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. 2023 May 11;39(8):769–778. doi: 10.1002/kjm2.12693

Sp1‐mediated miR‐193b suppresses atopic dermatitis by regulating HMGB1

Ying‐Ke Liu 1, Lei‐Shan Liu 1, Bo‐Chen Zhu 1, Xiu‐Fang Chen 1, Li‐Hong Tian 1,✉
PMCID: PMC11895914  PMID: 37166084

Abstract

Atopic dermatitis (AD) is a chronic and recurrent inflammatory skin disease. Keratinocyte dysfunction plays a central role in AD development. MicroRNA is a novel player in many inflammatory and immune skin diseases. In this study, we investigated the potential function and regulatory mechanism of miR‐193b in AD. Inflamed human keratinocytes (HaCaT) were established by tumor necrosis factor (TNF)‐α/interferon (IFN)‐γ stimulation. Cell viability was measured using MTT assay, while the cell cycle was analyzed using flow cytometry. The cytokine levels were examined by enzyme‐linked immunosorbent assay. The interaction between Sp1, miR‐193b, and HMGB1 was analyzed using dual luciferase reporter and/or chromatin immunoprecipitation (ChIP) assays. Our results revealed that miR‐193b upregulation enhanced the proliferation of TNF‐α/IFN‐γ‐treated keratinocytes and repressed inflammatory injury. miR‐193b negatively regulated high mobility group box 1 (HMGB1) expression by directly targeting HMGB1. Furthermore, HMGB1 knockdown promoted keratinocyte proliferation and inhibited inflammatory injury by repressing nuclear factor kappa‐B (NF‐κB) activation. During AD progression, HMGB1 overexpression abrogated increase of keratinocyte proliferation and repression of inflammatory injury caused by miR‐193b overexpression. Moreover, transcription factor Sp1 was identified as the biological partner of the miR‐193b promoter in promoting miR‐193b expression. Therefore, Sp1 upregulation promotes keratinocyte proliferation and represses inflammatory injury during AD development via promoting miR‐193b expression and repressing HMGB1/NF‐κB activation.

Keywords: atopic dermatitis, high mobility group box 1, keratinocyte, miR‐193b, Sp1

Abbreviations

AD

atopic dermatitis

ADEH

a history of eczema herpeticum

ANOVA

analysis of variance

BCA

bicinchoninic acid

ChIP

chromatin immunoprecipitation

DNFB

2,4‐dinitrofluorobenzene

ELISA

enzyme‐linked immunosorbent assay

HMGB1

high mobility group box 1

IgE

immunoglobulin E

IFN

interferon

IL

interleukin

miRNAs

microRNA

MTT

3‐(4, 5‐Dimethylthiazolyl2)‐2, 5‐diphenyltetrazolium bromide

nts

nucleotides

NF‐κB

nuclear factor kappa‐B

qRT‐PCR

quantitative real‐time polymerase chain reaction

SD

standard deviation

Sp1

specificity protein 1

TF

transcription factor

TNF

tumor necrosis factor

1. INTRODUCTION

Atopic dermatitis (AD) is a common inflammatory skin disease, 1 affecting 20% of children and 3% of adults worldwide, and this number is rapidly increasing. 2 AD is incurable and affects patients throughout their lives. 3 The complex pathogenesis of AD is related to many factors. The regulatory mechanisms of AD pathogenesis are still unclear. Recently, epidermal barrier dysfunction was found to trigger AD pathogenesis. 4 Keratinocytes, the primary constituent cells of the epidermis, forms a barrier against pathogens and allergens by releasing inflammatory cytokines. 5 Keratinocyte dysfunction is integral in AD development. 6 Therefore, improving keratinocyte function is a potential therapeutic strategy for AD.

