoprecipitation of AGO2 with miR-145 and STC1 (Figure 4C).Moreover, we investigated the effect of miR-145 overexpression in HaCaT cells.MiR-145 levels were markedly enhanced following transfection of miR-145 mimic under IL-22 stimulation, indicating successful transfection, and miR-145 overexpression significantly repressed STC1 mRNA levels (Figure 4D).From the perspective of protein expression, the overexpressed miR-145 also downregulated STC1 expression under IL-22 stimulation (Figure 4E and F).Collectively, the above experimental evidence revealed miR-145 bound to STC1, thereby repressing its expression in the cellular model of psoriasis.

SCT1 activated Notch1 via direct interaction
HaCaT cells received the overexpression of HA-tag STC1 protein.Then, the cells were treated with IL-22.Co-IP analysis data showed the interaction of STC1 with Notch1 (Figure 5A).Cells were transfected with si-STC1 or si-NC to create an STC1-silencing cell model, after which they were stimulated with IL-22.STC1 silencing significantly reversed the IL-22induced increase in STC1 protein expression (Figure 5B).Additionally, STC1 silencing decreased the levels of STC1 secretion under IL-22 stimulation (Figure 5C).Upregulated N1ICD expression has been reported as positively correlated with Notch1 activation [25].In our experiment, IL-22 significantly enhanced N1ICD expression, while STC1 silencing reversed the IL-22-induced increased expression of N1ICD (Figure 5D).Overall, these data demonstrated that STC1 activated Notch1 through direct interaction.

Overexpressed STC1 reversed the CAG-enhanced autophagy in IL-22-treated HaCaT cells
HaCaT cells pretreated with CAG were transfected with STC1 overexpression vectors and then underwent IL-22 simulation.CAG reversed the IL-22-induced increase in STC1 secretion levels, whereas STC1 overexpression abolished this effect of CAG under IL-22 stimulation (Figure 6A).Further, compared with IL-22 treatment alone, CAG downregulated STC1 and N1ICD protein expression in IL-22-treated HaCaT cells, and STC1 overexpression reversed the CAG-induced downregulation of STC1 and N1ICD protein expression (Figure 6B).CAG treatment overturned the IL-22-induced increase in cell viability, while STC1 overexpression recovered the cell viability (Figure 6C).Moreover, CAG reversed the decreased expression of LC3 II/LC3 I, the increased expression of p62, and the repression of autophagy in IL-22 treated cells.STC1 overexpression overturned the increase in autophagy modulated by CAG under IL-22 stimulation (Figure 6D and E).Similarly, CAG also abolished the inhibitory effects of IL-22 on apoptosis, while STC1 overexpression markedly overturned the regulatory effects of CAG in IL-22-treated HaCaT cells (Figure 6F).Collectively, our data further confirmed that STC1 overexpression reversed the CAG-induced promotion of autophagy and apoptosis and the inhibition of cell proliferation in IL-22stimulated HaCaT cells.

