Decreased miR-497-5p Suppresses IL-6 Induced Atrophy in Muscle Cells

Abstract

Interleukin-6 (IL-6) is a pro-inflammatory cytokine associated with skeletal muscle wasting in cancer cachexia. The control of gene expression by microRNAs (miRNAs) in muscle wasting involves the regulation of thousands of target transcripts. However, the miRNA-target networks associated with IL6-induced muscle atrophy remain to be characterized. Here, we show that IL-6 promotes the atrophy of C2C12 myotubes and changes the expression of 20 miRNAs (5 up-regulated and 15 down-regulated). Gene Ontology analysis of predicted miRNAs targets revealed post-transcriptional regulation of genes involved in cell differentiation, apoptosis, migration, and catabolic processes. Next, we performed a meta-analysis of miRNA-published data that identified miR-497-5p, a down-regulated miRNAs induced by IL-6, also down-regulated in other muscle-wasting conditions. We used miR-497-5p mimics and inhibitors to explore the function of miR-497-5p in C2C12 myoblasts and myotubes. We found that miR-497-5p can regulate the expression of the cell cycle genes CcnD2 and CcnE1 without affecting the rate of myoblast cellular proliferation. Notably, miR-497-5p mimics induced myotube atrophy and reduced Insr expression. Treatment with miR-497-5p inhibitors did not change the diameter of the myotubes but increased the expression of its target genes Insr and Igf1r. These genes are known to regulate skeletal muscle regeneration and hypertrophy via insulin-like growth factor pathway and were up-regulated in cachectic muscle samples. Our miRNA-regulated network analysis revealed a potential role for miR-497-5p during IL6-induced muscle cell atrophy and suggests that miR-497-5p is likely involved in a compensatory mechanism of muscle atrophy in response to IL-6.

Keywords: Interleukin-6; inflammation; microRNAs; muscle wasting.

Figure 1

IL-6 induces C2C12 myotube atrophy. (A–C) IL-6 and Myh7 mRNA levels in differentiated C2C12 myotubes treated with three different concentrations of IL-6. RT-qPCR data are represented as fold change (2^ΔΔCt) relative to Rpl13a. Data represent the average of three independent experiments with standard deviation. (D–F) Diameter of myotubes treated with IL-6 (D, 10 ng/mL; E, 50 ng/mL; F, 100 ng/mL) myotubes with their corresponding controls. (G), Representative images of myotubes from both control and IL-6 treated (100 ng/mL) myotubes. Immunofluorescence was performed using the antibody against Myh2 and Dapi (4′,6-Diamidine-2′-phenylindole dihydrochloride). (H) Quantitative analysis of the myotube area of control and IL-6 treated myotubes (100 ng/mL) myotubes. (I) mRNA levels of embryonic myosin (Myh-emb) and (J) mRNA levels of atrogenes MAFbx and MuRF1 in differentiated C2C12 myotubes treated with IL-6 (100 ng/mL). RT-qPCR data are presented as fold change (2^−ΔΔCt) relative to Rpl13a. Statistical difference was analyzed by Student’s t-test.

Figure 2

Global miRNA expression in IL-6-treated myotubes. (A) Volcano plot representing the log 2 fold change and p-value of all microRNAs differentially expressed. A set of 20 miRNAs displayed significant changes (p-value < 0.05 and fold change ≥ 1.5). The blue and red dots represent the down- and up-regulated microRNAs, respectively. (B) Interaction network representing the 100 interactions between 19 miRNAs and 116 predicted target genes. Node size indicates the number of the miRNA-target gene, and the gray edge width denotes overlapping miRNA-target gene transcripts. (C) Heatmap of the biological processes (GO) enriched by a predicted set of targets for each miRNA. The top five ranked annotations of GO and enriched in at least two datasets were included in the heatmap. Unsupervised clustering was performed according to the Kendall rank correlation coefficient.

