Background: The type-II-cytokine IFN-γ is a pivotal player in innate immune responses but also assumes functions in controlling tumor cell growth by orchestrating cellular responses against neoplastic cells. The role of IFN-γ in melanoma is not fully understood: it is a well-known growth inhibitor of melanoma cellsin vitro. On the other hand, IFN-γ may also facilitate melanoma progression. While interferon-regulated genes encoding proteins have been intensively studied since decades, the contribution of miRNAs to effects mediated by interferons is an emerging area of research.We recently described a distinct and dynamic regulation of a whole panel of microRNAs (miRNAs) after IFN-γ-stimulation. The aim of this study was to analyze the transcriptional regulation of miR-29 family members in detail, identify potential interesting target genes and thus further elucidate a potential signaling pathway IFN-γ → Jak→ P-STAT1 → miR-29 → miR-29 target genes and its implication for melanoma growth.Results: Here we show that IFN-γ induces STAT1-dependently a profound up-regulation of the miR-29 primary cluster pri-29a~b-1 in melanoma cell lines. Furthermore, expression levels of pri-29a~b-1 and mature miR-29a and miR-29b were elevated while the pri-29b-2~c cluster was almost undetectable. We observed an inverse correlation between miR-29a/b expression and the proliferation rate of various melanoma cell lines. This finding could be corroborated in cells transfected with either miR-29 mimics or inhibitors. The IFN-γ-induced G1-arrest of melanoma cells involves down-regulation of CDK6, which we proved to be a direct target of miR-29 in these cells. Compared to nevi and normal skin, and metastatic melanoma samples, miR-29a and miR-29b levels were found strikingly elevated in certain patient samples derived from primary melanoma.Conclusions: Our findings reveal that the miR-29a/b1 cluster is to be included in the group of IFN- and STAT-regulated genes. The up-regulated miR-29 family members may act as effectors of cytokine signalling in melanoma and other cancer cells as well as in the immune system.

Non-alcoholic fatty liver disease (NAFLD) is a complex pathology in which several dysfunctions, including alterations in metabolic pathways, mitochondrial functionality and unbalanced lipid import/export, lead to lipid accumulation and progression to inflammation and fibrosis. The enzyme glycine N-methyltransferase (GNMT), the most important enzyme implicated in S-adenosylmethionine catabolism in the liver, is downregulated during NAFLD progression. We have studied the mechanism involved in GNMT downregulation by its repressor microRNA miR-873-5p and the metabolic pathways affected in NAFLD as well as the benefit of recovery GNMT expression. miR-873-5p and GNMT expression were evaluated in liver biopsies of NAFLD/NASH patients. Different in vitro and in vivo NAFLD murine models were used to assess miR-873-5p/GNMT involvement in fatty liver progression through targeting of the miR-873-5p as NAFLD therapy. We describe a new function of GNMT as an essential regulator of Complex II activity in the electron transport chain in the mitochondria. In NAFLD, GNMT expression is controlled by miR-873-5p in the hepatocytes, leading to disruptions in mitochondrial functionality in a preclinical murine non-alcoholic steatohepatitis (NASH) model. Upregulation of miR-873-5p is shown in the liver of NAFLD/NASH patients, correlating with hepatic GNMT depletion. Importantly, NASH therapies based on anti-miR-873-5p resolve lipid accumulation, inflammation and fibrosis by enhancing fatty acid β-oxidation in the mitochondria. Therefore, miR-873-5p inhibitor emerges as a potential tool for NASH treatment. GNMT participates in the regulation of metabolic pathways and mitochondrial functionality through the regulation of Complex II activity in the electron transport chain. In NAFLD, GNMT is repressed by miR-873-5p and its targeting arises as a valuable therapeutic option for treatment.

The interleukin-6–type cytokine oncostatin M (OSM) acts via the Janus kinase/signal transducer and activator of transcription pathway as well as via activation of mitogen-activated protein kinases and is known to critically regulate processes such as liver development and regeneration, hematopoiesis, and angiogenesis, which are also determined by hypoxia with the hypoxia-inducible factor 1α (HIF1α) as a key component. Here we show that treatment of hepatocytes and hepatoma cells with OSM leads to an increased protein level of HIF1α under normoxic and hypoxic conditions. Furthermore, the OSM-dependent HIF1α increase is mediated via Janus kinase/signal transducer and activator of transcription 3 and mitogen-activated protein kinase kinase/extracellular signal-regulated kinase 1/2 pathways. OSM-mediated HIF1α up-regulation did not result from an increase in HIF1α protein stability but from increased transcription from the HIF1α gene. In addition, we show that the OSM-induced HIF1α gene transcription and the resulting enhanced HIF1α protein levels are important for the OSM-dependent vascular endothelial growth factor and plasminogen activator inhibitor 1 gene induction associated with several diseases. Conclusion: HIF1α levels increase significantly after treatment of hepatocytes and hepatoma cells with OSM, and HIF1α contributes to OSM downstream signaling events, pointing to a cross-talk between cytokine and hypoxia signaling in processes such as liver development and regeneration. (Hepatology 2009.)