MicroRNAs (miRNAs) are noncoding RNAs containing approximately 22 nucleotides (nts). 7 miRNAs regulate essential biological processes, such as cell proliferation 8 and inflammation. 9 Although studies have revealed alterations in miRNA expression in the skin or serum of AD patients, 10 their biological effect is yet to be fully elucidated. miR‐193b is involved in the progression of inflammatory skin diseases. Huang et al. revealed that the miR‐193b‐3p level was inversely correlated with disease severity in psoriasis patients, and its upregulation remarkably repressed keratinocyte proliferation and inflammatory cytokine secretion. 11 Although miR‐193b was found to be significantly downregulated in AD, 12 the molecular mechanism underlying miR‐193b‐mediated suppression of AD progression is still unclear. Nuclear transcription factor (TF) specificity protein 1 (Sp1) is involved in several cellular functions, 13 including inflammatory injury and antiviral response. Sp1 upregulation ameliorated lipopolysaccharide‐induced cardiomyocyte inflammatory injury. 14 Previous studies showed that Sp1 gene expression was markedly reduced in patients with a history of eczema herpeticum (ADEH)(+), and its knockdown promoted viral replication in keratinocytes, indicating that Sp1 is important for the skin's antiviral response during AD. 15 Furthermore, we predicted to find of binding sites between SP1 and miR‐193b promoter region. However, we could not fully elucidate the interaction between Sp1 and miR‐193b and the function of this signal axis in AD, which deserves further research.

High mobility group box 1 (HMGB1), a highly conserved nuclear protein found in all cell types, is essential during inflammation. 16 Although the role of HMGB1 in the nucleus is not fully understood, its extracellular function has been implicated in the inflammatory response. HMGB1 readily binds to other pro‐inflammatory molecules, including DNA, RNA, histones, and other factors, to exert pro‐inflammatory effects. 17 Previous studies showed that the HMGB1 level was significantly increased in the skin tissues of AD mice. 18 Wang et al. revealed that glycyrrhizin could ameliorate AD‐like symptoms in mice by inhibiting HMGB1. 19 Herein, we predicted that miR‐193b has a binding site with HMGB1, speculating that miR‐193b might regulate AD progression by targeting HMGB1.

Based on our results, we speculated that Sp1 represses AD development by regulating the miR‐193b/HMGB1 signaling axis, thus providing a potential therapeutic target for AD.

2. MATERIALS AND METHODS

2.1. Clinical sample collection

Skin tissues were collected from 10 AD patients and ten control subjects without any history of allergy. Elevated immunoglobulin E (IgE) or eosinophilia was detected in AD patients. All control individuals were free from allergic disorders and had negative skin prick tests for common allergens. They also had normal serum IgE for at least 5 years before inclusion into the study. None of the participants was under treatment with systemic medicines. The biopsies were snap‐frozen and kept at −80°C. This study was approved by XXX. All participants provided written informed consent.

2.2. Cell culture and treatment

Human epidermal keratinocyte cells (HaCaT cells) and 293T cells were obtained from the American Type Culture Collection. Normal primary human keratinocytes were purchased from PromoCell. Normal primary human keratinocytes were cultured in Keratinocyte Growth Medium 2 (KGM‐2, PromoCell) at 37°C in a humidified atmosphere with 5% CO2 as previously described. 20 HaCaT and 293T cells were cultured in Dulbecco's modified Eagle medium (Gibco) mixed with 10% fetal bovine serum (Gibco) at 37°C with 5% CO2. To establish an in vitro model of AD, the HaCaT cells and primary human keratinocytes were treated with 10 ng/mL each of recombinant tumor necrosis factor (TNF)‐α and interferon (IFN)‐γ (R&D systems) for 24 h.

2.3. Cell transfection

The short hairpin RNA of HMGB1 (sh‐HMGB1), overexpression plasmids of HMGB1 (OE‐HMGB1), and Sp1 overexpression (OE‐Sp1), miR‐193b mimics/inhibitor and their negative controls were acquired from GenePharma. They were transfected into the cells using Lipofectamine™ 3000 (Invitrogen).

2.4. 3‐(4, 5‐Dimethylthiazolyl2)‐2, 5‐diphenyltetrazolium bromide (MTT) assay

Cells were incubated with 5 mg/mL MTT (Beyotime) for 4 h at 37°C. The absorbance at 490 nm was measured after adding DMSO (Sigma‐Aldrich).

2.5. Cell cycle assay

Cells (1.5 × 106) were digested with 0.25% trypsin‐free EDTA, and the culture medium was added to terminate digestion. The cells were incubated with 70% ethanol for 30 min at 37°C, stained with 50 μg/mL PI (Sigma‐Aldrich), and analyzed using flow cytometry (Becton, Dickinson and Company).

