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TNF-α and IGF1 modify the microRNA signature in skeletal muscle cell differentiation.

Meyer SU, Thirion C, Polesskaya A, Bauersachs S, Kaiser S, Krause S, Pfaffl MW - Cell Commun. Signal (2015)

Bottom Line: Results reveal that i) TNF-α and IGF1 regulate miRNA expression during skeletal muscle cell differentiation in vitro, ii) microRNA targets can mediate the negative effect of TNF-α on fusion capacity of skeletal myoblasts by targeting genes associated with axon guidance, MAPK signalling, focal adhesion, and neurotrophin signalling pathway, iii) inhibition of miR-155 in combination with overexpression of miR-503 partially abrogates the inhibitory effect of TNF-α on myotube formation, and iv) MAPK/ERK inhibition might participate in modulating the effect of TNF-α and IGF1 on miRNA abundance.The inhibitory effects of TNF-α or the growth promoting effects of IGF1 on skeletal muscle differentiation include the deregulation of known muscle-regulatory miRNAs as well as miRNAs which have not yet been associated with skeletal muscle differentiation or response to TNF-α or IGF1.This study indicates that miRNAs are mediators of the inhibitory effect of TNF-α on myoblast differentiation.

View Article: PubMed Central - PubMed

Affiliation: Physiology Weihenstephan, ZIEL Research Center for Nutrition and Food Sciences, Technische Universität München, Weihenstephaner Berg 3, D-85354, Freising, Germany. meyers@wzw.tum.de.

ABSTRACT

Background: Elevated levels of the inflammatory cytokine TNF-α are common in chronic diseases or inherited or degenerative muscle disorders and can lead to muscle wasting. By contrast, IGF1 has a growth promoting effect on skeletal muscle. The molecular mechanisms mediating the effect of TNF-α and IGF1 on muscle cell differentiation are not completely understood. Muscle cell proliferation and differentiation are regulated by microRNAs (miRNAs) which play a dominant role in this process. This study aims at elucidating how TNF-α or IGF1 regulate microRNA expression to affect myoblast differentiation and myotube formation.

Results: In this study, we analyzed the impact of TNF-α or IGF1 treatment on miRNA expression in myogenic cells. Results reveal that i) TNF-α and IGF1 regulate miRNA expression during skeletal muscle cell differentiation in vitro, ii) microRNA targets can mediate the negative effect of TNF-α on fusion capacity of skeletal myoblasts by targeting genes associated with axon guidance, MAPK signalling, focal adhesion, and neurotrophin signalling pathway, iii) inhibition of miR-155 in combination with overexpression of miR-503 partially abrogates the inhibitory effect of TNF-α on myotube formation, and iv) MAPK/ERK inhibition might participate in modulating the effect of TNF-α and IGF1 on miRNA abundance.

Conclusions: The inhibitory effects of TNF-α or the growth promoting effects of IGF1 on skeletal muscle differentiation include the deregulation of known muscle-regulatory miRNAs as well as miRNAs which have not yet been associated with skeletal muscle differentiation or response to TNF-α or IGF1. This study indicates that miRNAs are mediators of the inhibitory effect of TNF-α on myoblast differentiation. We show that intervention at the miRNA level can ameliorate the negative effect of TNF-α by promoting myoblast differentiation. Moreover, we cautiously suggest that TNF-α or IGF1 modulate the miRNA biogenesis of some miRNAs via MAPK/ERK signalling. Finally, this study identifies indicative biomarkers of myoblast differentiation and cytokine influence and points to novel RNA targets.

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Functional analysis of miRNAs in human myoblast differentiation and TNF-α treatment. Relative fusion indices for miRNA mimics or miRNA inhibitor transfections into human LHCN myoblasts in the differentiation medium (black bars) or differentiation medium with TNF-α supplementation (grey bars). (A) Transfection of 25 nM miRNA mimics or scrambled control miRNA (scrbl), (B) 50 nM miRNA inhibitors or scrambled control inhibitors (anti-scrbl), and (C) a combination of 75 nM miRNA inhibitor and 25 nM miRNA mimic or respective controls. Samples with specific miRNAs or inhibitors are shown relative to the respective scrambled reference which was set to 100% fusion index. Significant differences (p > 0.05) of relative fusion indices are marked by an asterisk.
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Fig3: Functional analysis of miRNAs in human myoblast differentiation and TNF-α treatment. Relative fusion indices for miRNA mimics or miRNA inhibitor transfections into human LHCN myoblasts in the differentiation medium (black bars) or differentiation medium with TNF-α supplementation (grey bars). (A) Transfection of 25 nM miRNA mimics or scrambled control miRNA (scrbl), (B) 50 nM miRNA inhibitors or scrambled control inhibitors (anti-scrbl), and (C) a combination of 75 nM miRNA inhibitor and 25 nM miRNA mimic or respective controls. Samples with specific miRNAs or inhibitors are shown relative to the respective scrambled reference which was set to 100% fusion index. Significant differences (p > 0.05) of relative fusion indices are marked by an asterisk.

