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. 2012 Dec 17;10(1):41.
doi: 10.1186/1478-811X-10-41.

Interferon-γ-induced activation of Signal Transducer and Activator of Transcription 1 (STAT1) up-regulates the tumor suppressing microRNA-29 family in melanoma cells

Martina J Schmitt  1 , Demetra Philippidou, Susanne E Reinsbach, Christiane Margue, Anke Wienecke-Baldacchino, Dorothee Nashan, Iris Behrmann, Stephanie Kreis
Affiliations

Interferon-γ-induced activation of Signal Transducer and Activator of Transcription 1 (STAT1) up-regulates the tumor suppressing microRNA-29 family in melanoma cells

Martina J Schmitt et al. Cell Commun Signal. .

Abstract

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 cells in 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.

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Figures

Figure 1
Figure 1
Top 10 IFN-γ-up-regulated miRNAs. (A) Ten miRNAs with highest positive fold changes (as determined by previous microarray experiments [13]) include miR-29 family members (left). Detailed time course profiles are shown in Additional file 1: Figure S1. The miR-29 family is transcribed from the respective antisense strand from two genetic clusters of chromosomes 7 (pri-29a~b-1) and 1 (pri-29b-2~c) (right). The three mature forms miR-29a/29b/29c share the same seed region (grey box). Differences between the mature sequences are underlined; a nucleotide difference between miR-29a and miR-29c is shown in italics. (B) The presumed pri-29a~b-1 promoter region [22-24] contains five GAS-elements G1-5 (TT(C/A)CNNNAA(A/G)) and two ISRE-elements I1-2 ((G/A)(G/A)AANNGAAA(C/G)) (GRCh37/hg19).
Figure 2
Figure 2
Expression profiles of miR-29 clusters in melanoma cells. A375-STAT1(F), A375-STAT1(wt), A375 and MeWo melanoma cells were stimulated with IFN-γ for different time points. (A) Western Blot analysis (representative blots of biological triplicates) confirms activation of P-STAT1 and induction of STAT1 and IRF-1 after IFN-γ stimulation while dominant negative A375-STAT1(F) cells show minor effects. (B) Time course study of miRNA-expression after IFNγ-stimulation. Graphs show relative expression (REL) from quantitative qRT-PCR data for the pri-29a~b-1 and the pri-29b-2~c clusters, the precursors pre-29a/29b-1/29b-2/29c and mature miR-29a/29b/25/100. Fold expression was calculated relative to the untreated control and SDs are shown for biological triplicates. Statistical significance was tested with one-way ANOVA, followed by a Dunnett Post-Hoc test with * p<0.05, ** p<0.01 and *** p<0.001. (C) MiRNA and miRNA* expression profiles in A375 cells derived from a more detailed IFN-γ time course miRNA microarray experiment including cells treated with JI1 (IFN-γ stimulation for 72h after pre-treatment with JI1, black and grey dots). Depicted are log2-values of the mean of duplicate microarray experiments.
Figure 3
Figure 3
Inverse correlation of miR-29a expression levels and melanoma proliferation. Comparison of basal expression levels of (A) primary miRNA clusters pri-29a~b-1 (blue/red/black bars) and pri-29b-2~c (grey bars) and (B) mature miR-29a (blue/red/black bars) and miR-29b (grey bars) in NHEM-M2, eight melanoma cell lines and HaCaT keratinocytes. Graphs show 2-Δct x102 with SD of biological triplicates. (C) Mean growth curves of untreated melanoma cell lines over 4 days (biological quadruplicates). Melanoma cell lines with ‘low expression’ of pri-29a~b-1 and miR-29a show faster proliferation whereas cells with a relatively ‘high expression’ proliferate slower. (D,E) Proliferation assay of (black) mimic/inhibitor- and (grey) NC-mimic/NC-inhibitor-transfected cells over 72h in (D) A375 and (E) FM55P cells; representative graphs of four independent experiments. Error bars depict SDs of technical triplicates. The inserted graphs (upper left corners) show the mean confluence of 4 biological replicates at 0h and 72h time points of the proliferation assay. Depicted are ratios of confluence of 29ab-mimic/NC-mimic treated cells (D) and 29a-inhibitor/NC-inhibitor treated cells (E). Error bars show SEM. Significance was assessed by a two-tailed t-test with * p<0.05, ** p<0.01 and *** p<0.001.
Figure 4
Figure 4
Effects of miR-29 on target genes CDK6 and PI3K. (A,B) relative mRNA and protein expression levels (REL) of miR-29 target genes CDK6 (dark grey) and PI3K (light grey), assessed 24h, 48h and 72h after mimic/inhibitor transfection compared to NC-mimic/NC-inhibitor controls; graphs show means of biological triplicates ± SD. (C) Down-regulation of miR-29 target proteins CDK6 and PI3K is observed after IFN-γ stimulation of melanoma cells. (D) Schematic overview of CDK6 and PI3KR1 luciferase constructs with positions of conserved miR-29a binding sites predicted by TargetScan (bold) in the CDK6-3'UTR (BS1-3) and PI3KR1-3'UTR (BS) and corresponding miR-29a sequences (italics). (E) Luciferase activity in A375 cells transfected with constructs containing the positive control miR-29a full complementary sequence (29a-FC), parts of CDK6- or PI3K1-3'UTRs or CDK6 single binding sites (BS1-BS3) and miR-29a mimic or NC for 48h and 72h. Relative luciferase activity of miR-29a-transfected samples was normalized to NC-mimic-transfected control samples (luciferase activity of NC-mimic transfected samples was set to 100%). Bars show means of biological triplicates ± SD for each construct. (F) A375 and (G) FM55P cells transfected with CDK6 siRNA (black) show reduced proliferation compared to cells transfected with siRNA NC (grey). Results were reproduced at least in biological duplicates. Inserted bar diagrams show the mean confluence of at least biological triplicates at 0h and 72h. Shown are confluence ratios of si-CDK6/si-NC ± SEM. Significance was assessed by one-way ANOVA followed by a Bonferroni Post-Hoc test (A,B,E) or by a two-tailed t-test (F,G). * p<0.05, ** p<0.01 and *** p<0.001.
Figure 5
Figure 5
miR-29 expression in patient samples. Analysis of miR-29a (upper panel) and miR-29b (lower panel) basal expression of individual FFPE-patient samples from NS = normal skin, N = nevi, P = primary melanoma and M = metastatic melanoma. All graphs show 2-Δct with Δct= (ct (miR-29a/29b)– ct (RNU5A)). Primary and metastatic tumor samples were sorted according to patients (P1-5: P1-circles; P2-rectangles; P3-crosses; P4-triangles; P5-asterisks).
Figure 6
Figure 6
The miR-29 family is involved in multiple cellular processes. UV-radiation triggers the recruitment of macrophages to the skin, which secrete cytokines like interferon gamma (IFN-γ). By binding to its receptors, IFN-γ signals through the Jak/STAT pathway triggering subsequent activation of STAT1, which then binds to GAS-elements in the promoter region of target genes and initiates their transcription. IFN-γ-induced, STAT1-dependent up-regulation of miR-29 causes a down-regulation of CDK6, a novel miR-29 target gene in melanoma, which plays a crucial role in cell cycle G1/S-transition and thus growth control of cancer cells. The cell cycle inhibitor p16INK4A is often deleted in melanoma and its function (inhibition of CDK6) might be compensated by miR-29a/b. IFN-γ-activating transcription factors T-bet and Eomes and IFN-γ itself are also targeted by miR-29.
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