2.6. Enzyme‐linked immunosorbent assay

The levels of TNF‐α, interleukin (IL)‐1β, and IL‐6 were examined using ELISA kits for human TNF‐α (Beyotime, PT518), human IL‐1β (Beyotime, PI305) and human IL‐6 (Beyotime, PI330) according to the manufacturer's instructions.

2.7. Dual‐luciferase reporter gene assay

The human HMGB1 fragment was amplified by PCR. Site‐directed mutagenesis was performed using a site‐directed mutagenesis kit (Stratagene). The wild‐type (wt) and mutant (mut) reporter plasmids containing HMGB1 sequences were cloned into the pmirGLO vector (Promega). Then, the cells were co‐transfected with HMGB1‐wt or HMGB1‐mut plasmids and miR‐193b mimics or NC mimics using Lipofectamine™ 3000 (Invitrogen). Luciferase activity was examined using the dual luciferase reporter assay system (Promega).

For the miR‐193b promoter luciferase assay, the plasmids, including the miR‐193b promoter, were cloned into the pGL3 vector. Cells were co‐transfected with pGL3‐miR‐193b promoter and OE‐SP1 (SP1 overexpression). After 48 h, the cell lysate was collected, and the luciferase activity was detected using the dual luciferase reporter assay system.

2.8. Chromatin immunoprecipitation assay

Cells were fixed, quenched, and sonicated to produce 200–500 bp fragments. The cell lysate was cultured with anti‐Sp1 (Abcam, 1:100, ab231778) or anti‐IgG (Abcam, 1:100, ab172730) at 4°C overnight. Dynabeads protein G (Invitrogen) was added for 2 h to immunoprecipitate the DNA, which was analyzed using qRT‐PCR.

2.9. Animal experiments

A total of 18 female BALB/c mice (6‐week‐old) were purchased from SLACOM, and AD was induced by 2,4‐dinitrofluorobenzene (DNFB) treatment as previously described. 21 Briefly, 1% DNFB (Macklin) was dissolved in an acetone/olive oil (1:3) solution. Mice were randomized into three groups (n = 6/group): control, AD, and AD + miR‐193b groups. The surface of the abdomen lobes was gently stripped using surgical tape (Nichiban). Then, the abdomen was painted with 20 μL DNFB, repeated once a week for 4 weeks. Lentiviral vectors with negative control (NC) or miR‐193b mimics were generated by HanBio Biotechnology. Lentivirus (2 × 108 IFU/mL) harboring NC or miR‐193b mimics were injected into the AD and AD + miR‐193b mice through the tail vein. Abdomen swelling was detected 24 h after DFE/DNFB treatment by a micrometer (Mitutoyo). Then, the mice were euthanized, and the blood samples and skin tissues were collected.

2.10. Hematoxylin–eosin staining

The skin tissue block was fixed in 4% paraformaldehyde, paraffin‐embedded, and cut into 4 μm‐thick slices. The sections were dehydrated with different concentrations of alcohol, stained with HE (Sigma‐Aldrich), and observed and photographed under a microscope (Olympus).

2.11. Quantitative real‐time polymerase chain reaction (qRT‐PCR)

Total RNA was extracted with TRIzol (Thermo Fisher Scientific). For mRNA and miRNA, the cDNA was synthesized using the Reverse Transcription Kit (Toyobo) and first‐strand cDNA synthesis kit (Sangon), respectively. Then, qRT‐PCR was performed using SYBR (Thermo Fisher Scientific) using GAPDH and U6 as the reference gene for mRNA and miRNA. The data were analyzed with the 2−ΔΔCT method. The primers were listed as follows (5′–3′):

miR‐193b (human) (F): AACTGGCCCTCAAAGTCCCG.

miR‐193b (human) (R): GCGAGCACAGAATTAATACGAC.

HMGB1 (human) (F): ATATGGCAAAAGCGGACAAG.

HMGB1 (human) (R): GCAACATCACCAATGGACAG.

Sp1 (human) (F): GACAGGACCCCCTTGAGCTT.

Sp1 (human) (R): GGCACCACCACCATTACCAT.

U6 (human) (F): CTCGCTTCGGCAGCACA.

