Long noncoding RNA plasmacytoma variant translocation 1 is overexpressed in cutaneous squamous cell carcinoma and exon 2 is critical for its oncogenicity.

BACKGROUND: Cutaneous squamous cell carcinoma (cSCC) is one of the most common and fastest increasing forms of cancer worldwide with metastatic potential. Long noncoding RNAs (lncRNAs) are a group of RNA molecules with essential regulatory functions in both physiological and pathological processes. OBJECTIVES: To investigate the function and mode of action of lncRNA plasmacytoma variant translocation 1 (PVT1) in cSCC. METHODS: Quantitative reverse transcriptase polymerase chain reaction and single-molecule in situ hybridization were used to quantify the expression level of PVT1 in normal skin, premalignant skin lesions, actinic keratosis (AK) and primary and metastatic cSCCs. The function of PVT1 in cSCC was investigated both in vivo (tumour xenografts) and in vitro (competitive cell growth assay, 5-ethynyl-2'-deoxyuridine incorporation assay, colony formation assay and tumour spheroid formation assay) upon CRISPR-Cas9-mediated knockout of the entire PVT1 locus, the knockout of exon 2 of PVT1, and locked nucleic acid (LNA) gapmer-mediated PVT1 knockdown. RNA sequencing analysis was conducted to identify genes and processes regulated by PVT1. RESULTS: We identified PVT1 as a lncRNA upregulated in cSCC in situ and cSCC, associated with the malignant phenotype of cSCC. We showed that the expression of PVT1 in cSCC was regulated by MYC. Both CRISPR-Cas9 deletion of the entire PVT1 locus and LNA gapmer-mediated knockdown of PVT1 transcript impaired the malignant behaviour of cSCC cells, suggesting that PVT1 is an oncogenic transcript in cSCC. Furthermore, knockout of PVT1 exon 2 inhibited cSCC tumour growth both in vivo and in vitro, demonstrating that exon 2 is a critical element for the oncogenic role of PVT1. Mechanistically, we showed that PVT1 was localized in the cell nucleus and its deletion resulted in cellular senescence, increased cyclin-dependent kinase inhibitor 1 (p21/CDKN1A) expression and cell cycle arrest. CONCLUSIONS: Our study revealed a previously unrecognized role for exon 2 of PVT1 in its oncogenic role and that PVT1 suppresses cellular senescence in cSCC. PVT1 may be a potential biomarker and therapeutic target in cSCC.

miR-378a regulates keratinocyte responsiveness to interleukin-17A in psoriasis.

BACKGROUND: Psoriasis is an immune-mediated inflammatory skin disease, in which an interplay between infiltrating immune cells and keratinocytes sustains chronic skin inflammation. Interleukin (IL)-17A is a key inflammatory cytokine in psoriasis and its main cellular targets are keratinocytes. OBJECTIVES: To explore the role of miR-378a in psoriasis. METHODS: Keratinocytes obtained from psoriatic skin and healthy epidermis were separated by magnetic sorting, and the expression of miR-378a was analysed by quantitative polymerase chain reaction. The regulation and function of miR-378a was studied using primary human keratinocytes. The expression of miR-378a was modulated by synthetic mimics, and nuclear factor kappa B (NF-κB) activity and transcriptomic changes were studied. Synthetic miR-378a was delivered to mouse skin in conjunction with induction of psoriasiform skin inflammation by imiquimod. RESULTS: We show that miR-378a is induced by IL-17A in keratinocytes through NF-κB, C/EBP-ß and IκBζ and that it is overexpressed in psoriatic epidermis. In cultured keratinocytes, ectopic expression of miR-378a resulted in the nuclear translocation of p65 and enhanced NF-κB-driven promoter activity even in the absence of inflammatory stimuli. Moreover, miR-378a potentiated the effect of IL-17A on NF-κB nuclear translocation and downstream activation of the NF-κB pathway. Finally, injection of miR-378a into mouse skin augmented psoriasis-like skin inflammation with increased epidermal proliferation and induction of inflammatory mediators. Mechanistically, miR-378a acts as a suppressor of NFKBIA/IκBζ, an important negative regulator of the NF-κB pathway in keratinocytes. CONCLUSIONS: Collectively, our findings identify miR-378a as an amplifier of IL-17A-induced NF-κB signalling in keratinocytes and suggest that increased miR-378a levels contribute to the amplification of IL-17A-driven skin inflammation in psoriasis.

