Validation of the prognostic value of a three-gene signature and clinical parameters-based risk score in prostate cancer patients.

BACKGROUND: The study aimed to validate the prognostic value of the Prostatype® risk score (P-score), which includes a three-gene signature and conventional risk factors, in a retrospective cohort. METHODS: All 716 patients diagnosed with prostate cancer from 2008 to 2010 at Skåne University Hospital, Sweden, were included. After excluding patients based on pathological and clinical eligibility criteria, RNA quality, and presence of metastases at diagnosis, a final cohort comprising 316 patients was further analyzed. Expression levels of three genes (IGFBP3, F3, and VGLL3) were measured in archived formalin-fixed paraffin-embedded core needle biopsies. The gene expression data were combined with clinical parameters (Gleason score, prostate-specific antigen, and clinical tumor stage) to calculate the P-score for each patient. Predictive performance of the P-score in terms of prostate cancer-specific mortality (PCSM), distant metastasis and adverse pathological outcomes were investigated. RESULTS: The P-score predicted both PCSM (hazard ratio [HR] = 1.6) and metastasis (HR = 1.46). The P-score had an area under curve (AUC) of 0.93 when predicting the PCSM risk at 10 years (95% confidence interval [CI]: 0.89-0.98), which was significantly better than both D'Amico (AUC: 0.81, 95% CI: 0.72-0.90, p < 0.001) and UCSF-CAPRA (AUC: 0.88, 95% CI: 0.80-0.96, p < 0.05). Decision curve analysis showed a higher net benefit of the P-score compared to both D'Amico and CAPRA. All three risk scores performed similarly in the prediction of distant metastases. For patients who underwent radical prostatectomy (RP), a higher P-score correlated with adverse pathological features such as pathologic tumor stage T3-4 (p < 0.0001) and ≥International Society of Urological Pathology grade group 3 (p < 0.0001). CONCLUSIONS: Our findings provide evidence for the prognostic value of the P-score. The P-score predicted the risk for PCSM more accurately than the D'Amico and CAPRA scores. Performance was similar when predicting the risk for development of distant metastases within 10 years. Moreover, the P-score correlated with adverse pathological outcomes in RP specimens. Thus, the P-score could provide useful information for patients and their doctors to make informed decisions at the time of diagnosis.

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.

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.

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.

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.

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.

MiR-21 is up-regulated in psoriasis and suppresses T cell apoptosis.

MicroRNAs are short non-coding RNAs that regulate gene expression. Previously, in a genome-wide screen, we found deregulation of microRNA expression in psoriasis skin. MicroRNA-21 (miR-21) is one of the microRNAs significantly up-regulated in psoriasis skin lesions. To identify the cell type responsible for the increased miR-21 level, we compared expression of miR-21 in epidermal cells and dermal T cells between psoriasis and healthy skin and found elevated levels of miR-21 in psoriasis in both cell types. In cultured T cells, expression of miR-21 increased markedly upon activation. To explore the function of miR-21 in primary human T helper cells, we inhibited miR-21 using a tiny seed-targeting LNA-anti-miR. Specific inhibition of miR-21 increased the apoptosis rate of activated T cells. Our results suggest that miR-21 suppresses apoptosis in activated T cells, and thus, overexpression of miR-21 may contribute to T cell-derived psoriatic skin inflammation.

MiR-155 is overexpressed in patients with atopic dermatitis and modulates T-cell proliferative responses by targeting cytotoxic T lymphocyte-associated antigen 4.

BACKGROUND: MicroRNAs (miRNAs) are short noncoding RNAs that suppress gene expression at the posttranscriptional level. Atopic dermatitis is a common chronic inflammatory skin disease characterized by the presence of activated T cells within the skin. OBJECTIVE: We sought to explore the role of miRNAs in the pathogenesis of atopic dermatitis. METHODS: Global miRNA expression in healthy and lesional skin of patients with atopic dermatitis was compared by using TaqMan MicroRNA Low Density Arrays. miR-155 expression in tissues and cells was quantified by means of quantitative real-time PCR. The cellular localization of miR-155 was analyzed by means of in situ hybridization. The regulation of cytotoxic T lymphocyte-associated antigen (CTLA-4) by miR-155 was investigated by using luciferase reporter assays and flow cytometry. CTLA-4 expression and functional assays were performed on T(H) cells overexpressing miR-155. RESULTS: miR-155 was one of the highest-ranked upregulated miRNAs in patients with atopic dermatitis. In the skin miR-155 was predominantly expressed in infiltrating immune cells. miR-155 was upregulated during T-cell differentiation/activation and was markedly induced by T-cell activators in PBMCs in vitro and by superantigens and allergens in the skin in vivo. CTLA-4, an important negative regulator of T-cell activation, was identified as a direct target of miR-155. Overexpression of miR-155 in T(H) cells resulted in decreased CTLA-4 levels accompanied by an increased proliferative response. CONCLUSION: miR-155 is significantly overexpressed in patients with atopic dermatitis and might contribute to chronic skin inflammation by increasing the proliferative response of T(H) cells through the downregulation of CTLA-4.