U6 (human) (R): AACGCTTCACGAATTTGCGT.

GAPDH (human) (F): CCAGGTGGTCTCCTCTGA.

GAPDH (human) (R): GCTGTAGCCAAATCGTTGT.

IL‐4 (mouse) (F): CCGTAACAGACATCTTTGCTGCC.

IL‐4 (mouse) (R): GAGTGTCCTTCTCATGGTGGCT.

IL‐6 (mouse) (F): CCGGAGAGGAGACTTCACAG.

IL‐6 (mouse) (R): CAGAATTGCCATTGCACAAC.

IL‐13 (mouse) (F): ACGGTCATTGCTCTCACTTGCC.

IL‐13 (mouse) (R): CTGTCAGGTTGATGCTCCATACC.

GAPDH (mouse) (F): AGGTCGGTGTGAACGGATTTG.

GAPDH (mouse) (R): GGGGTCGTTGATGGCAACA.

2.12. Western blot

The proteins were isolated using RIPA buffer, and the concentration was measured by bicinchoninic acid (BCA) kit (Beyotime). Equal amounts of protein were separated using a 12% polyacrylamide gel electrophoresis gel and transferred onto a polyvinylidene fluoride membrane (Millipore). The membranes were blocked with 5% skimmed milk and incubated overnight with antibodies against HMGB1 (Abcam, 1:1000, ab79823), p65 nuclear factor kappa‐B (NF‐κB) (Abcam, 1:1000, ab131100), Sp1 (Abcam, 1:1000, ab231778), and GAPDH (Abcam, 1:10000, ab8245). Then, they were incubated with the corresponding secondary antibody (Abcam, 1:5000, ab7090) for 60 min following washing with PBST. The protein bands were analyzed using enhanced chemiluminescence detection kit (Beyotime).

2.13. Statistical analysis

All the data were obtained from three independent experiments. Statistical data were analyzed by SPSS 19.0 and expressed as means ± standard deviation (SD). The differences between the two groups were analyzed using Student's t‐tests. One‐way analysis of variance was performed to assess the differences among multiple groups. The p‐values < 0.05 were considered significant.

3. RESULTS

3.1. miR‐193b overexpression promoted cell proliferation and repressed inflammatory injury in TNF‐α/IFN‐γ‐treated keratinocytes

First, we observed that miR‐193b was markedly downregulated in the skin tissues of AD patients compared to that of control subjects (Figure 1A), indicating a potential role of miR‐193b in AD development. As previously reported, keratinocytes secrete high concentrations of inflammatory cytokines after TNF‐α/IFN‐γ stimulation. 22 Therefore, we used TNF‐α/IFN‐γ‐treated HaCaT cells and primary human keratinocytes to probe the role of miR‐193b in vitro. It was observed that miR‐193b expression in HaCaT cells was markedly reduced by TNF‐α/IFN‐γ stimulation (Figure 1B). We induced miR‐193b overexpression in HaCaT cells and primary human keratinocytes by transfecting miR‐193b mimics into these cells (Figures 1C and S1A). The viability of the HaCaT cells and primary human keratinocyte were markedly suppressed by TNF‐α/IFN‐γ stimulation (Figures 1D and S1B). These changes were reversed by miR‐193b overexpression. Additionally, TNF‐α/IFN‐γ‐induced cell arrest in the G1 phase, which was abrogated by miR‐193b overexpression (Figures 1E and S1C). The ELISA results subsequently demonstrated that TNF‐α/IFN‐γ stimulation increased the secretion levels of inflammatory cytokines (TNF‐α, IL‐1β, and IL‐6) (Figures 1F and S1D), which were all abolished by miR‐193b overexpression. Collectively, miR‐193b overexpression remarkably promoted the proliferation and inhibited inflammatory injury of HaCaT cells induced by TNF‐α/IFN‐γ treatment.

FIGURE 1.