Discussion
Psoriasis is a characteristic skin inflammatory disease affecting approximately 0.1%-0.4% of the global population [26].Psoriasis involves the disruption of autophagy, resulting in abnormal inflammation and keratinocyte hyperproliferation due to decreased levels of keratinocyte apoptosis [27,28].Our study revealed that CAG-enhanced autophagy to augment apoptosis and ameliorate the hyperproliferation of IL-22treated keratinocytes via the miR-145/STC1/Notch1 axis.Thus,   CAG shows high potential as a candidate drug for treating psoriasis.
CAG is a triterpenoid saponin compound from Astragalus membranaceus membranaceus, one traditional herb medicine of China.CAG is an active molecule, and its pharmacological effects include repressing inflammation, oxidative stress, tumor progression, and aging [29].Additionally, CAG ameliorated psoriasis-like skin inflammation in a mouse model by inhibiting the inflammasome-regulated pyroptosis process [8].However, its modulatory role in keratinocyte hyperproliferation in models of psoriasis was unclear.Our study demonstrated that CAG markedly inhibited the hyperproliferation of IL-22stimulated keratinocytes in an in vitro model of psoriasis by promoting autophagy.This finding is in partial agreement with a report that Astragalus membranaceus polysaccharide extracts inhibited the hyperplastic growth of IL-1β-treated synoviocytes via autophagy enhancement in a rheumatoid arthritis model [30].Regarding epigenetic mechanisms, we found that miR-145 expression was downregulated in IL-22-stimulated HaCaT cells, which was coincident with the effects of miR-145 in a previous study [31].Our current study was the first to demonstrate that CAG activated autophagy and promoted apoptosis in IL-22treated keratinocytes by upregulating miR-145.
STC1 is a secreted glycoprotein hormone with a wide spectrum of biological functions.Notably, STC1 has been reported to exert modulatory effects on cell proliferation and differentiation, such as the processes of adipose tissue maintenance and wound healing [32].STC1 promotes MMP1 expression to potentially regulate the dermal-epidermal interaction of psoriasis, and MMP1 levels are used as a diagnostic marker of psoriasis [16].Our study is the first to validate the abnormal expression of STC1 in the skin tissue of patients with psoriasis and the precise function of STC1 in an in vitro model.Our findings revealed that IL-22 stimulation markedly enhanced STC1 expression in keratinocytes, and STC1 inhibition restrained keratinocyte hyperproliferation and promoted cell autophagy and apoptosis under IL-22 stimulation.This is in line with the role of STC1 reported in an acute myocardial infarction model, where STC1 inhibition was accompanied by apoptosis promotion in myocardial tissues [33].STC1 is involved in the regulatory mechanisms between miRNAs and relevant modulations, such as STC1 and miR-382-5p [33].The online prediction from starBase that STC1 contained miR-145 binding sites led us to investigate and subsequently provide the first verification of miR-145 binding with STC1, leading to STC1's repression.This evidence revealed the exact role of STC1 in keratinocyte proliferation, autophagy, and apoptosis in psoriasis and verified the regulatory interaction between miR-145 and STC1 in the abovementioned processes.
Notch1 signaling is a highly conserved pathway that regulates epidermal cell proliferation, differentiation, and apoptosis to modulate skin disease development [34].Additionally, STC1 binds with and activates Notch1 signaling in glioblastoma cells [20].Our study is the first to demonstrate that STC1 binds with Notch1 and enhances N1ICD expression, which indicated the activation of Notch1 signaling in psoriasis model.IL-22 treatment significantly promoted N1ICD expression, which was reversed by STC1 silencing.This further validated the interaction between STC1 and Notch1 signaling.Notch1 signaling has been reported as involved in the activation and invasion processes of endothelial cells to enhance psoriasis progression [35].Our study was the first to reveal that CAG treatment inhibited STC1 and N1ICD expression, whereas STC1 overexpression abolished the regulation of CAG.Further, our findings showed that STC1 overexpression reversed the CAG-induced increase of autophagy and apoptosis and the inhibition of keratinocyte proliferation under IL-22 stimulation.Notch1 signaling inhibition was reported to partially promote autophagy in lipopolysaccharidetreated intestinal epithelial cells [36].Additionally, it was reported that the Notch1 pathway positively interacted with autophagy in Klebsiella pneumoniae-infected alveolar epithelial cells [37].At the least, our study data indicated that STC1 partially regulated autophagy through Notch1 signaling.However, further investigation is necessary to determine whether STC1 can modulate the autophagy process through other molecules or pathways.Future studies should explore the downstream pathways of STC1 in autophagy regulation by transcriptome sequencing after interfering with STC1.
In summary, our study uncovered CAG as a promoter of autophagy that moderated the hyperproliferation of keratinocytes via the miR-145/STC1/Notch1 axis in psoriasis.It provides experimental evidence that CAG may be clinically utilized as a potential anti-psoriasis drug.However, the mechanisms identified in this study lack relevant validation in vivo and should be verified in an animal model.Overall, our results revealed the repressive function and mechanism of CAG in psoriasis and its potential as a clinical treatment.Furthermore, the miR-145/STC1/Notch1 axis is a potential treatment target.

Figure 3 .
Figure 3. Silenced Stc1 ameliorated autophagy repression in Il-22-treated hacat cells.hacat cells were transfected with si-Stc1 or si-nc and further stimulated with Il-22.(a) qrt-pcr determined the levels of Stc1 mrna.(B) Western blot analysis determined the protein levels of Stc1.(c) Western blot analysis determined the protein level of Stc1.(d) ccK-8 assays were performed to investigate cell viability.(E) Western blot analysis was used to determine the protein levels of lc3 II/lc3 I and p62.(F) autophagic flux was observed with autophagic flux assays (mrFp-gFp-lc3) under confocal microscopy.(g) Flow cytometry assessed the extent of cell apoptosis following double-staining with annexin V-FItc and pI.Each experiment was performed in triplicate.Values are expressed as means ± Sd, n = 3 per group.*P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4 .
Figure 4. mir-145 bound with Stc1 and downregulated its expression.hEK-293t cells were co-transfected with Stc1-Wt/Stc1-mut reporter gene plasmid and mir-145 mimics or mimic ncs (a-c).hacat cells were transfected with mir-145 mimics or mimic ncs and further stimulated with Il-22 (d-F).(a) the starBase database predicted potential binding sites for mir-145 on Stc1.(B) a dual-luciferase reporter gene assay examined the interaction between Stc1 and mir-145.(c) rIp analysis verified the interaction between Stc1 and mir-145.(d) qrt-pcr determined the expression levels of mir-145 and Stc1.(E) Western blot analysis determined the protein levels of Stc1.(F) ElISa evaluated the Stc1 levels in the culture supernatant.Each experiment was performed in triplicate.Values are expressed as means ± Sd, n = 3 per group.*P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5 .
Figure 5. Sct1 activated notch1 via direct interaction.hacat cells were received with ha-Stc1 protein overexpression and further stimulated with Il-22 (a).hacat cells were transfected with si-Stc1 or si-nc and further stimulated with Il-22 (B-d).(a) co-Ip assays verified direct interaction between Stc1 and notch1.(B) Western blot analysis determined the level of Stc1.(c) ElISa evaluated the Stc1 level in the culture supernatant.(d) Western blot analysis determined the level of n1Icd.Each experiment was performed in triplicate.Values are expressed as means ± Sd, n = 3 per group.*P < 0.05, **P < 0.01, ***P < 0.001.