Mentions: Functional miRNA analyses were performed in a human skeletal muscle precursor cell line, LHCN [26] which showed strong sensitivity to the repressive effect of TNF-α upon myotube formation in vitro (Additional file 5). We aimed at rescuing the inhibitory effect of TNF-α on myoblast fusion efficiency by i) overexpression of miRNAs which were upregulated during differentiation in murine PMI28 cells, primary human skeletal muscle cells (Table 1B, Additional file 1A,B) or in human LHCN muscle cells [10] or ii) by inhibition of miRNAs which are downregulated during differentiation but upregulated due to TNF-α or iii) by promoting differentiation-associated miRNA patterns by a combination of overexpression and inhibition of miRNAs which are inversely regulated during control differentiation and TNF-α treatment. However, overexpression or inhibition of selected miRNAs did not outweigh the negative effect of TNF-α on fusion capacity (Figure 3A,B). However, combined inhibition of miR-155 and overexpression of miR-503 ameliorated the negative effect of TNF-α on differentiation (Figures 3C, 4A). Overexpression of hsa-miR-361, hsa-miR-486 or inhibition of hsa-miR-98 alone or in combination with hsa-miR-133a overexpression enhanced fusion capacity in control myoblasts significantly but was not powerful enough to rescue the TNF-α effect (Figure 3A,B,C). Some miRNAs or inhibitors resulted in partial detachment of the cell layer, leading to high standard deviations.Figure 3


TNF-α and IGF1 modify the microRNA signature in skeletal muscle cell differentiation.

Meyer SU, Thirion C, Polesskaya A, Bauersachs S, Kaiser S, Krause S, Pfaffl MW - Cell Commun. Signal (2015)

Functional analysis of miRNAs in human myoblast differentiation and TNF-α treatment. Relative fusion indices for miRNA mimics or miRNA inhibitor transfections into human LHCN myoblasts in the differentiation medium (black bars) or differentiation medium with TNF-α supplementation (grey bars). (A) Transfection of 25 nM miRNA mimics or scrambled control miRNA (scrbl), (B) 50 nM miRNA inhibitors or scrambled control inhibitors (anti-scrbl), and (C) a combination of 75 nM miRNA inhibitor and 25 nM miRNA mimic or respective controls. Samples with specific miRNAs or inhibitors are shown relative to the respective scrambled reference which was set to 100% fusion index. Significant differences (p > 0.05) of relative fusion indices are marked by an asterisk.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4325962&req=5

Fig3: Functional analysis of miRNAs in human myoblast differentiation and TNF-α treatment. Relative fusion indices for miRNA mimics or miRNA inhibitor transfections into human LHCN myoblasts in the differentiation medium (black bars) or differentiation medium with TNF-α supplementation (grey bars). (A) Transfection of 25 nM miRNA mimics or scrambled control miRNA (scrbl), (B) 50 nM miRNA inhibitors or scrambled control inhibitors (anti-scrbl), and (C) a combination of 75 nM miRNA inhibitor and 25 nM miRNA mimic or respective controls. Samples with specific miRNAs or inhibitors are shown relative to the respective scrambled reference which was set to 100% fusion index. Significant differences (p > 0.05) of relative fusion indices are marked by an asterisk.
Mentions: Functional miRNA analyses were performed in a human skeletal muscle precursor cell line, LHCN [26] which showed strong sensitivity to the repressive effect of TNF-α upon myotube formation in vitro (Additional file 5). We aimed at rescuing the inhibitory effect of TNF-α on myoblast fusion efficiency by i) overexpression of miRNAs which were upregulated during differentiation in murine PMI28 cells, primary human skeletal muscle cells (Table 1B, Additional file 1A,B) or in human LHCN muscle cells [10] or ii) by inhibition of miRNAs which are downregulated during differentiation but upregulated due to TNF-α or iii) by promoting differentiation-associated miRNA patterns by a combination of overexpression and inhibition of miRNAs which are inversely regulated during control differentiation and TNF-α treatment. However, overexpression or inhibition of selected miRNAs did not outweigh the negative effect of TNF-α on fusion capacity (Figure 3A,B). However, combined inhibition of miR-155 and overexpression of miR-503 ameliorated the negative effect of TNF-α on differentiation (Figures 3C, 4A). Overexpression of hsa-miR-361, hsa-miR-486 or inhibition of hsa-miR-98 alone or in combination with hsa-miR-133a overexpression enhanced fusion capacity in control myoblasts significantly but was not powerful enough to rescue the TNF-α effect (Figure 3A,B,C). Some miRNAs or inhibitors resulted in partial detachment of the cell layer, leading to high standard deviations.Figure 3