Figure 3

miR-497-5p is deregulated in skeletal muscle cells in different atrophic conditions. (A) Venn diagram showing the miR-497-5p as a common element between our data, cancer cachexia, primary muscle disorders, and other catabolic conditions. (B) miR-497 expression levels after 1, 3, 4, 7, and 21 days of cardiotoxin injury (CTX). Relative miRNA expression versus baseline (GSE37479). (C) MiR-497-5p expression levels in differentiated C2C12 myotubes treated with three different concentrations of IL-6 vs. control (PBS). RT-qPCR data are presented as fold change (2^−ΔΔCt) relative to MammU6. (D) Interactome between the predicted miR-497-5p target genes. Nodes represent miR-497-5p target genes according to the biological process. The larger the nodes, the higher the degree of interactions identified. Nodes with no connections were excluded. STRING v10.5.1 was used to generate protein interactions, and the network interactions were visualized using Cytoscape v3.4.0.

Figure 4

miR-497-5p effects in differentiated C2C12 myotubes. (A) Relative log10 transformed miR-497-5p levels measured by RT-qPCR in C2C12 myotubes transfected with mimic miR-497-5p, compared to C2C12 myotubes transfected with mimic control. (B) RT-qPCR showing decreased expression of miR-497 in C2C12 myotubes transfected with miRNA inhibitor against miR-497-5p. (C) Representative images of myotubes transfected with negative control or with miR-497-5p mimic. The immunofluorescence was performed using the antibody against Myh2 (red) and Dapi (blue). (D) Diameter of myotubes transfected with negative control or miR-497-5p mimic. (E) Representative images of myotubes from the negative control or transfected with miR-497-5p inhibitor. (F) Diameter of myotubes transfected with negative control or miR-497-5p inhibitor. (G), mRNA levels of atrogenes MAFbx and MuRF1 C2C12 myotubes transfected with mimic miR-497-5p and (H) in C2C12 myotubes transfected with miRNA inhibitor against miR-497-5p. Statistical difference was analyzed by Student’s t-test.

Figure 5

miR-497-5p target genes enrich the insulin-growth factor pathway. (A) Biological processes terms (GO) enriched for miR-497-5p target genes (p-value ≤ 1.82 × 10⁻²). Insr, Igf1r, Pik3r1, and Mapk8ip2 mRNA levels in differentiated C2C12 myotubes transfected with miR-497-5p mimic (B) or inhibitor (C). (D) Insr and Ingf1r expression levels in differentiated C2C12 myotubes treated with IL-6 and control (PBS). RT-qPCR data are represented as fold change (2^−ΔΔCt) relative to Rpl13a. Data represent the average of three independent experiments with standard deviation. Statistical difference was analyzed by Student’s t-test. Insr: insulin receptor; Igf1r: insulin-like growth factor 1 receptor; Pik3r1: phosphoinositide-3-kinase regulatory subunit 1; Mapk8ip2: mitogen-activated protein kinase 8 interacting protein 2.

Figure 6

miR-497-5p and cellular proliferation. mRNA levels of the cyclins CcnD2 and CcnE1 in C2C12 myoblasts treated with IL-6 [100 ng/mL] or PBS (A) transfected with miR-497-5p mimic or negative control (B) and transfected with miR-497-5p inhibitor or negative control. (C) RT-qPCR data are presented as fold change (2^−ΔΔCt) relative to Rpl13a. (D) Representative images of myoblasts transfected with miR-497-5p mimic or negative control. Cells were stained with EdU (red). (E) Percentage of Dapi (blue), and Edu+ cells from the total Dapi cells. (F) Representative images of myoblasts treated with IL-6 (100 ng/mL) or PBS (control) and stained with EdU (red). (G) Percentage of Dapi (blue), and Edu+ cells out of the total Dapi cells. Data represent the average of three independent experiments with standard deviation. Statistical difference was analyzed by Student’s t-test.

Figure 7

miR-497-5p involves the regulation of Igf1r and Insr genes and the activation of a compensatory molecular mechanism potentially able to reduce cell muscle wasting. (a) Heatmap of the expression levels (log2 fold change) of miR-497-5p target genes in four different cancer cachexia datasets and myotubes transfected with inhibitor miR-497-5p. Both rows (target genes) and columns (datasets) were clustered using the Kendall rank correlation coefficient. (b) Circos plot showing the overlapping genes among the studies. (c) Schematic diagram of the IL-6–miR-497–Igf1r/Insr pathway and its role in a compensatory mechanism during skeletal muscle atrophy.