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. 2023 Mar 6;32(6):934-947.
doi: 10.1093/hmg/ddac250.

miRNA analysis reveals novel dysregulated pathways in amyotrophic lateral sclerosis

Junguk Hur  1 , Ximena Paez-Colasante  2 , Claudia Figueroa-Romero  2   3 , Ting-Wen Lo  4 , Sami J Barmada  2 , Michelle T Paulsen  5 , Mats Ljungman  5   6 , Fadhl M Alakwaa  2 , Masha G Savelieff  3 , Stephen A Goutman  2   3 , Eva L Feldman  2   3
Affiliations

miRNA analysis reveals novel dysregulated pathways in amyotrophic lateral sclerosis

Junguk Hur et al. Hum Mol Genet. .

Abstract

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease. Its complex pathogenesis and phenotypic heterogeneity hinder therapeutic development and early diagnosis. Altered RNA metabolism is a recurrent pathophysiologic theme, including distinct microRNA (miRNA) profiles in ALS tissues. We profiled miRNAs in accessible biosamples, including skin fibroblasts and whole blood and compared them in age- and sex-matched healthy controls versus ALS participants with and without repeat expansions to chromosome 9 open reading frame 72 (C9orf72; C9-ALS and nonC9-ALS), the most frequent ALS mutation. We identified unique and shared profiles of differential miRNA (DmiRNA) levels in each C9-ALS and nonC9-ALS tissues versus controls. Fibroblast DmiRNAs were validated by quantitative real-time PCR and their target mRNAs by 5-bromouridine and 5-bromouridine-chase sequencing. We also performed pathway analysis to infer biological meaning, revealing anticipated, tissue-specific pathways and pathways previously linked to ALS, as well as novel pathways that could inform future research directions. Overall, we report a comprehensive study of a miRNA profile dataset from C9-ALS and nonC9-ALS participants across two accessible biosamples, providing evidence of dysregulated miRNAs in ALS and possible targets of interest. Distinct miRNA patterns in accessible tissues may also be leveraged to distinguish ALS participants from healthy controls for earlier diagnosis. Future directions may look at potential correlations of miRNA profiles with clinical parameters.