Cross-talk between IFN-γ and TWEAK through miR-149 amplifies skin inflammation in psoriasis.

BACKGROUND: Psoriasis is a chronic inflammatory skin disease with disturbed interplay between immune cells and keratinocytes. A strong IFN-γ signature is characteristic for psoriasis skin, but the role of IFN-γ has been elusive. MicroRNAs are short RNAs regulating gene expression. OBJECTIVE: Our aim was to investigate the role of miR-149 in psoriasis and in the inflammatory responses of keratinocytes. METHODS: miR-149 expression was measured by quantitative RT-PCR in keratinocytes isolated from healthy skin and lesional and nonlesional psoriasis skin. Synthetic miR-149 was injected intradermally into the back skin of mice, and imiquimod was applied to induce psoriasis-like skin inflammation, which was then evaluated at the morphologic, histologic, and molecular levels. miR-149 was transiently overexpressed or inhibited in keratinocytes in combination with IFN-γ- and/or TNF-related weak inducer of apoptosis (TWEAK)-treatment. RESULTS: Here we report a microRNA-mediated mechanism by which IFN-γ primes keratinocytes to inflammatory stimuli. Treatment with IFN-γ results in a rapid and long-lasting suppression of miR-149 in keratinocytes. Depletion of miR-149 in keratinocytes leads to widespread transcriptomic changes and induction of inflammatory mediators with enrichment of the TWEAK pathway. We show that IFN-γ-mediated suppression of miR-149 leads to amplified inflammatory responses to TWEAK. TWEAK receptor (TWEAKR/Fn14) is identified as a novel direct target of miR-149. The in vivo relevance of this pathway is supported by decreased miR-149 expression in psoriasis keratinocytes, as well as by the protective effect of synthetic miR-149 in the imiquimod-induced mouse model of psoriasis. CONCLUSION: Our data define a new mechanism, in which IFN-γ primes keratinocytes for TWEAK-induced inflammatory responses through suppression of miR-149, promoting skin inflammation.

Chromatin interactions in differentiating keratinocytes reveal novel atopic dermatitis- and psoriasis-associated genes.

BACKGROUND: Hundreds of variants associated with atopic dermatitis (AD) and psoriasis, 2 common inflammatory skin disorders, have previously been discovered through genome-wide association studies (GWASs). The majority of these variants are in noncoding regions, and their target genes remain largely unclear. OBJECTIVE: We sought to understand the effects of these noncoding variants on the development of AD and psoriasis by linking them to the genes that they regulate. METHODS: We constructed genomic 3-dimensional maps of human keratinocytes during differentiation by using targeted chromosome conformation capture (Capture Hi-C) targeting more than 20,000 promoters and 214 GWAS variants and combined these data with transcriptome and epigenomic data sets. We validated our results with reporter assays, clustered regularly interspaced short palindromic repeats activation, and examination of patient gene expression from previous studies. RESULTS: We identified 118 target genes of 82 AD and psoriasis GWAS variants. Differential expression of 58 of the 118 target genes (49%) occurred in either AD or psoriatic lesions, many of which were not previously linked to any skin disease. We highlighted the genes AFG1L, CLINT1, ADO, LINC00302, and RP1-140J1.1 and provided further evidence for their potential roles in AD and psoriasis. CONCLUSIONS: Our work focused on skin barrier pathology through investigation of the interaction profile of GWAS variants during keratinocyte differentiation. We have provided a catalogue of candidate genes that could modulate the risk of AD and psoriasis. Given that only 35% of the target genes are the gene nearest to the known GWAS variants, we expect that our work will contribute to the discovery of novel pathways involved in AD and psoriasis.

EGFR/Ras-induced CCL20 production modulates the tumour microenvironment.

