MicroRNA let-7b inhibits keratinocyte differentiation by targeting IL-6 mediated ERK signaling in psoriasis
The extensive involvement of microRNA (miRNA) in the pathophysiology of psoriasis is well documented. However, in order for this information to be useful in therapeutic manipulation of miRNA levels, it is essential that detailed functional mechanisms are elucidated. This study aimed to explore the effects of IL-6 targeting by let-7b and ERK1/2 mediated signaling on keratinocyte differentiation in psoriasis.
Following imiquimod cream (IMQ) application to let-7bTG (keratinocyte-specific let-7b overexpression mouse) and control mice for 7 days, we analyzed erythema, scaling and thickening of skin. A dual luciferase reporter assay and bioinformatics was carried out to detect target gene of let-7b. Additionally, the differentiation markers were measured. Immunohistochemistry analyses demonstrate a relationship of let-7b with IL-6 and ERK signaling.
we found let-7bTG inhibits acanthosis and reduces the disease severity by treatment with IMQ compared to wild-type mice. Further study illustrated that let-7b promotes differentiation of keratinocytes in vivo and in vitro. Using bioinformatics and reporter gene assays, we found that IL-6 is a target gene of let-7b. In psoriasis, high expression levels of IL-6 lead to increased acivation of p-ERK1/2. High levels of let-7bTG transgene expression suppresses IL-6 expression and leads to increased keratinocyte differentiation. Moreover, let-7b acts as an upstream negative regulator of the ERK signaling pathway in keratinocytes of psoriasis.
Our result reveals a previously unknown mechanism for regulation of IL-6 levels during psoriasis by let-7b and highlights a critical role for the ERK1/2 signaling pathway in epidermal differentiation during psoriasis.
The ethical approval for this study was from the Affiliated Hospital of Medical University of Anhui _ Fast_ PJ2017–11–14.
KeywordsPsoriasis Let-7b IL-6 ERK1/2 Differentiation
Insulin-like growth factor 2 mRNA-binding protein 2
Keratinocyte-specific let-7b overexpression mouse
Psoriasis Area and Severity Index of mouse
Inhibitors of MEK/ERK
Extracellular signal-regulated kinase phosphorylation
Reverse transcription polymerase chain reaction
Psoriasis is a long-lasting autoimmune disease characterized by patches of abnormal skin that affects 2–3% of the world’s population . Psoriasis undergoes three different processes of cellular alteration in skin: abnormal differentiation of keratinocytes, hyperproliferation of keratinocytes, followed by infiltration of immune cells into the dermis and epidermis . Some common molecular components, genetic alterations of genes that participate in inflammatory pathways, and environmental risks can contribute to the pathogenesis of psoriasis . However, the underlying mechanisms regulating these epidermal defects and immunological dysfunction remain largely unknown.
Results from recent studies have indicated that microRNAs (miRNAs), as a potential regulator of gene expression, play important roles in psoriasis . miRNAs are small endogenous RNA molecules with 22–24 bases long, which are often expressed in cell lineages and play critical roles in nearly all biological processes, including cell differentiation, development, and metabolism, as well as complex diseases . Previous studies have identified a distinct miRNA expression profile in psoriasis skin compared to healthy skin . Several of these deregulated miRNAs have been shown to regulate keratinocyte proliferation and differentiation, such as miR-21 , miR-217 , miR-194 , miR-99a  and miR-330 . Although a large number of miRNAs have been identified, their roles in the biology of psoriasis and the underlying mechanisms have not been fully explored.
The miRNA let-7 is the first known human miRNA and second found in Coenorhabditis elegans (C. elegans) . Let-7b, a member of the let-7 family, has been shown to play a role in proliferation and differentiation of neural stem cells , and also suppresses the odonto/osteogenic differentiation capacity of stem cells from apical papilla by targeting MMP1 . Recent study demonstrated that let-7b stimulates differentiation and prevents metaplasia by regulating HNF6 expression in pancreatic acinar cells . It is also becoming increasingly recognized that let-7b plays an important role in hair growth [15, 16], cutaneous wound healing  and skin disease . Recently, it was found that let-7b inhibits keratinocyte migration through targeting IGF2BP2 . As well as, let-7b has been also implicated in other malignacies e.g. liver and thyroid cancer and it exerts a tumorsuppressive role by inhibiting proliferation [19, 20]. However, the biological role of let-7b in the pathogenesis of psoriasis, and especially with regard to its influence on keratinocyte differentiation, is not fully understood.