FIGURE 1

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miR‐193b overexpression promotes cell proliferation and represses inflammatory injury in TNF‐α/IFN‐γ‐treated keratinocytes. (A) Skin tissues were collected from 10 AD patients and 10 control subjects without any history of allergy and used for detecting miR‐193b expression by qRT‐PCR. (B) miR‐193b expression in HaCaT cells after TNF‐α/IFN‐γ treatment was detected using qRT‐PCR. (C) miR‐193b expression was determined using qRT‐PCR after transfecting HaCaT cells with NC or miR‐193b mimics. miR‐193b overexpression plasmid was transfected into the TNF‐α/IFN‐γ‐treated HaCaT cells. (D) The viability of HaCaT cells was analyzed using the MTT assay. (E) The cell cycle was analyzed using flow cytometry. (F) TNF‐α, IL‐1β and IL‐6 levels were analyzed using ELISA. All data were expressed as mean ± SD and were obtained from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

3.2. HMGB1 functioned as the target of miR‐193b

HMGB1 acts as crucial role for regulating inflammatory response, and its level is significantly increased in the skin tissues of AD mice. 18 Herein, using starbase (http://starbase.sysu.edu.cn/index.php), we predicted that miR‐193b has potential HMGB1‐binding sites (Figure 2A). Transfection of miR‐193b mimics suppressed the luciferase activity after co‐transfection with HMGB1‐wt plasmid but had no significant effect on that of HMGB1‐mut, suggesting that miR‐193b directly binds with HMGB1 (Figure 2B ). We subsequently induced miR‐193b overexpression or miR‐193b knockdown in HaCaT cells. We observed that miR‐193b expression in HaCaT cells was reduced and elevated after transfection with miR‐193b inhibitor and mimics, respectively, indicating successful transfection (Figure 2C). We also observed that miR‐193b knockdown markedly increased HMGB1 mRNA levels in HaCaT cells, while miR‐193b overexpression exhibited the opposite effect (Figure 2C). miR‐193b inhibition also significantly enhanced HMGB1 and p‐p65 NF‐κB protein levels in HaCaT cells, while miR‐193b overexpression exerted the opposite effects (Figure 2D). Therefore, HMGB1 might function as a direct miR‐193b target.

FIGURE 2.

FIGURE 2

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HMGB1 functions as a miR‐193b target. (A) The binding sites between HMGB1 and miR‐193b were predicted using starBase. (B) The interaction between miR‐193b and HMGB1 was analyzed using a dual luciferase reporter gene assay. (C) miR‐193b and HMGB1 expressions in HaCaT cells following miR‐193b upregulation/inhibition were detected by qRT‐PCR (D) Western blot was performed to examine HMGB1 and p‐p65 NF‐κB levels in cells. Data were expressed as mean ± SD and were obtained from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

3.3. HMGB1 knockdown promoted TNF‐α/IFN‐γ‐treated keratinocyte proliferation and repressed inflammatory injury by regulating the NF‐κB signaling axis

We observed marked upregulation of HMGB1 in the skin tissues of AD patients compared to that of control subjects (Figure 3A). To investigate the role of HMGB1 in regulating keratinocyte function during AD, we induced HMGB1 knockdown in HaCaT cells by transfecting sh‐HMGB1 into these cells (Figure 3B, C). The MTT assay results demonstrated that HMGB1 knockdown remarkably increased TNF‐α/IFN‐γ‐treated keratinocyte viability (Figure 3D). Moreover, TNF‐α/IFN‐γ‐induced cell arrest in the G1 phase was eliminated by HMGB1 silencing (Figure 3E). HMGB1 silencing also abolished the induction of TNF‐α, IL‐1β, and IL‐6 due to TNF‐α/IFN‐γ stimulation in HaCaT cells (Figure 3F ). Moreover, we observed that TNF‐α/IFN‐γ stimulation increased HMGB1 and p‐p65 NF‐κB protein levels in keratinocytes, which was abrogated by HMGB1 knockdown (Figure 3G). In summary, HMGB1 silencing facilitated TNF‐α/IFN‐γ‐treated keratinocyte proliferation and attenuated inflammatory injury by repressing the NF‐κB signaling axis.

FIGURE 3.