BACKGROUND: The activation of the EGFR/Ras-signalling pathway in tumour cells induces a distinct chemokine repertoire, which in turn modulates the tumour microenvironment. METHODS: The effects of EGFR/Ras on the expression and translation of CCL20 were analysed in a large set of epithelial cancer cell lines and tumour tissues by RT-qPCR and ELISA in vitro. CCL20 production was verified by immunohistochemistry in different tumour tissues and correlated with clinical data. The effects of CCL20 on endothelial cell migration and tumour-associated vascularisation were comprehensively analysed with chemotaxis assays in vitro and in CCR6-deficient mice in vivo. RESULTS: Tumours facilitate progression by the EGFR/Ras-induced production of CCL20. Expression of the chemokine CCL20 in tumours correlates with advanced tumour stage, increased lymph node metastasis and decreased survival in patients. Microvascular endothelial cells abundantly express the specific CCL20 receptor CCR6. CCR6 signalling in endothelial cells induces angiogenesis. CCR6-deficient mice show significantly decreased tumour growth and tumour-associated vascularisation. The observed phenotype is dependent on CCR6 deficiency in stromal cells but not within the immune system. CONCLUSION: We propose that the chemokine axis CCL20-CCR6 represents a novel and promising target to interfere with the tumour microenvironment, and opens an innovative multimodal strategy for cancer therapy.

The Keratinocyte Transcriptome in Psoriasis: Pathways Related to Immune Responses, Cell Cycle and Keratinization.

Psoriasis is a common immune-mediated disease resulting from altered cross-talk between keratinocytes and immune cells. Previous transcriptomic studies have identified thousands of deregulated genes in psoriasis skin; however, the transcriptomic changes confined to the epidermal compartment remained poorly characterized. The aim of this study was to characterize the transcriptomic landscape of psoriatic keratinocytes, using sorted CD45neg epidermal cells. Genes with functions in innate immunity, type I interferon response, cell cycle and keratinization were enriched among deregulated genes in psoriatic keratinocytes. Gene set enrichment analysis indicated the dominance of interleukin (IL)-22/IL-17A signatures in the epidermal psoriasis-signature. A set of deregulated genes overlapped with psoriasis-associated genetic regions, suggesting that genetic variations affecting gene expression in keratinocytes contribute to susceptibility to psoriasis. Several psoriasis-susceptibility genes, which were previously believed to be expressed preferentially or exclusively in immune cells, were identified as having altered expression in psoriatic keratinocytes. These results highlight the role of keratinocytes in the pathogenesis of psoriasis, and indicate that both genetic factors and an inflammatory microenvironment contribute to epidermal alterations in psoriasis.

Tofacitinib Represses the Janus Kinase-Signal Transducer and Activators of Transcription Signalling Pathway in Keratinocytes.

Tofacitinib is a Janus kinase (JAK) inhibitor, which has shown efficacy in treating psoriasis. The mode of action of tofacitinib is not completely understood but it has been thought to be mediated by the inhibition of CD4+ T-cell activation. Here, we investigated whether the molecular targets of tofacitinib are expressed in keratinocytes, and whether tofacitinib can modulate the activity of the JAK/Signal Transducer and Activators of Transcription (STAT)-pathway in keratinocytes. Transcriptomic profiling of human keratinocytes treated with IL-22 in combination with tofacitinib revealed that tofacitinib could prevent the majority of IL-22-mediated gene expression changes. Pathway analysis of tofacitinib-regulated genes in keratinocytes revealed enrichment of genes involved in the JAK/STAT signalling pathway. Quantitative real-time-PCR confirmed the upregulation of S100A7 and downregulation of EGR1 expression by IL-22, which was prevented by tofacitinib pre-treatment. These results indicate a direct effect of tofacinitib on keratinocytes, which can have relevance for systemic as well as for topical treatment of psoriasis with tofacitinib.

MicroRNA-146a suppresses IL-17-mediated skin inflammation and is genetically associated with psoriasis.