Bottom Line: Results reveal that i) TNF-α and IGF1 regulate miRNA expression during skeletal muscle cell differentiation in vitro, ii) microRNA targets can mediate the negative effect of TNF-α on fusion capacity of skeletal myoblasts by targeting genes associated with axon guidance, MAPK signalling, focal adhesion, and neurotrophin signalling pathway, iii) inhibition of miR-155 in combination with overexpression of miR-503 partially abrogates the inhibitory effect of TNF-α on myotube formation, and iv) MAPK/ERK inhibition might participate in modulating the effect of TNF-α and IGF1 on miRNA abundance.The inhibitory effects of TNF-α or the growth promoting effects of IGF1 on skeletal muscle differentiation include the deregulation of known muscle-regulatory miRNAs as well as miRNAs which have not yet been associated with skeletal muscle differentiation or response to TNF-α or IGF1.This study indicates that miRNAs are mediators of the inhibitory effect of TNF-α on myoblast differentiation.

View Article: PubMed Central - PubMed

Affiliation: Physiology Weihenstephan, ZIEL Research Center for Nutrition and Food Sciences, Technische Universität München, Weihenstephaner Berg 3, D-85354, Freising, Germany. meyers@wzw.tum.de.

ABSTRACT

Background: Elevated levels of the inflammatory cytokine TNF-α are common in chronic diseases or inherited or degenerative muscle disorders and can lead to muscle wasting. By contrast, IGF1 has a growth promoting effect on skeletal muscle. The molecular mechanisms mediating the effect of TNF-α and IGF1 on muscle cell differentiation are not completely understood. Muscle cell proliferation and differentiation are regulated by microRNAs (miRNAs) which play a dominant role in this process. This study aims at elucidating how TNF-α or IGF1 regulate microRNA expression to affect myoblast differentiation and myotube formation.

Results: In this study, we analyzed the impact of TNF-α or IGF1 treatment on miRNA expression in myogenic cells. Results reveal that i) TNF-α and IGF1 regulate miRNA expression during skeletal muscle cell differentiation in vitro, ii) microRNA targets can mediate the negative effect of TNF-α on fusion capacity of skeletal myoblasts by targeting genes associated with axon guidance, MAPK signalling, focal adhesion, and neurotrophin signalling pathway, iii) inhibition of miR-155 in combination with overexpression of miR-503 partially abrogates the inhibitory effect of TNF-α on myotube formation, and iv) MAPK/ERK inhibition might participate in modulating the effect of TNF-α and IGF1 on miRNA abundance.

Conclusions: The inhibitory effects of TNF-α or the growth promoting effects of IGF1 on skeletal muscle differentiation include the deregulation of known muscle-regulatory miRNAs as well as miRNAs which have not yet been associated with skeletal muscle differentiation or response to TNF-α or IGF1. This study indicates that miRNAs are mediators of the inhibitory effect of TNF-α on myoblast differentiation. We show that intervention at the miRNA level can ameliorate the negative effect of TNF-α by promoting myoblast differentiation. Moreover, we cautiously suggest that TNF-α or IGF1 modulate the miRNA biogenesis of some miRNAs via MAPK/ERK signalling. Finally, this study identifies indicative biomarkers of myoblast differentiation and cytokine influence and points to novel RNA targets.

Show MeSH
Related in: MedlinePlus