In this study, we aimed to investigate the biological role of let-7b and the underlying molecular mechanism in regulating the pathogenesis of psoriasis. We found the expression of let-7b was markedly down-regulated in psoriasis keratinocytes in a psoriatic mouse model. We demonstrate a previously unrecognized role of let-7b in regulating the keratinocyte differentiation by using a keratinocyte-specific let-7b expression mouse model (let-7bTG). We have revealed that the let-7b overexpression in basal keratinocytes inhibits acanthosis and reduces the disease severity in two mouse models of psoriasis. Moreover, interleukin (IL)-6 was identified as a direct target of let-7b by which let-7b regulates keratinocyte differentiation. Taken together, our study demonstrates that let-7b regulates keratinocyte differentiation through targeting IL-6-dependent ERK1/2 signaling that may play an important role in the pathogenesis of psoriasis.
Four patients with psoriasis and four healthy subjects were enrolled in the present study. Psoriasis patients were recruited from the Department of Dermatology, Chongqing First People’s Hospital (Chongqing, China). The psoriasis patients enrolled in this study had neither received any local treatment for two weeks nor had used any systemically immunosuppressive medications for at least one month before study participation. Punch biopsies (4 mm) were collected from psoriasis patients at the lesional site. Punch biopsies collected from the healthy subjects were used as control. The specimens were immediately frozen in liquid nitrogen and stored at − 80 °C for further use. The present study was reviewed and approved by Institutional Human Experiment and Ethic Committee of the Affiliated Hospital of Medical University of Anhui. The written informed consents were obtained from all participants.
Keratinocyte-specific let-7b expressing transgenic mice (let-7bTG) were kindly provided by Dr. Xiao Yang (Institute of Biotechnology, The Academy of Military Medical Sciences, Beijing, China). Because male mice are aggressive and easy to fight each other and torn the back skin. Keeping in view this aspect, we decided to choose only female mice (8–12 weeks of age) as the experimental model . IMQ-induced mouse model of psoriasis: The mice were applied a daily topical dose of 62.5 mg of IMQ cream (5%) (MedShine, #120503; China) on the shaved back for seven consecutive days. At the days indicated, skin thickness was measured using a micrometer (Mitutoyo). Control mice were treated with a same dose of vehicle cream. PD98059 treatment in vivo: when mice treated with IMQ after 4 h, a daily topical dose of PD98059 (5 mg/kg, APExBIO) dissolve the 10% dimethylsulfoxide (DMSO) or DMSO directly drop on the skin to treatment psoriasis. All animal procedures were performed under the ethical guidelines of Laboratory Animal Welfare and Ethics Committee Of the Third Military Medical University and according to the recommendations of the Chongqing Experimental Animal Regulation Board (SCXK-PLA-20140011); the approval number is SYXK-PLA-20140031.
Cell culture and transfection
HaCaT cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 1% Non-Essential Amino acid and 10% fetal bovine serum (Hyclone) under 5% CO2 at 37 °C. Transfection of HaCaT cells were done with let-7b mimic, scrambled miRNA (control group), let-7b antisense oligonucleotides or scrambled oligonucleotides (sham control group) transiently (Genepharm) with lipofectamine 2000 reagent (Invitrogen).
Luciferase reporter assays
Firefly luciferase reporter plasmids containing 3′ -UTR of the human IL6 gene and empty luciferase vectors were obtained from Promega. HaCaT cells were co-transfected with the luciferase reporters (50 ng per well) together with 10 nM let-7b mimic or scrambled miRNA using Lipofectamine 2000 (Invitrogen). Luciferase activity was analyzed 48 h post-transfection using Dual-Luciferase Reporter Assay System (Berthold Centro) following the manufacturer’s instructions.
Mouse skin tissues were embedded in paraffin, 6 μm sections were prepared and stained with K14 antibody (1:1000, Covance), IL-6 antibody (1:250, Cell Signaling Technology) and p-ERK1/2 antibody (1:250, Cell Signaling Technology) analyzed by immunohistochemistry methods. Signals were detected by DAB (Zhongshan) staining. Slides were observed under a EVOS FL Auto Microscope (Thermo Fisher).
Culture of keratinocytes
Primary mouse keratinocytes were isolated from normal and transgenic newborn mice skin. 5 mg/ml Dispase (Invitrogen) solution was applied to the full thickness skin overnight (14–16 h) at 4 °C. The epidermis was softly separated from the dermis and transferred onto the surface of the drop of TrypLE Select with the basal layer downward (Invitrogen). The epidermis was incubated for 20–30 min at room temperature, followed by suspension in Keratinocyte-SFM medium with supplements (Invitrogen). Cells were separated from the epidermis and incubated in dishes coated with a solution containing 1% v/v Invitrogen 100 collagen, 10 mM HEPES, 10 μg /ml fibronectin, and 100 μg /ml BSA, at 34 °C in 5% CO2 . Cells were further cultured in fresh Keratinocyte-SFM medium with supplements and 5% fetal bovine serum (Hyclone), which was replaced once every two days.