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Figures

Figure 1
Figure 1
Study design. miRNA levels in fibroblasts (FB) and WB were evaluated from two cohorts of C9-ALS and nonC9-ALS participants versus control samples using NanoString. Differential miRNAs (DmiRNAs) were identified by NanoStringDiff and compared across tissue type and in C9-ALS and nonC9-ALS versus controls. DmiRNAs were validated by qPCR and their predicted target mRNAs by BruChase-Seq. Biological meaning from DmiRNAs was inferred by pathway analysis, and random forest was applied to leverage DmiRNAs as biomarkers. KEGG, Kyoto Encyclopedia of Genes and Genomes. Generated in part using BioRender.com.
Figure 2
Figure 2
Fibroblast and WB DmiRNAs from C9-ALS and nonC9-ALS versus control participants. Fold-change (x-axis) of differential miRNAs (DmiRNAs; P < 0.05; y-axis) identified by NanoStringDiff. DmiRNAs that increased in ALS versus controls in yellow; DmiRNAs that decreased in ALS versus controls in blue. Plots for fibroblasts (FB) in (A) C9-ALS and (B) nonC9-ALS versus controls; plots for WB in (C) C9-ALS and (D) nonC9-ALS versus controls; yellow, upregulated in ALS versus controls; blue downregulated in ALS versus controls. (E) Venn diagram of the number of shared and unique DmiRNAs between fibroblasts and WB for C9-ALS and nonC9-ALS groups.
Figure 3
Figure 3
KEGG pathway analysis of DmiRNAs. Heat-map of significantly enriched KEGG pathways identified for each of the DmiRNA datasets represented by a log10-based color and number index. FB, fibroblasts.
Figure 4
Figure 4
KEGG pathway association networks. Significantly enriched KEGG pathways were combined and visualized in a network for (A) C9-ALS and (B) nonC9-ALS samples. KEGG pathways are represented by nodes; shared gene content between pathways are represented by edges. Within the network, node shape indicates the tissue source of the enriched pathways: diamond, fibroblasts only; circle, WB only; square, both fibroblasts and WB. Node color is based on –log10 (P-value). Node size corresponds to the number of connections each node has. All networks were organized by the inverted self-organizing map layout with minimal manual node rearrangement for visibility. Highly inter-connected subnetworks were identified by Cytoscape MCODE and are highlighted by various colors. Single nodes, which are not connected to other nodes, were excluded from this network visualization.
Figure 5
Figure 5
Identifying potential DmiRNA as regulators of mRNA stability in fibroblasts. miRNA fold-change (FC) for differential miRNAs (DmiRNAs; top row) in C9-ALS fibroblast. Predicted target mRNA (first column) stability values (mature/nascent; second column) are from our previously published study (21). Values in other table cells are the significant Pearson correlation coefficients between each pair of DmiRNA and its predicted mRNA targets; color represents the degree of differential expression in fold-changes (red up-regulation in ALS, blue down-regulation in ALS). Stability-FC and miRNA-FC were scaled independently.
Figure 6
Figure 6
Validation of target mRNA in fibroblasts. Target mRNA in fibroblasts were validated by qPCR. Results were normalized to yWHAZ and presented as fold-change calculated by the 2-∆CT method for GADD45 [C9-ALS, n = 5; Control (Ctrl), n = 6], IP6K2 (C9-ALS, n = 5; Ctrl, n = 6). Transcript stability was determined as the ratio between transcript abundance at 6 h (pulse/chase) to 0.5 h (pulse) and compared with Ctrl. Experiment performed in duplicate; analysis by Student’s t-test; data represented as mean ± standard error of the mean.
Figure 7
Figure 7
Random forest analysis using overlapping DmiRNAs. ROC curves for random forest analysis of (A) overlap DmiRNAs (shared by NanoStringDiff and nSolver) with an AUC of 0.831 (95% CI 0.734–0.929), (B) nSolver AUC 0.778 (95%CI 0.664–0.892) and (C) NanoStringDiff AUC 0.761 (95%CI 0.645–0.877). (D) Variable importance in projection plot ranking importance of DmiRNAs to all three classifiers, overlap (blue), NanoStringDiff (orange) and nSolver (grey). miR-26a-5p was the top candidate, followed by miR-30c-5p, across all methods.
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References

    1. Goutman, S.A., Hardiman, O., Al-Chalabi, A., Chio, A., Savelieff, M.G., Kiernan, M.C. and Feldman, E.L. (2022) Recent advances in the diagnosis and prognosis of amyotrophic lateral sclerosis. Lancet Neurol., 21, 480–493. - PMC - PubMed
    1. Goutman, S.A., Hardiman, O., Al-Chalabi, A., Chio, A., Savelieff, M.G., Kiernan, M.C. and Feldman, E.L. (2022) Emerging insights into the complex genetics and pathophysiology of amyotrophic lateral sclerosis. Lancet Neurol., 21, 465–479. - PMC - PubMed
    1. Bensimon, G., Lacomblez, L. and Meininger, V. (1994) A controlled trial of riluzole in amyotrophic lateral sclerosis. ALS/Riluzole Study Group. N. Engl. J. Med., 330, 585–591. - PubMed
    1. Writing, G. and Edaravone, A.L.S.S.G. (2017) Safety and efficacy of edaravone in well defined patients with amyotrophic lateral sclerosis: a randomised, double-blind, placebo-controlled trial. Lancet Neurol., 16, 505–512. - PubMed
    1. Kaji, R., Imai, T., Iwasaki, Y., Okamoto, K., Nakagawa, M., Ohashi, Y., Takase, T., Hanada, T., Shimizu, H., Tashiro, K. et al. (2019) Ultra-high-dose methylcobalamin in amyotrophic lateral sclerosis: a long-term phase II/III randomised controlled study. J. Neurol. Neurosurg. Psychiatry, 90, 451–457. - PMC - PubMed

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