Modulation of the IL-6-Signaling Pathway in Liver Cells by miRNAs Targeting gp130, JAK1, and/or STAT3

Abstract

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.

Keywords: JAK1; STAT3; cancer; cytokine; gp130; inflammation; interleukin-6; liver; microRNAs; signal transduction.

Graphical abstract

Figure 1

Effect of Selected miRNAs on STAT3 and pSTAT3 Protein Levels Hep3B cells were transfected with 34 selected mimics and 2 days later stimulated for 24 h with hy-IL-6 to activate the JAK-STAT pathway. pSTAT3 and STAT3 protein levels were quantified using the LI-COR device and normalized to total protein staining (data not shown). The normalized signal obtained for each sample was divided by the signal obtained for the transfection control (HiPerFect). Vinculin staining is shown as an additional control. Error bars represent the SD of 3 biological replicates. Mimics selected for luciferase-3′ UTR reporter assays are in bold and marked with a star. A representative western blot result for pSTAT3, STAT3, and Vinculin is shown.

Figure 2

Identification of miRNAs Targeting the Co-receptor gp130 and Affecting Its Surface Availability (A) Graphic representations of the gp130 3′ UTR as well as the predicted binding sites (based on TargetScan website). Sites conserved among mammals are underlined. (B) HEK293T cells were co-transfected with mimics specifically selected for one of the vectors harboring the Gaussia luciferase cDNA followed by overlapping 3′ UTR sequences of the gene encoding gp130 (gp130a/b/c). Mimics selected for further functional assays are shown in gray, and negative control mimic 1 (NCM1) is in white. Error bars represent the SD of 3 biological replicates. Kruskal-Wallis followed by Dunn’s Post hoc test were performed to assess statistical significance represented with *p < 0.05 and **p < 0.01. (C–H) Selected miRNA candidates affect the surface availability of gp130 in various cell lines. (C) Hep3B hepatoma cells, (D) non-neoplastic PH5CH8 liver cells, (E) A549 lung adenocarcinoma cells, (F) DU145 prostate cancer cells, (G) HEK293T cells, and (H) IGR39 melanoma cells were either left untreated (Ctrl, in purple) or transfected with a negative control (NCM1, in gray) or one of the selected miRNA mimics and analyzed for gp130 surface expression level by flow cytometry. Candidate miRNAs are depicted in orange, the isotype control is in red, and the transfection control is in blue.

Figure 3

miR-16-1-3p, miR-4473, and miR-520f-3p Partially Prevent the Activation of STAT3 upon hy-IL-6 Stimulation by Targeting gp130 mRNA, Reducing the Expression of a hy-IL-6 Target Gene in PH5CH8 Cells Cells were left untreated, reverse transfected with NCM1 or miR-16-1-3p, miR-4473, and miR-520f-3p mimics (20 nM), and were stimulated with hy-IL-6 (20 ng/mL). Single mimics as well as combinations were tested and compared to the respective NCM1 control (in black). For the double or triple combinations, 40 or 60 nM NCM1 was used as the respective control. (A) Normalized mRNA expression level of gp130. (B) Representative western blot analysis of both the activated and total forms of STAT3 and of the Vinculin as loading control. (C) Normalized mRNA expression level of Lipopolysaccharide-binding protein (LBP). The mRNA levels were monitored upon mimic transfection and hy-IL-6 stimulation (depicted with dashed lines). Error bars represent the SD of at least 3 biological replicates. Bottom indicates the applied treatments per lane.

Figure 4

Identification of miRNAs Targeting JAK1 and STAT3 and Affecting Their Endogenous Protein Levels (A and C) Graphic representations of (A) JAK1 and (C) STAT3 3′ UTRs as well as predicted binding sites (TargetScan). Sites conserved among mammals are underlined. HEK293T cells were co-transfected with the mimics specifically selected for each construct and one of the vectors harboring the Gaussia luciferase cDNA followed by the 3′ UTR of the gene encoding (A) JAK1 or (C) STAT3. Mimics selected for further functional assays are shown in gray, and NCM1 is in white. Kruskal-Wallis followed by Dunn’s post hoc test were performed to assess statistical significance represented with *p < 0.05, **p < 0.01, and ***p < 0.001. (B and D) Selected miRNAs downregulate endogenous JAK1 and STAT3 protein levels in PH5CH8 cells. (B) miR-299-3p, miR-3677-5p, and miR-520f-3p reduce JAK1 protein levels. (D) miR-3677-5p and miR-506-3p reduce STAT3 protein levels. A representative western blot as well as quantifications are depicted. Error bars represent the SD of at least 3 biological replicates.

Figure 5

miR-124-3p, miR-16-1-3p, and miR-299-3p Partially Prevent the Activation of STAT3 upon IL-6 Stimulation by Targeting STAT3, IL6R, gp130, and JAK1 mRNA, and They Reduce the Induction of the Expression of IL-6 Target Genes in Hep3B Cells Cells were left untreated, reverse transfected with NCM1 or miR-124-3p, miR-16-1-3p, and miR-299-3p mimics (20 nM), and stimulated with IL-6 (20 ng/mL). Single mimics as well as combinations were tested and compared to the respective NCM1 control (in black). For the double or triple combinations, 40 or 60 nM NCM1 was used as the respective control. (A) Western blot analysis for both the activated and total forms of STAT3 and of Vinculin as loading control. (B) Normalized mRNA expression levels of LBP, Leucine-rich alpha-2 glycoprotein (LRG1), and SOCS3 were monitored upon mimic transfection and IL-6 stimulation (depicted with dashed lines). Error bars represent the SD of at least 3 biological replicates. Bottom indicates the applied treatments per lane.

Figure 6

Summary of the Results Obtained in This Study (A) Graphical overview of effects of the different miRNA mimics on the IL-6-type cytokine-signaling pathway. For STAT3 phosphorylation, the mimics that reduce the western blot signal to below 75% or below 50% are listed. For JAK1 and STAT3 mimics, those that reduce the western blot signal to below 60% are shown. For gp130, the mimics that reduce surface expression efficiently (below 70%) are listed. The colored dots behind the name of the mimics in the lists indicate additional target genes of the respective miRNA (red, JAK1; blue, gp130; yellow, STAT3; and green, phosphorylation of STAT3). miRNAs labeled with a “#” have already been characterized before. Interestingly, downregulation of STAT3 protein alone was never associated with a reduction of phosphorylated STAT3. (B) Summary of the results obtained upon transfection with each of the 15 selected miRNAs for pSTAT3 (Figure 1, Hep3B), JAK1, and STAT3 (Figure 4, PH5CH8) protein levels, for gp130 surface availability (Figure S5, PH5CH8) and for luciferase-3′ UTR reporter assays (Figures 2, 4, and S7, HEK293T). Results represent the protein and luciferase signals measured for the corresponding target compared to the control (expressed in %). The SD of at least 3 biological replicates is shown. A dash indicates that no data were obtained for this specific miRNA and target. For miR-494-3p, no target could be identified from the ones tested. The list of miRNA effects is colorcoded for the protein levels in Hep3B and PH5CH8 cells (reduction depicted in red and increase in green). In addition, the miRNAs are sorted according to their inhibitory effects on STAT3 activation (from the strongest to the lowest).