RNA extraction and qRT-PCR
Total cellular RNA was extracted using TRIzol reagent (TaKaRa) following the manufacturer’s protocol. RNA was reversed transcribed to cDNA using SuperScriptIII (Promega). qRT-PCR was carried out in 20 μl volumes containing 1× Eva Green qPCR master mix (Applied Biological Materials Inc., Richmond, BC, Canada) on CFX Connect™ Real-Time PCR Detection System (USA). Primers are listed in Supplement Table S1. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a reference gene. The relative expression levels of mRNA were quantified using the ΔΔCt method.
Western blot analysis
Western blotting was performed as follows: the protein lysates were collected from the cells. The tissue lysates were separated on 12% SDS-polyacrylamide gels and transferred onto polyvinylidene fluoride membranes (Bio-Rad). Antibodies used included CK10, CK1 (1:1000, Abcam), Involucrin, IL-6, p-ERK and ERK antibody (1:1000, Cell Signaling Technology)), while GAPDH (1:2000) was used as a loading control. The Western blot results were further analyzed using BIO-RAD ChenmiDoc™ XRS+.
All values were expressed as mean ± S.D. Statistical analysis of the data was performed by two-tailed Student’s t test (*, p < 0.05; **, p < 0.01).
Reduction of disease severity in let-7bTG mice treated with IMQ
Accelerated cell differentiation in let-7bTG mice
Let-7b promotes keratinocyte differentiation in HaCaT
The 3’ UTR of the IL-6 gene is a target of let-7b
Let-7b blocks ERK phosphorylation signaling
Psoriasis is a chronic relapsing-remitting inflammatory skin disease that develops in genetically predisposed individuals after an unknown initial environmental, pathogenic or internal trigger. For decades, efforts have been made to explore the inflammation and keratinocytes proliferation or apoptosis role of miRNA in psoriasis while little is known about the roles of miRNAs in the development of psoriasis [28, 29]. In the present study we observed that let-7b directly acts on IL-6 and ERK signaling to affect keratinocyte differentiation. Notably, our findings confirm by in vitro and in vivo analyses in keratinocytes and in transgenic mice, respectively, that let-7b serves an essential role during psoriasis.
Previously, our group had established a keratinocyte-specific let-7b expression in a mouse model and explored a significant role let-7b in wound healing mediated migration in keratinocytes . The sequential over-lapping phases during cutaneous wound healing closely resemble psoriasis lesional micro-environment. This led us to the question whether let-7b is instrumental in the pathogenesis of psoriasis. Among the numerous mouse models used for studying human psoriasis , the repetitive epicutaneous application of IMQ-containing imiquimod cream to the shaved mouse skin represents a versatile and efficient experimental regimen to study early events of human disease . The results of our study confirm that let-7bTG mice have less acanthosis and a reduced disease severity when treated with IMQ. It is well known that hyper-proliferation, altered differentiation of epidermal keratinocytes, and subepidermal angiogenesis are also observed in psoriatic lesions. However, previous study illustrated that let-7b does not affect keratinocyte proliferation and apoptosis, so we focused on the contribution of let-7b to cell differentiation in psoriasis.
The epidermis is a dynamic tissue mainly constituted by keratinocytes that undergo a tightly controlled differentiation program, moving from the basal to the suprabasal layers . In the epidermal basal layer, cells express keratin 5 (CK5) and keratin 14 (CK14), while in cells of the upper (spinous) layers these are progressively down-regulated and replaced by keratin 1 (CK1) and keratin 10 (CK10) . Thus, expression of KRT1 and KRT10 is the first indication of differentiation. In psoriasis skin that is characterized by reduced levels of differentiation markers (K10) and the balance between proliferation and differentiation is impaired . During cornification, additional proteins such as involucrin, loricrin and filaggrin are required to form corneodesmosomes and cornified envelopes . Consistently, the results of the present study revealed an obvious decrease in CK10 production and increased Involucrin expression in skin biopsies of psoriasis patients, which is likely to be responsible for a significant suppression of keratinocyte differentiation. Interestingly, we observed a complete reversal of the expression of differentiation markers in let-7bTG mice treated with IMQ, and we illustrate that let-7b affects keratinocyte differentiation in vitro.