FIGURE 3

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HMGB1 knockdown promotes TNF‐α/IFN‐γ‐treated keratinocyte proliferation and represses inflammatory injury by regulating the NF‐κB signaling axis. (A) Skin tissues were collected from 10 AD patients and 10 control subjects without any history of allergy. HMGB1 expression in skin tissues was detected by qRT‐PCR. (B), (C) HMGB1 expression in HMGB1silenced‐HaCaT cells was assessed using qRT‐PCR and Western blot. HMGB1 knockdown was induced in inflamed HaCaT cells. (D) HaCaT cell viability was detected using the MTT assay. (E) The cell cycle was analyzed using flow cytometry. (F) TNF‐α, IL‐1β and IL‐6 levels were analyzed using ELISA. (G) HMGB1 and p‐p65 NF‐κB levels were analyzed by Western blot. Data were expressed as mean ± SD and were obtained from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

3.4. HMGB1 overexpression reversed the effect of miR‐193b overexpression on TNF‐α/IFN‐γ‐induced keratinocyte inflammatory injury

To investigate the effect of HMGB1 on miR‐193b overexpression‐mediated biological effects in AD, we overexpressed HMGB1 and miR‐193b in inflamed HaCaT cells. The HMGB1 expression level was significantly increased by OE‐HMGB1 transfection (Figure 4A, B), suggesting successful transfection. Functional experiments subsequently demonstrated that HMGB1 overexpression reversed the cell viability of TNF‐α/IFN‐γ‐treated HaCaT cells facilitated by miR‐193b overexpression (Figure 4C). The cell cycle assay results revealed that miR‐193b overexpression reduced the induction of HaCaT cell arrest in the G1 phase stimulated by TNF‐α/IFN‐γ, which was eliminated by HMGB1 overexpression (Figure 4D). Additionally, the inhibitory effect of miR‐193b overexpression on TNF‐α/IFN‐γ‐induced TNF‐α, IL‐1β, and IL‐6 levels in HaCaT cells was partially reversed by HMGB1 overexpression (Figure 4E). Moreover, HMGB1 abrogated the inhibition of miR‐193b overexpression on HMGB1 and p‐p65 NF‐κB protein levels in TNF‐α/IFN‐γ‐treated HaCaT cells (Figure 4F). These results indicated that miR‐193b promoted the cell viability of inflamed HaCaT and HMGB1 overexpression reversed the inhibition of inflammatory injury.

FIGURE 4.

FIGURE 4

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HMGB1 overexpression reversed the effect of miR‐193b overexpression on TNF‐α/IFN‐γ‐induced keratinocyte inflammatory injury. (A), (B) HMGB1 expression in HMGB1‐overexpressing HaCaT cells was examined by qRT‐PCR and Western blot. HMGB1 and miR‐193b overexpression were induced in inflamed HaCaT cells. (C) HaCaT cell viability was determined by MTT assay. (D) The cell cycle was examined using flow cytometry. (E) TNF‐α, IL‐1β and IL‐6 levels were analyzed using ELISA. (F) HMGB1 and p‐p65 NF‐κB levels in cells were detected using Western blot. Data were expressed as mean ± SD and were obtained from three independent experiments. *p < 0.05, **p < 0.01.

3.5. Sp1 positively regulated miR‐193b expression by directly binding to miR‐193b promoter

We subsequently probed the upstream regulatory element of miR‐193b in regulating AD development. Using the TransmiR v2.0 database, a comprehensive database for regulating human TFs and their targets (http://www.cuilab.cn/transmir), and the JASPAR database, we found that Sp1 has potential miR‐193b promoter binding sites (Figure 5A). The subsequent molecular interaction results indicated that Sp1 directly binds with miR‐193b promoter (Figure 5B, C). Furthermore, TNF‐α/IFN‐γ stimulation significantly reduced the Sp1 mRNA level in HaCaT cells (Figure 5D). Additionally, we induced Sp1 knockdown or Sp1 overexpression in HaCaT cells (Figure 5E, F). And Sp1 knockdown markedly reduced miR‐193b expression in HaCaT cells, Sp1 overexpression showed the opposite effect (Figure 5G). Overall, Sp1 functioned as the TF of miR‐193b to positively regulate miR‐193b expression.

FIGURE 5.