MicroRNA-203 Inversely Correlates with Differentiation Grade, Targets c-MYC, and Functions as a Tumor Suppressor in cSCC.

Cutaneous squamous cell carcinoma (cSCC) is the second most common cancer and a leading cause of cancer mortality among solid organ transplant recipients. MicroRNAs (miR) are short RNAs that regulate gene expression and cellular functions. Here, we show a negative correlation between miR-203 expression and the differentiation grade of cSCC. Functionally, miR-203 suppressed cell proliferation, cell motility, and the angiogenesis-inducing capacity of cSCC cells in vitro and reduced xenograft tumor volume and angiogenesis in vivo. Transcriptomic analysis of cSCC cells with ectopic overexpression of miR-203 showed dramatic changes in gene networks related to cell cycle and proliferation. Transcription factor enrichment analysis identified c-MYC as a hub of miR-203-induced transcriptomic changes in squamous cell carcinoma. We identified c-MYC as a direct target of miR-203. Overexpression of c-MYC in rescue experiments reversed miR-203-induced growth arrest in cSCC, which highlights the importance of c-MYC within the miR-203-regulated gene network. Together, miR-203 acts as a tumor suppressor in cSCC, and its low expression can be a marker for poorly differentiated tumors. Restoration of miR-203 expression may provide a therapeutic benefit, particularly in poorly differentiated cSCC.

MicroRNA-31 Promotes Skin Wound Healing by Enhancing Keratinocyte Proliferation and Migration.

Wound healing is a basic biological process restoring the integrity of the skin. The role of microRNAs during this process remains largely unexplored. By using an in vivo human skin wound healing model, we show here that the expression of miR-31 is gradually upregulated in wound edge keratinocytes in the inflammatory (1 day after injury) through the proliferative phase (7 days after injury) in comparison with intact skin. In human primary keratinocytes, overexpression of miR-31 promoted cell proliferation and migration, whereas inhibition of miR-31 had the opposite effects. Moreover, we identified epithelial membrane protein 1 (EMP-1) as a direct target of miR-31 in keratinocytes. The expression of EMP-1 in the skin was negatively correlated with the level of miR-31 during wound healing. Silencing of EMP-1 mimicked the effects of overexpression of miR-31 on keratinocyte proliferation and migration, indicating that EMP-1 is a critical target mediating the functions of miR-31 in keratinocytes. Finally, we demonstrated that transforming growth factor-ß2, which is highly expressed in skin wounds, upregulated miR-31 expression in keratinocytes. Collectively, we identify miR-31 as a key regulator for promoting keratinocyte proliferation and migration during wound healing.

MicroRNA-132 enhances transition from inflammation to proliferation during wound healing.

Wound healing is a complex process that is characterized by an initial inflammatory phase followed by a proliferative phase. This transition is a critical regulatory point; however, the factors that mediate this process are not fully understood. Here, we evaluated microRNAs (miRs) in skin wound healing and characterized the dynamic change of the miRNome in human skin wounds. miR-132 was highly upregulated during the inflammatory phase of wound repair, predominantly expressed in epidermal keratinocytes, and peaked in the subsequent proliferative phase. TGF-ß1 and TGF-ß2 induced miR-132 expression in keratinocytes, and transcriptome analysis of these cells revealed that miR-132 regulates a large number of immune response- and cell cycle-related genes. In keratinocytes, miR-132 decreased the production of chemokines and the capability to attract leukocytes by suppressing the NF-κB pathway. Conversely, miR-132 increased activity of the STAT3 and ERK pathways, thereby promoting keratinocyte growth. Silencing of the miR-132 target heparin-binding EGF-like growth factor (HB-EGF) phenocopied miR-132 overexpression in keratinocytes. Using mouse and human ex vivo wound models, we found that miR-132 blockade delayed healing, which was accompanied by severe inflammation and deficient keratinocyte proliferation. Together, our results indicate that miR-132 is a critical regulator of skin wound healing that facilitates the transition from the inflammatory to the proliferative phase.

MicroRNA-31 is overexpressed in cutaneous squamous cell carcinoma and regulates cell motility and colony formation ability of tumor cells.

Cutaneous squamous cell carcinoma (cSCC) is a malignancy of epidermal keratinocytes that is responsible for approximately 20% of skin cancer-related death yearly. We have previously compared the microRNA (miRNA) expression profile of cSCC to healthy skin and found the dysregulation of miRNAs in human cSCC. In this study we show that miR-31 is overexpressed in cSCC (n = 68) compared to healthy skin (n = 34) and precancerous skin lesions (actinic keratosis, n = 12). LNA in situ hybridization revealed that miR-31 was specifically up-regulated in tumor cells. Mechanistic studies of inhibition of endogenous miR-31 in human metastatic cSCC cells revealed suppressed migration, invasion and colony forming ability, whereas overexpression of miR-31 induced these phenotypes. These results indicate that miR-31 regulates cancer-associated phenotypes of cSCC and identify miR-31 as a potential target for cSCC treatment.