BACKGROUND: Psoriasis is an immune-mediated inflammatory skin disease with a strong genetic background in which activation of IL-17 signaling is central in the pathogenesis. Little has been known about the role of noncoding RNAs, including microRNAs (miRNAs), in predisposition to the disease. OBJECTIVE: We sought to investigate the genetic association of single nucleotide polymorphisms in microRNA-146a (miR-146a) to psoriasis and to explore its function in the initiation and resolution of the disease. METHODS: Analysis of the genetic association of miR-146a rs2910164 and psoriasis was carried out on 1546 patients with psoriasis and 1526 control subjects. The role of miR-146a in patients with psoriasis was assessed by using miR-146a-/- mice in conjunction with the imiquimod-induced mouse model of psoriasis. The severity of psoriasis-like skin inflammation was evaluated at morphologic, histologic, and molecular levels. miR-146a was ectopically overexpressed and inhibited in keratinocytes treated with IL-17. Synthetic miR-146a was injected intradermally into mice. RESULTS: Here we report protective association of a functional polymorphism in the miR-146a precursor (rs2910164). Genetic deficiency in miR-146a leads to earlier onset and exacerbated pathology of skin inflammation, with increased expression of IL-17-induced keratinocyte-derived inflammatory mediators, epidermal hyperproliferation, and increased neutrophil infiltration. Moreover, miR-146a-deficient mice do not resolve inflammation after discontinuation of imiquimod challenge. The overexpression of miR-146a suppressed, whereas its inhibition enhanced, IL-17-driven inflammation in keratinocytes. Functionally, miR-146a impairs the neutrophil chemoattractant capacity of keratinocytes. Finally, delivery of miR-146a mimics into the skin leads to amelioration of psoriasiform skin inflammation, decreased epidermal proliferation, and neutrophil infiltration. CONCLUSIONS: Our results define a crucial role for miR-146a in modulating IL-17-driven inflammation in the skin.

Next-generation sequencing identifies microRNAs that associate with pathogenic autoimmune neuroinflammation in rats.

MicroRNAs (miRNAs) are known to regulate most biological processes and have been found dysregulated in a variety of diseases, including multiple sclerosis (MS). In this study, we characterized miRNAs that associate with susceptibility to develop experimental autoimmune encephalomyelitis (EAE) in rats, a well-established animal model of MS. Using Illumina next-generation sequencing, we detected 544 miRNAs in the lymph nodes of EAE-susceptible Dark Agouti and EAE-resistant Piebald Virol Glaxo rats during immune activation. Forty-three miRNAs were found differentially expressed between the two strains, with 81% (35 out of 43) showing higher expression in the susceptible strain. Only 33% of tested miRNAs displayed differential expression in naive lymph nodes, suggesting that a majority of regulated miRNAs are EAE dependent. Further investigation of a selected six miRNAs indicates differences in cellular source and kinetics of expression. Several of the miRNAs, including miR-146a, miR-21, miR-181a, miR-223, and let-7, have previously been implicated in immune system regulation. Moreover, 77% (33 out of 43) of the miRNAs were associated with MS and other autoimmune diseases. Target genes likely regulated by the miRNAs were identified using computational predictions combined with whole-genome expression data. Differentially expressed miRNAs and their targets involve functions important for MS and EAE, such as immune cell migration through targeting genes like Cxcr3 and cellular maintenance and signaling by regulation of Prkcd and Stat1. In addition, we demonstrated that these three genes are direct targets of miR-181a. Our study highlights the impact of multiple miRNAs, displaying diverse kinetics and cellular sources, on development of pathogenic autoimmune inflammation.

MicroRNA-31 is overexpressed in psoriasis and modulates inflammatory cytokine and chemokine production in keratinocytes via targeting serine/threonine kinase 40.

Psoriasis is characterized by a specific microRNA expression profile, distinct from that of healthy skin. MiR-31 is one of the most highly overexpressed microRNAs in psoriasis skin; however, its biological role in the disease has not been studied. In this study, we show that miR-31 is markedly overexpressed in psoriasis keratinocytes. Specific inhibition of miR-31 suppressed NF-κB-driven promoter luciferase activity and the basal and TNF-α-induced production of IL-1ß, CXCL1/growth-related oncogene-α, CXCL5/epithelial-derived neutrophil-activating peptide 78, and CXCL8/IL-8 in human primary keratinocytes. Moreover, interference with endogenous miR-31 decreased the ability of keratinocytes to activate endothelial cells and attract leukocytes. By microarray expression profiling, we identified genes regulated by miR-31 in keratinocytes. Among these genes, we identified serine/threonine kinase 40 (STK40), a negative regulator of NF-κB signaling, as a direct target for miR-31. Silencing of STK40 rescued the suppressive effect of miR-31 inhibition on cytokine/chemokine expression, indicating that miR-31 regulates cytokine/chemokine expression via targeting STK40 in keratinocytes. Finally, we demonstrated that TGF-ß1, a cytokine highly expressed in psoriasis epidermis, upregulated miR-31 expression in keratinocytes in vitro and in vivo. Collectively, our findings suggest that overexpression of miR-31 contributes to skin inflammation in psoriasis lesions by regulating the production of inflammatory mediators and leukocyte chemotaxis to the skin. Our data indicate that inhibition of miR-31 may be a potential therapeutic option in psoriasis.