FIGURE 5

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Sp1 positively regulates miR‐193b expression by directly binding to the miR‐193b promoter. (A) The binding sites between Sp1 and miR‐193b promoter were predicted by TransmiR v2.0 and JASPAR database. (B), (C) The interaction between Sp1 and miR‐193b promoter was analyzed by ChIP and dual luciferase reporter gene assays. (D) Sp1 mRNA levels in TNF‐α/IFN‐γ‐treated HaCaT cells were analyzed using qRT‐PCR. (E), (F) Sp1 expression in HaCaT cells following Sp1 knockdown or Sp1 overexpression was assessed using qRT‐PCR and Western blot. (G) miR‐193b expression in HaCaT cells following Sp1 knockdown or Sp1 overexpression was analyzed using qRT‐PCR. Data were expressed as mean ± SD and were obtained from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

3.6. miR‐193b overexpression inhibited AD in vivo

To further probe the role of miR‐193b in AD, miR‐193b overexpression was induced in the AD mouse model. The HE staining results revealed that DNFB treatment induced DNFB inflammatory cell infiltration and hyperkeratosis in the skin of the AD mice. These changes were remarkably ameliorated by miR‐193b overexpression (Figure 6A), which also reduced the severity score (Figure 6B). Moreover, DNFB treatment significantly increased the serum IgE levels (Figure 6C) and IL‐6, IL‐13, and IL‐4 expression levels, these changes were reversed by miR‐193b overexpression (Figure 6D). These results suggested that DNFB‐induced inflammatory response and histopathological changes in mice were alleviated by miR‐193b overexpression.

FIGURE 6.

FIGURE 6

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miR‐193b overexpression inhibited AD in vivo. DNFB was applied on the abdomen of BALB/c mice to induce AD before inducing miR‐193b overexpression. (A) Representative photomicrographs of skin sections were stained with HE. (B) The severity score was analyzed. (C) Serum IgE levels were analyzed using ELISA. (D) IL‐6, IL‐13, and IL‐4 expression levels were determined using qRT‐PCR. Data were expressed as mean ± SD. N = 6. *p < 0.05, **p < 0.01, ***p < 0.001.

4. DISCUSSION

The current drugs for AD, including cortisol and immunosuppressants, have shown good therapeutic effects. However, these drugs might be inappropriate for long‐term use because of their adverse effects. 23 Therefore, finding novel treatment strategies for AD is urgent and necessary. Keratinocytes are the skin's barrier against external damage. Keratinocyte dysfunction caused by various external stimuli contributes to the occurrence and development of AD inflammatory response. 5 , 24 Therefore, improving keratinocyte function is a potentially effective treatment strategy for AD. Our current results revealed that Sp1 upregulation promoted TNF‐α/IFN‐γ‐treated keratinocyte proliferation and repressed inflammatory injury by acting on the miR‐193b/HMGB1 signaling axis. miRNA dysfunction has been considered to result in AD onset. 10 A previous study identified 34 downregulated miRNAs in AD skin tissues, including miR‐193b. 25 miR‐193b overexpression suppressed inflammatory factor secretion in activated keratinocytes. 11 Considering the role of miR‐193b in regulating keratinocyte inflammatory response and its differential expression in AD, we further investigated its specific role in AD. In this study, we observed low miR‐193b expression levels in the skin tissues of AD patients and TNF‐α/IFN‐γ‐treated HaCaT cells, a commonly used inflamed keratinocyte model. 22 Further experiments revealed that miR‐193b upregulation promoted TNF‐α/IFN‐γ‐treated keratinocyte proliferation and repressed inflammatory injury. Meanwhile, animal experiments showed that DNFB‐induced inflammatory response and histopathological changes in mice were alleviated by miR‐193b overexpression. Therefore, our research suggested that miR‐193b upregulation might inhibit inflammatory injury in keratinocytes during AD.