MiR-146a negatively regulates TLR2-induced inflammatory responses in keratinocytes.

Keratinocytes represent the first line of defense against pathogens in the skin and have important roles in initiating and regulating inflammation during infection and autoimmunity. Here we investigated the role of miR-146a in the regulation of the innate immune response of keratinocytes. Toll-like receptor 2 (TLR2) stimulation of primary human keratinocytes resulted in an NF-κB- and mitogen-activated protein kinase-dependent upregulation of miR-146a expression, which was surprisingly long lasting, contrasting with the rapid and transient induction of inflammatory mediators. Overexpression of miR-146a significantly suppressed the production of IL-8, CCL20, and tumor necrosis factor-α, which functionally suppressed the chemotactic attraction of neutrophils by keratinocytes. Inhibition of endogenous miR-146a induced the production of inflammatory mediators even in nonstimulated keratinocytes, and potentiated the effect of TLR2 stimulation. Transcriptomic profiling revealed that miR-146a suppresses the expression of a large number of immune-related genes in keratinocytes. MiR-146a downregulated interleukin-1 receptor-associated kinase 1 and TNF receptor-associated factor 6, two key adapter molecules downstream of TLR signaling, and suppressed NF-κB promoter-binding activity as shown by promoter luciferase experiments. Together, these data identify miR-146a as a regulatory element in keratinocyte innate immunity, which prevents the production of inflammatory mediators under homeostatic conditions and serves as a potent negative feedback regulator after TLR2 stimulation.

Interleukin-8 is regulated by miR-203 at the posttranscriptional level in primary human keratinocytes.

Background: MicroRNAs are important posttranscriptional regulators of gene expression. MiR-203 is a miRNA preferentially expressed in the skin, and an important regulator of keratinocyte differentiation. MiR-203 has been implicated in skin diseases, in particular in psoriasis in which it is overexpressed, and in basal cell carcinoma where it acts as a tumor suppressor miRNA. Objectives: To identify novel targets for miR-203 that may be relevant in skin physiology and diseases. Materials & Methods: Bioinformatics was used to identify putative miR-203 targets among genes expressed in keratinocytes. Interleukin-8 (IL-8) gene expression and concentration in keratinocyte medium was measured by quantitative real-time PCR and ELISA, respectively. For miRNA overexpression, resting or TNF-α-treated primary human keratinocytes were transfected with synthetic precursor of miR-203, or scramble miRNA precursors using Lipofectamine 2000. 3'UTR luciferase reporter experiments were performed to prove the direct miRNA:mRNA interaction. Site-specific mutagenesis was used to mutate the predicted miR-203 binding sites in the 3'UTR of IL-8 gene. Results: Bioinformatic analysis indentified two putative miR-203 binding sites in the 3'UTR of IL-8. MiR-203 suppressed IL-8 mRNA and protein expression in primary human keratinocytes both under resting conditions and after TNF-α treatment. Overexpression of miR-203 suppressed the luciferase activity of a reporter gene fused with the IL-8 3'UTR. The suppressive effect was abolished when the predicted binding sites of miR-203 on IL-8 3'UTR were mutated. Conclusion: We identify IL-8 as a novel target of miR-203 for posttranscriptional suppression. These findings may have relevance in diseases in which miR-203 and IL-8 expression are deregulated.

MiR-125b, a microRNA downregulated in psoriasis, modulates keratinocyte proliferation by targeting FGFR2.

MicroRNAs (miRNAs) are short, single-stranded, noncoding RNAs that play important roles in the regulation of gene expression. We previously identified a characteristic miRNA expression profile in psoriasis, distinct from that of healthy skin. One of the most downregulated miRNAs in psoriasis skin was microRNA-125b (miR-125b). In this study, we aimed to identify the potential role(s) of miR-125b in psoriasis pathogenesis. In situ hybridization results showed that the major cell type responsible for decreased miR-125b levels in psoriasis lesions was the keratinocyte. Overexpression of miR-125b in primary human keratinocytes suppressed proliferation and induced the expression of several known differentiation markers. Conversely, inhibition of endogenous miR-125b promoted cell proliferation and delayed differentiation. Fibroblast growth factor receptor 2 (FGFR2) was identified as one of the direct targets for suppression by miR-125b by luciferase reporter assay. The expression of miR-125b and FGFR2 was inversely correlated in both transfected keratinocytes and in psoriatic skin. Knocking down FGFR2 expression by siRNA suppressed keratinocyte proliferation, but did not enhance differentiation. Altogether, our results demonstrate a role for miR-125b in the regulation of keratinocyte proliferation and differentiation, partially through the regulation of FGFR2. Loss of miR-125b in psoriasis skin may contribute to hyperproliferation and aberrant differentiation of keratinocytes.