MiR-449a antagonizes EMT through IL-6-mediated trans-signaling in laryngeal squamous cancer

Abstract

MicroRNAs (miRNAs) are involved in post-transcriptional gene expression regulation and in mechanisms of cancer growth and metastases. In this light, miRNAs could be promising therapeutic targets and biomarkers in clinical practice. Therefore, we investigated if specific miRNAs and their target genes contribute to laryngeal squamous cell carcinoma (LSCC) development. We found a significant decrease of miR-449a in LSCC patients with nodal metastases (63.3%) compared with patients without nodal involvement (44%). The AmpliSeq Transcriptome of HNO-210 miR-449a-transfected cell lines allowed the identification of IL6-R as a potential target. Moreover, the downregulation of IL6-R and the phosphorylation reduction of the downstream signaling effectors, suggested the inhibition of the IL-6 trans-signaling pathway. These biochemical effects were paralleled by a significant inhibition of invasion and migration in vitro and in vivo, supporting an involvement of epithelial-mesenchymal transition. These findings indicate that miR-449a contributes to suppress the metastasization of LSCC by the IL-6 trans-signaling block and affects sensitivity to external stimuli that mimic pro-inflammatory conditions.

Keywords: IL-6 trans-signaling; LSCC; MT: non-coding RNAs; gene expression; metastases miR-449a; microRNAs.

Graphical abstract

Figure 1

Downregulation of miR-449a was associated with nodal involvement in LSCC Validation of miR-449a expression in primary cancer tissues from patients with LSCC with N⁺ lymph node involvement (n = 60) and without N^– lymph node involvement (n = 72) by qRT-PCR (chi square test p = 0.030).

Figure 2

Expression profile of miR-449a in LSCC cell lines (A) Relative basal miR-449a expression levels (mean ± SD) in FaDu, UPCI:SCC152,HNO-210 cell lines vs. human hypopharyngeal normal cell. (B) miR-449a relative expression levels in HNO-210 cells. (C) miR-449a relative expression levels in FaDu cells. Each data point was obtained in triplicate and data are shown as mean ± SD (Student’s t test) ∗p < 0.05, ∗∗∗p < 0.001.

Figure 3

miR-449a genes modulation in HNO-210 transduced cells (A) PCA of the AmpliSeq Transcriptome data. (B) Volcano plot shows the comparison between HNO-210 cells over-expressing miR-449a and the empty-backbone. (C) Boxplot of log2 expression for IL-6R and SNAI2 (p value of Student's test). The y axis represents log10 (false discovery rate [FDR]), while the x axis represents log2 HR. (D) Dotplot of enriched GO:BP categories selected for genes down-regulated by miR-449a compared with the empty-backbone. Dot sizes represent the number of downregulated genes associated with the GO term and dot colors represent the p value from the over-represented Fisher’ test. (E) Map of enrichment from GSEA in the comparison between miR-449a and empty-backbone. The sizes of the dots represent the number of major genes and the colors of the dots represent the normalized enrichment score (NES).

Figure 4

miR-449a leads to decrease invasion and migration in vitro and in vivo and downregulates SNAI2, N-cadherin, and Vimentin expression levels (A) Graphs representing the effect of miR-449a expression on cell migration and invasion of HNO-210 and FaDu cells using the transwell assay (left graph panel: HNO-210; right graph panel: FaDu). The data were presented as means ± SD from three biological replicates. ∗p < 0.05; ∗∗p < 0.001; ∗∗∗p < 0.0005; ∗∗∗∗p < 0.0001; Student’s t test. (B) Immunofluorescence localization of E-cadherin and N-cadherin antibodies in HNO-210 cell line overexpressing miR-449a. (C) Relative expression levels of SNAI2, E-cadherin, N-cadherin and Vimentin by qRT-PCR HNO-210 transduced cells. A technical and experimental triplicate was carried out ∗p < 0.05; ∗∗p < 0.001; ∗∗∗p < 0.0005; ∗∗∗∗p < 0.0001; Student’s t test. (D) Transduced cells: HNO-210-empty-backbone and HNO-210-miR-449a cells were injected into the perivitelline space (PVS) of 48 h post fertilization (hpf) Tg (fli1:EGFP) zebrafish larvae. BV, blood vessels. Cells were fluorescently labeled with the CM-Dil cell tracker and the zebrafish tumor xenograft was analyzed at 24 and 72 hpi. The number of metastases was analyzed in the head and tail of the animals. Metastatic tumor cells are indicated in zebrafish with white arrows (left). Boxplots represent the number of metastatic larynx cancer cells in the head and tail of zebrafish Tg (fli1:eGFP) larvae at 24 and 72 hpi N, the number of larvae with metastases (≥2 in any position) divided by the total number of larvae analyzed (N = met/total). Scale bars, 100 μm. The p value was calculated from the linear longitudinal-log Poisson model (right). The results come from two independent experiments. All data are representative of three independent experiments.

Figure 5

miR-449a directly targets IL-6R and switches off IL-6 trans-signaling (A) The putative miR-449a binding sites in IL-6R were predicted using online bioinformatics tools. The interaction between miR-449a and IL-6R was verified using a dual luciferase reporter assay. ∗p < 0.05; ∗∗p < 0.001; ∗∗∗p < 0.0005; ∗∗∗∗p < 0.0001 Student’s t test vs. control group. Each experiment was repeated three times. Bars indicate SDs. (B) RIP assay of the enrichment of Ago2 on IL-6R and miR-449a transcripts normalized to a negative control irrelevant IgG antibody (CTR) in HNO-210 cell line overexpressing miR-449a. (C) Levels of sIL-6R and IL-6R in HNO-210 cell culture medium by quantitative ELISA (left and right) and expression levels of sIL-6R in HNO-210 cells by qRT-PCR (last panel). The experiments were repeated three times in triplicate. Bars, SDs. (D) Western blots analysis of transduced HNO-210 cell models in different experimental conditions: (i) 0.1% serum starvation, (ii) Hyper IL-6 stimulation (20 ng/mL for 30 min), (iii) sgp130fc treatment (1,000 ng/mL for 1 h), and sgp130fc pretreatment followed by Hyper IL-6 stimulation. miR-449a modulation of IL-6 trans-signaling mediated by pStat3 Tyr705 – pERK (Thr202; Tyr204)—pAKT Ser473. Vinculin was used as loading control. The experiments were repeated at least three times giving always similar results. Columns represent the intensity of the different bands evaluated as arbitrary units. Bars are SDs. ANOVA ∗p < 0.01; ∗∗p < 0.05; ∗∗∗p < 0.005; ∗∗∗∗p < 0.0001. (E) Relative expression levels of SNAI2, E-cadherin, and N-cadherin of transduced HNO-210 cell models in different experimental conditions: (i) 0.1% serum starvation, (ii) Hyper IL-6 stimulation (20 ng/mL for 30 min), (iii) sgp130fc treatment (1,000 ng/mL for 1 h) and sgp130fc pretreatment followed by Hyper IL-6 stimulation. ANOVA ∗<0.01 ∗∗p < 0.05; ∗∗∗p < 0.005; ∗∗∗∗p < 0.0001. All data are representative of three independent experiments.