miR-193b/365a cluster controls progression of epidermal squamous cell carcinoma.

Incidence of cutaneous squamous cell carcinomas (cSCCs) constantly increases in the Caucasian population. Developing preferentially on precancerous lesions such as actinic keratoses due to chronic sunlight exposure, cSCCs result from the malignant transformation of keratinocytes. Although a resection of the primary tumor is usually curative, a subset of aggressive cSCCs shows a high risk of recurrence and metastases. The characterization of the molecular dysfunctions involved in cSCC development should help to identify new relevant targets against these aggressive cSCCs. In that context, we have used small RNA sequencing to identify 100 microRNAs (miRNAs) whose expression was altered during chemically induced mouse skin tumorigenesis. The decreased expression of the miR-193b/365a cluster during tumor progression suggests a tumor suppressor role. Ectopic expression of these miRNAs in tumor cells indeed inhibited their proliferation, clonogenic potential and migration, which were stimulated in normal keratinocytes when these miRNAs were blocked with antisense oligonucleotides. A combination of in silico predictions and transcriptome analyses identified several target genes of interest. We validated KRAS and MAX as direct targets of miR-193b and miR-365a. Repression of these targets using siRNAs mimicked the effects of miR-193b and miR-365a, suggesting that these genes might mediate, at least in part, the tumor-suppressive action of these miRNAs.

Genetic polymorphisms altering microRNA activity in psoriasis--a key to solve the puzzle of missing heritability?

Psoriasis is a chronic immune-mediated skin disease in which the balance in the interplay of immune cells and keratinocytes is disturbed. MicroRNAs (miRNAs) are endogenous small regulatory RNAs that stabilize cellular phenotypes and fine-tune signal transduction feedback loops through the regulation of gene networks. Through the regulation of their multiple target genes, miRNAs regulate the development of inflammatory cell subsets and have a significant impact on the magnitude of inflammatory responses. Since the discovery of deregulated miRNA expression in psoriasis, we have learned that they can regulate differentiation, proliferation and cytokine response of keratinocytes, activation and survival of T cells and the crosstalk between immunocytes and keratinocytes through the regulation of chemokine production. In recent years, it became apparent that genetic polymorphisms in miRNA genes and/or in miRNA binding sites of target genes can affect miRNA activity and contribute to disease susceptibility. Psoriasis has a strong genetic background; however, the contribution of genetic variants involving miRNAs is largely unexplored in psoriasis. We propose that changes in miRNA-mediated gene regulation may be a major contributor to the disturbed balance in immune regulation that results in chronic skin inflammation. In this viewpoint essay, we focus on the emerging new aspects of the role of miRNAs in psoriasis and propose that genetic polymorphisms that affect miRNA activity might be important in the pathogenesis of psoriasis.

Activation of toll-like receptors alters the microRNA expression profile of keratinocytes.

Keratinocytes recognize invading pathogens by various receptors, among them Toll-like receptors (TLRs), and provide the first line of defense in skin immunity. The role of microRNAs in this important defense mechanism has not been explored yet. Our aim was to identify microRNAs involved in the innate immune response of keratinocytes. MicroRNA expression profiling revealed that the TLR2 ligand zymosan, the TLR3 ligand poly(I:C) or the TLR5 ligand flagellin significantly altered the microRNA expression in keratinocytes. The regulation of microRNAs was concentration-dependent and it could be neutralized by siRNAs specific for TLR2, TLR3 and TLR5, respectively, confirming the specificity of the TLR response. Interestingly, one microRNA, miR-146a, was strongly induced by all studied TLR ligands, while other microRNAs were regulated in a TLR- or time point-specific manner. These findings suggest an important role for microRNAs in the innate immune response of keratinocytes and provide a basis for further investigations.

