Journal: Stem Cell Reports

Article Title: Upregulation of β-catenin due to loss of miR-139 contributes to motor neuron death in amyotrophic lateral sclerosis

doi: 10.1016/j.stemcr.2022.05.019

Figure Lengend Snippet: MiR-139-5p targets WNT signaling in FUS ALS MNs (A) PCA using the top 500 variable genes from miR-139 overexpression (OE) in FUS H517Q MNs. RNA collected at day 35. (B) Cumulative distribution function (CDF) plot comparing the fold changes of predicted miR-139 targets (blue curve) relative to all genes (black curve) in response to miR-139 OE. p values were estimated using the Kolmogorov-Smirnov test. (C) Enrichment of 7mers starting at positions 1–15 of miR-139-5p in the 3′ UTR of genes downregulated upon miR-139-5p OE. The miRNA sequence is shown at top for reference. p values were estimated using a hypergeometric distribution and corrected for multiple hypothesis testing using the Benjamini-Hochberg method. A p value threshold of 0.01 is indicated by the vertical dotted line. (D) Left panel: GSEA analysis of miR-139 OE using curated gene sets from MSigDB. X axis displays the log transformed p values. Genes downregulated upon CTNNB1 deletion in intestinal epithelial cells (FEVR_CTNNB1_TARGETS_DN) were identified in the top 10 gene sets with a significant negative enrichment score (i.e., these gene sets were downregulated after miR-139-5p OE). Right panel: GSEA enrichment plot for the gene set FEVR_CTNNB1_TARGETS_DN. X axis shows genes sorted from most upregulated (left-hand side) to most downregulated (right-hand side). Enrichment p values for the gene sets were estimated using 1,000 random simulations and adjusted using the Benjamini-Hochberg method. (E) MiR-139-5p is predicted to directly target several WNT-associated genes, including the WNT transcriptional mediator CTNNB1. Predicted targets were obtained from 3 databases: miRWalk, Targetscan, and miRDB ( Sticht et al., 2018 ; Agarwal et al., 2015 ; ). Score indicates the number of databases supporting each interaction. (F) In-cell western blot analysis to measure β-catenin protein levels in response to miR-139-5p overexpression (OE) in FUS H517Q/H517Q ALS (N = 5) and healthy 80a MNs (N = 3). (G) Relative luminescence of the Renilla luciferase gene, carrying the CTNNB1 3′ UTR with the predicted miR-139 binding site (wild type [WT]) or with the mutated site (mutant). Mutation of the miRNA binding site rescues luciferase activity when transfected in human MNs. N = 5. (H) SuperTopFlash dual luciferase assay to measure β-catenin activity upon inhibition of miR-139 in SH-SY5Y cells. N = 3. (I) qRT-PCR analysis of miR-139-5p expression upon activation of WNT signaling using CHIR09921 in SH-SY5Y cells. N = 3. (J) Schematic of proposed negative feedback loop between miR-139 and the WNT pathway. (K) CDF plot of miR-139-5p WNT targets (blue) and all expressed genes (black) in differentially expressed genes in laser capture microdissected sALS MN relative to healthy controls. GSE18920 : MNs were derived from lumbar spinal cords (12 ALS and 10 controls) and gene expression analyzed using microarrays. GSE76220 : MN were derived from lumbar spinal cords (13 ALS and 8 controls) and gene expression analyzed by RNA-seq. The p value was calculated using the Kolmogorov-Smirnov test. N: number of independent differentiations. Error bars indicate SEMs. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 by Student’s t test, unless otherwise stated.

Article Snippet: Cells were transfected with 700 ng STF19 plasmid (Psicheck2, Promega) and 100 ng Renilla luciferase plasmid (pRLcmv, Promega).

Techniques: Over Expression, Sequencing, Transformation Assay, In-Cell ELISA, Luciferase, Binding Assay, Mutagenesis, Activity Assay, Transfection, Inhibition, Quantitative RT-PCR, Expressing, Activation Assay, Derivative Assay, Gene Expression, RNA Sequencing