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See detailModulation of the IL-6 signaling pathway in liver cells by miRNAs targeting gp130, JAK1 and/or STAT3
Servais, Florence UL; Kirchmeyer, Mélanie UL; Hamdorf, Matthias UL et al

in Molecular Therapy: Nucleic Acids (2019), 16

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 ... [more ▼]

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. [less ▲]

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See detailThe PD-L1- and IL6-mediated dampening of the IL27/STAT1 anticancer responses are prevented by a-PD-L1 or a-IL6 antibodies
Rolvering, Catherine UL; Zimmer, Andreas David UL; Ginolhac, Aurélien UL et al

in Journal of Leukocyte Biology (2018), 104

Interleukin-27 (IL27) is a type-I cytokine of the IL6/IL12 family and is predominantly secreted by activated macrophages and dendritic cells.We show that IL27 induces STAT factor phosphorylation in ... [more ▼]

Interleukin-27 (IL27) is a type-I cytokine of the IL6/IL12 family and is predominantly secreted by activated macrophages and dendritic cells.We show that IL27 induces STAT factor phosphorylation in cancerous cell lines of different tissue origin. IL27 leads to STAT1 phosphorylation and recapitulates an IFN-𝛾-like response in the microarray analyses, with up-regulation of genes involved in antiviral defense, antigen presentation, and immune suppression. Like IFN-𝛾, IL27 leads to an up-regulation of TAP2 and MHC-I proteins, which mediate increased tumor immune clearance. However, both cytokines also upregulate proteins such as PD-L1 (CD274) and IDO-1, which are associatedwith immune escape of cancer. Interestingly, differential expression of these geneswas observed within the different cell lines and when comparing IL27 to IFN-𝛾. In coculture experiments of hepatocellular carcinoma (HCC) cells with peripheral blood mononuclear cells, pre-treatment of the HCC cells with IL27 resulted in lowered IL2 production by anti-CD3/-CD28 activated T-lymphocytes. Addition of anti-PD-L1 antibody, however, restored IL2 secretion. The levels of other TH1 cytokines were also enhanced or restored upon administration of anti-PD-L1. In addition, we show that the suppression of IL27 signaling by IL6-type cytokine prestimulation— mimicking a situation occurring, for example, in IL6-secreting tumors or in tumor inflammation–induced cachexia—can be antagonized by antibodies against IL6-type cytokines or their receptors. Therapeutically, the antitumor effects of IL27 (mediated, e.g., by increased antigen presentation) might thus be increased by combining IL27with blocking antibodies against PD-L1 or/and IL6-type cytokines. [less ▲]

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See detailCytokine-mediated modulation of the hepatic miRNome: miR-146b-5p is an IL-6-inducible miRNA with multiple targets.
Kirchmeyer, Melanie; Servais, Florence UL; Hamdorf, Matthias et al

in Journal of leukocyte biology (2018)

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 ... [more ▼]

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). [less ▲]

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See detailInflammatory signaling in liver cells: Cross-regulation of the IL-6/JAK/STAT3 pathway and microRNAs
Servais, Florence UL

Doctoral thesis (2017)

Interleukin-6 (IL-6) plays important roles in the regulation of liver functions and promotes the development of hepatocellular carcinoma (HCC), the most common primary liver cancer. Whereas protein ... [more ▼]

Interleukin-6 (IL-6) plays important roles in the regulation of liver functions and promotes the development of hepatocellular carcinoma (HCC), the most common primary liver cancer. Whereas protein-encoding genes are well-studied, the contribution of IL-6-regulated microRNAs (miRNAs) to its effects is largely unexplored. Similarly, little is known about miRNAs regulating key molecules of the IL-6/JAK/STAT3 signaling pathway. In the main part of this thesis, cell-based high-throughput screenings systems were developed, allowing the identification of miRNAs interfering with the IL-6/JAK/STAT3 signaling. Out of 538 miRNA mimics, this dual screening approach followed by various validation steps (luciferase-3’UTR-reporter assays, western blot and flow cytometry analyses) allowed us to identify twelve miRNAs targeting STAT3, JAK1, SOCS3, IL6R and/or gp130, ten of them had not yet been reported as regulators of this pathway before. In the second part of this work, microarray analyses revealed that primary hepatocytes are considerably more responsive to IL-6 stimulation regarding changes in their miRNomes than liver-derived cell lines. Despite their weaker response, one of the top regulated miRNA found in the primary hepatocytes, miR-146b-5p, could also be induced by IL-6-type cytokines (IL-6 and Oncostatin M) in cell lines. Its functional role(s) in hepatocytes are currently investigated. A larger, comparative study included additionally Interferon gamma and IL-27, cytokines with a STAT1-dominated response, and supplementary cell lines originating not only from the liver but also from other tissues. While all cytokines had profound effects on the mRNA transcriptomes, only very small to moderate changes were observed at the miRNome level, suggesting that the regulation mediated by cytokines mainly happens through the modulation of protein-coding genes rather than by fine-tuning via miRNAs. Additionally, we analyzed expression of a broad panel of cytokines in sera of 125 patients with liver diseases (steatosis, non-alcoholic steatohepatitis, HCC) by bead-based multiplex immunoassays. We observed that, for example, serum HGF, IL-6 and IL-8 levels increased in relation with the severeness of liver pathology while PDGF-BB and RANTES decreased. When investigating the impact of the patatin-like phospholipase domain-containing protein 3 variant I148M (PNPLA3 I148M), known to be a risk factor for the development of liver diseases, on the serum levels of cytokines, no significant differences could be observed between the different genotypes. In this PhD thesis, we identified and characterized miRNAs relevant for the IL-6/JAK/STAT3 signaling pathway, including miR-146b-5p as an IL-6-inducible miRNA. Others like miR-194-5p, miR-4473 and miR-548k were demonstrated to be negative regulators of this pathway and may be of potential therapeutic significance. In addition, we showed that, while miRNAs can modulate this pathway, IL-6 seems to rather regulate the mRNA transcriptome than the miRNome. [less ▲]

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