Reduced levels of intratumoural oxygen are associated with hypoxia-induced pro-oncogenic events such as invasion, metabolic reprogramming, epithelial–mesenchymal transition, metastasis and resistance to therapy, all favouring cancer progression. Small extracellular vesicles (EV) shuttle various cargos (proteins, miRNAs, DNA and others). Tumour-derived EVs can be taken up by neighbouring or distant cells in the tumour microenvironment, thus facilitating intercellular communication. The quantity of extracellular vesicle secretion and their composition can vary with changing microenvironmental conditions and disease states. Here, we investigated in melanoma cells the influence of hypoxia on the content and number of secreted EVs. Whole miRNome and proteome profiling revealed distinct expression patterns in normoxic or hypoxic growth conditions. Apart from the well-known miR-210, we identified miR-1290 as a novel hypoxia-associated microRNA, which was highly abundant in hypoxic EVs. On the other hand, miR-23a-5p and -23b-5p were consistently downregulated in hypoxic conditions, while the protein levels of the miR-23a/b-5p-predicted target IPO11 were concomitantly upregulated. Furthermore, hypoxic melanoma EVs exhibit a signature consisting of six proteins (AKR7A2, DDX39B, EIF3C, FARSA, PRMT5, VARS), which were significantly associated with a poor prognosis for melanoma patients, indicating that proteins and/or miRNAs secreted by cancer cells may be exploited as biomarkers.

Interleukin-6 (IL-6)-type cytokines share the common receptor glycoprotein 130 (gp130), which activates a signaling cascade involving Janus kinases (JAKs) and signal transducer and activator of transcription (STAT) transcription factors. IL-6 and/or its signaling pathway is often deregulated in diseases, such as chronic liver diseases and cancer. Thus, the identification of compounds inhibiting this pathway is of interest for future targeted therapies. We established novel cellular screening systems based on a STAT-responsive reporter gene (Cypridina luciferase). Of a library containing 538 microRNA (miRNA) mimics, several miRNAs affected hyper-IL-6-induced luciferase activities. When focusing on candidate miRNAs specifically targeting 3' UTRs of signaling molecules of this pathway, we identified, e.g., miR-3677-5p as a novel miRNA affecting protein expression of both STAT3 and JAK1, whereas miR-16-1-3p, miR-4473, and miR-520f-3p reduced gp130 surface expression. Interestingly, combination treatment with 2 or 3 miRNAs targeting gp130 or different signaling molecules of the pathway did not increase the inhibitory effects on phospho-STAT3 levels and STAT3 target gene expression compared to treatment with single mimics. Taken together, we identified a set of miRNAs of potential therapeutic value for cancer and inflammatory diseases, which directly target the expression of molecules within the IL-6-signaling pathway and can dampen inflammatory signal transduction.

Interleukin-6 (IL-6)-type cytokines play important roles in liver (patho-)biology. For instance, they regulate the acute phase response to inflammatory signals and are involved in hepatocarcinogenesis. Much is known about the regulation of protein-coding genes by cytokines whereas their effects on the miRNome is less well understood. We performed a microarray screen to identify microRNAs (miRNAs) in human hepatocytes which are modulated by IL-6-type cytokines. Using samples of 2 donors, 27 and 68 miRNAs (out of 1,733) were found to be differentially expressed upon stimulation with hyper-IL-6 (HIL-6) for up to 72 h, with an overlap of 15 commonly regulated miRNAs. qPCR validation revealed that miR-146b-5p was also consistently up-regulated in hepatocytes derived from 2 other donors. Interestingly, miR-146b-5p (but not miR-146a-5p) was induced by IL-6-type cytokines (HIL-6 and OSM) in non-transformed liver-derived PH5CH8 and THLE2 cells and in Huh-7 hepatoma cells, but not in HepG2 or Hep3B hepatoma cells. We did not find evidence for a differential regulation of miR-146b-5p expression by promoter methylation, also when analyzing the TCGA data set on liver cancer samples. Inducible overexpression of miR-146b-5p in PH5CH8 cells followed by RNA-Seq analysis revealed effects on multiple mRNAs, including those encoding IRAK1 and TRAF6 crucial for Toll-like receptor signaling. Indeed, LPS-mediated signaling was attenuated upon overexpression of miR-146b-5p, suggesting a regulatory loop to modulate inflammatory signaling in hepatocytes. Further validation experiments suggest DNAJC6, MAGEE1, MPHOSPH6, PPP2R1B, SLC10A3, SNRNP27, and TIMM17B to be novel targets for miR-146b-5p (and miR-146a-5p).IL-6-type cytokines up-regulate miR-146b-5p expression in liver cells, an anti-inflammatory miRNA with multiple targets shared with miR-146a-5p.