With regard to the mechanism of let-7b in keratinocytes, potential target genes were determined. Among all potential target genes revealed by bioinformatics prediction software, IL-6 was selected as a suitable candidate for further analyses. It is known that stressed keratinocytes release IL-6 that recruits macrophages and neutrophils to sites of inflammation. Cytokines lead to abnormal keratinocyte maturation and activation of dendritic cells (DCs) . Plasmacytoid DCs that are known to be involved in antiviral responses have been implicated in pathogenesis of psoriasis . Although let-7b was reported to modulate inflammatory cytokines such as IL-6 in cholangiocarcinoma by short-term oxidative stress responses , the in vivo function of let-7b and the underlying mechanisms by which let-7b regulates cell differentiation in psoriasis is poorly understood. Here we identified the IL-6 gene as a direct target of let-7b to promote keratinocyte differentiation. Four separate bioinformatics tools predict that let-7b targets a sequence in the 3’UTR of IL-6 mRNA. In addition, IL-6 expression is significantly decreased in diseased epidermis of let-7bTG mice compared to control mice treated with IMQ. Moreover, let-7b overexpression in HaCaT cells results in significantly decreased luciferase activity after transfection of HaCaT cells with a construct expressing the target sequence in the 3’UTR of IL-6. Lastly, the administration of a let-7b mimic amplifies let-7b function by suppressing IL-6 mRNA and protein levels in vivo. IL-6 has been shown to regulate inflammatory response and angiogenesis in a series of cell lines [37, 38]. The role of IL-6 in keratinocyte differentiation and in the pathogenesis of psoriasis was so far unknown; here we showed that IL-6 expression is augmented in the epidermis of lesioned skin from psoriasis patients. Overexpression as realized in let-7bTG mice reduces IL-6 expression that leads to acanthosis and enhanced disease severity that is reduced by IMQ treatment. The data described above indicate that modulation of IL-6 expression in keratinocytes provide a basis for further studies to unravel the detailed mechanism of its differentiation regulation and a potential for future psoriasis therapies.
Furthermore, IL-6 mediated differentiation via signalling cascades, including ERK1/2, STAT3 and NF-κB signaling [39, 40]. The inhibition of ERK1/2 phosphorylation is associated with cell differentiation in certain cell lines . In this study, we found that ERK1/2 phosphorylation was demonstrated in lesioned psoriatic skin compared to non-lesioned psoriatic skin, which is consistent with other reports and shows that active ERK1/2 suppresses differentiation of keratinocytes . To our knowledge, no prior studies have addressed the possible direct intrinsic role of let-7b in keratinocyte differentiation and in the pathogenesis of psoriasis. Here we demonstrate that the overexpression of let-7b in transgenic mice leads to increased keratinocyte differentiation via ERK1/2 signaling, while inhibiting acanthosis and reducing disease severity in psoriasis mouse models.
In conclusion, the present study has shown that let-7b overexpression increases epidermal differentiation and attenuates disease severity in mouse models of psoriasis. Let-7b directly targets IL-6, an essential cytokine regulating cell differentiation, which has been shown to be induced in the epidermis of lesioned skin from psoriasis patients. Our findings reveal a previously unknown mechanism for an homoeostatic let-7b-IL-6 axis whilst highlighting a critical role of ERK1/2 signaling in epidermal differentiation of psoriasis.
We thank Dr. Yongpin Bao, University of East Anglia, UK, for useful discussions and suggestions that help to improve this manuscript.
Availability of data and material
All data generated in this study are included in the manuscript.
This work was supported by the National Natural Science Foundation of China under Grant 81570703, Fundamental Research Funds for the Central Universities (No.106112017CDJ), the National Science Foundation for Young Scholars (81704090) and Key projects of Chongqing Municipal Science and Technology Commission (cstc2017jcyjbx0044). Both projects are not in a financial or personal relationship to this study.
YW, LL and CXB performed the experiments. MJZ helped with mouse breeding and flow cytometry. XYH provided specific reagents and human skin samples. YW, MFN, QCD, JLZ designed the conceptual idea for this study. YW, XMJ, JWB, JLZ and MFN wrote the manuscript. All the authors approved the submission of this manuscript in its final form.
Skin tissues were drawn from healthy and psoriasis volunteers with informed consent in concordance with the ethical of the Affiliated Hospital of Medical University of Anhui Human Research Ethics Committee (approval number _ Fast_ PJ2017–11–14). All animal procedures were performed under the ethical guidelines of Laboratory Animal Welfare and Ethics Committee Of the Third Military Medical University and according to the recommendations of the Chongqing Experimental Animal Regulation Board (SCXK-PLA-20140011); the approval number is SYXK-PLA-20140031.
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- 4.Liu Y, Liu Q. MicroRNAs as regulatory elements in psoriasis. Open Med (Wars). 2016;11:336–40.Google Scholar
- 23.Young PM, Parsi KK, Schupp CW, Armstrong AW. Psoriasis and wound healing outcomes: a retrospective cohort study examining wound complications and antibiotic use. Dermatol Online J. 2017;23:11.Google Scholar
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