MicroRNA-125b down-regulates matrix metallopeptidase 13 and inhibits cutaneous squamous cell carcinoma cell proliferation, migration, and invasion.

Cutaneous squamous cell carcinoma (cSCC) is the second most common human cancer. Although dysregulation of microRNAs (miRNAs) is known to be involved in a variety of cancers, the role of miRNAs in cSCC is unclear. In this study, we aimed to identify tumor suppressive and oncogenic miRNAs involved in the pathogenesis of cSCC. MiRNA expression profiles in healthy skins (n = 4) and cSCCs (n = 4) were analyzed using MicroRNA Low Density Array. MiR-125b expression was analyzed by quantitative real-time PCR and in situ hybridization in skin biopsies from 40 healthy donors, 13 actinic keratosis, and 74 cSCC patients. The effect of miR-125b was analyzed in wound closure, colony formation, migration, and invasion assays in two cSCC cell lines, UT-SCC-7 and A431. The genes regulated by miR-125b in cSCC were identified by microarray analysis and its direct target was validated by luciferase reporter assay. Comparing cSCC with healthy skin, we identified four up-regulated miRNAs (miR-31, miR-135b, miR-21, and miR-223) and 54 down-regulated miRNAs, including miR-125b, whose function was further examined. We found that miR-125b suppressed proliferation, colony formation, migratory, and invasive capacity of cSCC cells. Matrix metallopeptidase 13 (MMP13) was identified as a direct target suppressed by miR-125b, and there was an inverse relationship between the expression of miR-125b and MMP13 in cSCC. Knockdown of MMP13 expression phenocopied the effects of miR-125b overexpression. These findings provide a novel molecular mechanism by which MMP13 is up-regulated in cSCCs and indicate that miR-125b plays a tumor suppressive role in cSCC.

Constraints for monocyte-derived dendritic cell functions under inflammatory conditions.

The activation of TLRs expressed by macrophages or DCs, in the long run, leads to persistently impaired functionality. TLR signals activate a wide range of negative feedback mechanisms; it is not known, however, which of these can lead to long-lasting tolerance for further stimulatory signals. In addition, it is not yet understood how the functionality of monocyte-derived DCs (MoDCs) is influenced in inflamed tissues by the continuous presence of stimulatory signals during their differentiation. Here we studied the role of a wide range of DC-inhibitory mechanisms in a simple and robust model of MoDC inactivation induced by early TLR signals during differentiation. We show that the activation-induced suppressor of cytokine signaling 1 (SOCS1), IL-10, STAT3, miR146a and CD150 (SLAM) molecules possessed short-term inhibitory effects on cytokine production but did not induce persistent DC inactivation. On the contrary, the LPS-induced IRAK-1 downregulation could alone lead to persistent MoDC inactivation. Studying cellular functions in line with the activation-induced negative feedback mechanisms, we show that early activation of developing MoDCs allowed only a transient cytokine production that was followed by the downregulation of effector functions and the preservation of a tissue-resident non-migratory phenotype.

RNA editing of the GLI1 transcription factor modulates the output of Hedgehog signaling.

The Hedgehog (HH) signaling pathway has important roles in tumorigenesis and in embryonal patterning. The Glioma-associated oncogene 1 (GLI1) is a key molecule in HH signaling, acting as a transcriptional effector and, moreover, is considered to be a potential therapeutic target for several types of cancer. To extend our previous focus on the implications of alternative splicing for HH signal transduction, we now report on an additional post-transcriptional mechanism with an impact on GLI1 activity, namely RNA editing. The GLI1 mRNA is highly edited at nucleotide 2179 by adenosine deamination in normal cerebellum, but the extent of this modification is reduced in cell lines from the cerebellar tumor medulloblastoma. Additionally, basal cell carcinoma tumor samples exhibit decreased GLI1 editing compared with normal skin. Interestingly, knocking down of either ADAR1 or ADAR2 reduces RNA editing of GLI1. This adenosine to inosine substitution leads to a change from Arginine to Glycine at position 701 that influences not only GLI1 transcriptional activity, but also GLI1-dependent cellular proliferation. Specifically, the edited GLI1, GLI1-701G, has a higher capacity to activate most of the transcriptional targets tested and is less susceptible to inhibition by the negative regulator of HH signaling suppressor of fused. However, the Dyrk1a kinase, implicated in cellular proliferation, is more effective in increasing the transcriptional activity of the non-edited GLI1. Finally, introduction of GLI1-701G into medulloblastoma cells confers a smaller increase in cellular growth relative to GLI1. In conclusion, our findings indicate that RNA editing of GLI1 is a regulatory mechanism that modulates the output of the HH signaling pathway.