However, it is important to elucidate the specific mechanism underlying miR‐193b‐mediated regulation of inflammatory injury in keratinocytes during AD. Although miRNA mainly achieves its role through base‐pairing to the mRNA 3'UTR regions, 26 the target of miR‐193b in regulating AD progression is largely unknown. Herein, we proposed a new regulatory network involving HMGB1 as the downstream target of miR‐193b. As a multifunctional protein, HMGB1 plays different biological roles under different stimuli. Dysregulation of HMGB1 is associated with many diseases, especially inflammatory diseases. 16 HMGB1 is an important pro‐inflammatory cytokine that binds to specific receptors to induce the production of pro‐inflammatory cytokines, leading to inflammation and injury. 27 Activation of these receptors, in turn, activates NF‐κB, which accelerates pro‐inflammatory cytokines production. A previous study revealed that HMGB1 and NF‐κB were significantly upregulated in the skin tissues of AD mice, and their inhibition contributed to AD recovery. 18 Herein, we found that HMGB1 knockdown promoted TNF‐α/IFN‐γ‐treated keratinocyte proliferation and repressed inflammatory injury by suppressing p65 NF‐κB activation. Additionally, miR‐193b negatively regulated HMGB1 by directly targeting HMGB1. The rescue experiment results also proved that HMGB1 overexpression abrogated the proliferation of HaCaT cells and repression of TNF‐α/IFN‐γ induced inflammatory injuries mediated by miR‐193b overexpression. To the best of our knowledge, this is the first study demonstrating that miR‐193b upregulation inhibited inflammatory injury in keratinocytes during AD by repressing the HMGB1/NF‐κB axis.

Several TFs have been known to mediate miRNA expression in various diseases. 28 , 29 Therefore, it is vital to explore the upstream TF of miR‐193b to establish a complete regulatory network in AD. Bioinformatic studies have shown that TF Sp1 has potential miR‐193b binding sites. Sp1 had also been reported to influence keratinocyte development positively 30 by contributing to their early differentiation. 31 Moreover, Sp1 promoted the skin's antiviral ability during AD. 15 Here, we showed that Sp1 was downregulated in TNF‐α/IFN‐γ‐ stimulated HaCaT cells. Consistently, ChIP and dual luciferase reporter gene assays showed that Sp1 promoted miR‐193b expression in human keratinocytes by directly binding with the miR‐193b promoter. Collectively, Sp1 facilitated keratinocyte proliferation and repressed inflammatory injury during AD via regulating the miR‐193b/HMGB1 signaling axis. Therefore, our work provides a theoretical basis for developing novel treatments for AD.

However, our research also has some shortcomings. Our results revealed that IL‐4 and IL‐13 in skin tissues were markedly increased by DNFB treatment, suggesting that IL‐4 or IL‐13 stimulation might be the possible model for in vitro AD study. However, we did not elucidate IL‐4 or IL‐13 stimulation effects, which will be addressed in our future studies.

CONFLICT OF INTEREST STATEMENT

All authors declare no conflict of interest.

Supporting information

Figure S1. miR‐193b overexpression promoted cell proliferation and repressed inflammatory injury in TNF‐α/IFN‐γ‐treated primary human keratinocytes. (A) miR‐193b expression in primary human keratinocytes transfected with NC or miR‐193b mimics was determined using qRT‐PCR. miR‐193b overexpression plasmid was transfected into TNF‐α/IFN‐γ‐treated primary human keratinocytes. (B) The viability of primary human keratinocytes was assessed by MTT assay. (C) The cell cycle was analyzed using flow cytometry. (D) TNF‐α, IL‐1β, and IL‐6 levels were analyzed using ELISA. Data were expressed as mean ± SD and were obtained from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

KJM2-39-769-s001.tif (1.2MB, tif)

ACKNOWLEDGMENTS

We would like to give our sincere gratitude to the reviewers for their constructive comments.

Liu Y‐K, Liu L‐S, Zhu B‐C, Chen X‐F, Tian L‐H. Sp1‐mediated miR‐193b suppresses atopic dermatitis by regulating HMGB1 . Kaohsiung J Med Sci. 2023;39(8):769–778. 10.1002/kjm2.12693

Ying‐Ke Liu and Bo‐Chen Zhu are the co‐first authors.

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Associated Data

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Supplementary Materials

Figure S1. miR‐193b overexpression promoted cell proliferation and repressed inflammatory injury in TNF‐α/IFN‐γ‐treated primary human keratinocytes. (A) miR‐193b expression in primary human keratinocytes transfected with NC or miR‐193b mimics was determined using qRT‐PCR. miR‐193b overexpression plasmid was transfected into TNF‐α/IFN‐γ‐treated primary human keratinocytes. (B) The viability of primary human keratinocytes was assessed by MTT assay. (C) The cell cycle was analyzed using flow cytometry. (D) TNF‐α, IL‐1β, and IL‐6 levels were analyzed using ELISA. Data were expressed as mean ± SD and were obtained from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

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