MYCN-regulated microRNAs repress estrogen receptor-alpha (ESR1) expression and neuronal differentiation in human neuroblastoma.

MYCN, a proto-oncogene normally expressed in the migrating neural crest, is in its amplified state a key factor in the genesis of human neuroblastoma (NB). However, the mechanisms underlying MYCN-mediated NB progression are poorly understood. Here, we present a MYCN-induced miRNA signature in human NB involving the activation and transrepression of several miRNA genes from paralogous clusters. Several family members derived from the miR-17 approximately 92 cluster, including miR-18a and miR-19a, were among the up-regulated miRNAs. Expression analysis of these miRNAs in NB tumors confirmed increased levels in MYCN-amplified samples. Specifically, we show that miR-18a and miR-19a target and repress the expression of estrogen receptor-alpha (ESR1), a ligand-inducible transcription factor implicated in neuronal differentiation. Immunohistochemical staining demonstrated ESR1 expression in human fetal sympathetic ganglia, suggesting a role for ESR1 during sympathetic nervous system development. Concordantly, lentiviral restoration of ESR1 in NB cells resulted in growth arrest and neuronal differentiation. Moreover, lentiviral-mediated inhibition of miR-18a in NB cells led to severe growth retardation, outgrowth of varicosity-containing neurites, and induction of neuronal sympathetic differentiation markers. Bioinformatic analyses of microarray data from NB tumors revealed that high ESR1 expression correlates with increased event-free survival in NB patients and favorable disease outcome. Thus, MYCN amplification may disrupt estrogen signaling sensitivity in primitive sympathetic cells through deregulation of ESR1, thereby preventing the normal induction of neuroblast differentiation. Collectively, our findings demonstrate the molecular consequences of abnormal miRNA transcription in a MYCN-driven tumor and offer unique insights into the pathology underlying MYCN-amplified NB.

The Long Noncoding RNA LINC00958 Is Induced in Psoriasis Epidermis and Modulates Epidermal Proliferation.

Psoriasis is a common, immune-mediated skin disease characterized by epidermal hyperproliferation and chronic skin inflammation. Long noncoding RNAs are >200 nucleotide-long transcripts that possess important regulatory functions. To date, little is known about the contribution of long noncoding RNAs to psoriasis. In this study, we identify LINC00958 as a long noncoding RNA overexpressed in keratinocytes (KCs) from psoriasis skin lesions, in a transcriptomic screen performed on KCs sorted from psoriasis and healthy skin. Increased levels of LINC00958 in psoriasis KCs were confirmed by RT-qPCR and single-molecule in situ hybridization. Confocal microscopy and analysis of subcellular fractions showed that LINC00958 is mainly localized in the cytoplasm of KCs. IL-17A, a key psoriasis cytokine, induced LINC00958 in KCs through C/EBP-ß and the p38 pathway. The inhibition of LINC00958 led to decreased proliferation as measured by Ki-67 expression, live cell analysis imaging, and 5-ethynyl-2-deoxyuridine assays. Transcriptomic analysis of LINC00958-depleted KCs revealed enrichment of proliferation- and cell cycle‒related genes among differentially expressed transcripts. Moreover, LINC00958 depletion led to decreased basal and IL-17A‒induced phosphorylation of p38. Furthermore, IL-17A‒induced KC proliferation was counteracted by the inhibition of LINC00958. In summary, our data support a role for the IL-17A‒induced long noncoding RNA, LINC00958, in the pathological circuits of psoriasis by reinforcing IL-17A‒induced epidermal hyperproliferation.