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Role of miR-146a in neural stem cell differentiation and neural lineage determination: relevance for neurodevelopmental disorders

Nguyen, Lam Son, Fregeac, Julien, Bole-Feysot, Christine, Cagnard, Nicolas, Iyer, Anand, Anink, Jasper, Aronica, Eleonora, Alibeu, Olivier, Nitschke, Patrick, Colleaux, Laurence·2018·Molecular Autism
DOI 10.1186/s13229-018-0219-3PMC PMC6011198
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Fig. 1 Generation and characterization of H9 hNSC overexpressing miR-146a. a Expression of miR-146a in the brain of ASD patients. Expression was measured in the temporal lobe of ASD patients (white box) and age-matched controls (gray box). Box plot showing relative expression of miR-146a measured by Taqman RT-qPCR for two house keeping miRNAs (miR-106a and miR-17) and the average of all controls. ***Fold change > 1.2 and P < 0.001 by Mann Whitney Test. b Phase contrast image showing morphology of hNSC lines in undifferentiated (day 0) and differentiated condition upon withdrawal of GF (days 7 and 14). c Expression of miR-146a (±S.D.) in H9 hNSC with stable construct compared to untransduced cells. Analyses were measured by Taqman assay, normalized against U6. d Expression of GFP transgene (±S.D.) in the two newly established hNSC H9 overexpressing miR-146a or miR-146a-Mut. Relative expression was measured by RT-qPCR, normalized against GPBP1. e Repression ratio (miR-146a/miR-146a mut) of known targets of miR-146a in undifferentiated H9 hNSC by RNA-Seq analyses. *P < 0.05, **P < 0.01, ***P > 0.001 by EdgeR analysis
Fig. 1 Generation and characterization of H9 hNSC overexpressing miR-146a. a Expression of miR-146a in the brain of ASD patients. Expression was measured in the temporal lobe of ASD patients (white box) and age-matched controls (gray box). Box plot showing relative expression of miR-146a measured by Taqman RT-qPCR for two house keeping miRNAs (miR-106a and miR-17) and the average of all controls. ***Fold change > 1.2 and P < 0.001 by Mann Whitney Test. b Phase contrast image showing morphology of hNSC lines in undifferentiated (day 0) and differentiated condition upon withdrawal of GF (days 7 and 14). c Expression of miR-146a (±S.D.) in H9 hNSC with stable construct compared to untransduced cells. Analyses were measured by Taqman assay, normalized against U6. d Expression of GFP transgene (±S.D.) in the two newly established hNSC H9 overexpressing miR-146a or miR-146a-Mut. Relative expression was measured by RT-qPCR, normalized against GPBP1. e Repression ratio (miR-146a/miR-146a mut) of known targets of miR-146a in undifferentiated H9 hNSC by RNA-Seq analyses. *P < 0.05, **P < 0.01, ***P > 0.001 by EdgeR analysisCC BY · PubMed Central
Fig. 2 miR-146a controls responses of H9 hNSC to neurogenic cues. a Cell proliferation rates measured using the Incucyte machine over 48 h after induction of differentiation. Graph shows the average ratio (±S.D.) of proliferation slopes from 3 independent experiments. *P < 0.05 by Student’s 2-tail unpaired T test. b Apoptotic cell rates measured by the number of Annexin V labeled cells over the confluence percentage using the Incucyte machine. Graph shows average ratio (±S.D.) of four technical replicates and are representative of two independent repeats showing the same results. c Average number of neurite branching (±S.D.) and average neurite length d (±S.D.) in cells undergoing differentiation over 2 weeks. Analyses were performed using an analysis mask (see Additional file 2: Fig. S1a) on images taken at 25 different spots every 3 h over 15 days by the Incucyte machine. Results are representative of three independent repeats showing similar results. ***P < 0.001 by Student’s 2-tailed unpaired T test. e FACS analysis was performed on differentiated cells labeled by TUB-III or NESTIN antibodies (see Additional file 2: Figure S2 for details). Graph shows average ratio (±S.D.) of three independent repeats. *P < 0.05 by Student’s 2-tail unpaired T test
Fig. 2 miR-146a controls responses of H9 hNSC to neurogenic cues. a Cell proliferation rates measured using the Incucyte machine over 48 h after induction of differentiation. Graph shows the average ratio (±S.D.) of proliferation slopes from 3 independent experiments. *P < 0.05 by Student’s 2-tail unpaired T test. b Apoptotic cell rates measured by the number of Annexin V labeled cells over the confluence percentage using the Incucyte machine. Graph shows average ratio (±S.D.) of four technical replicates and are representative of two independent repeats showing the same results. c Average number of neurite branching (±S.D.) and average neurite length d (±S.D.) in cells undergoing differentiation over 2 weeks. Analyses were performed using an analysis mask (see Additional file 2: Fig. S1a) on images taken at 25 different spots every 3 h over 15 days by the Incucyte machine. Results are representative of three independent repeats showing similar results. ***P < 0.001 by Student’s 2-tailed unpaired T test. e FACS analysis was performed on differentiated cells labeled by TUB-III or NESTIN antibodies (see Additional file 2: Figure S2 for details). Graph shows average ratio (±S.D.) of three independent repeats. *P < 0.05 by Student’s 2-tail unpaired T testCC BY · PubMed Central
Fig. 3 miR-146a targets key genes to promote hNSC differentiation. a Distribution of DEGs identified by RNA-Seq in undifferentiated (Undiff) and differentiated (Diff) conditions. b Graph shows expression ratios of DEGs measured by RNA-Seq and RT-qPCR. Circle indicates only incompatible/non-validated DEGs. c Protein interaction network of all DEGs identified in differentiated cells mapped by STRING. Two modules are identified: Neuronal Module (top) contains 350 genes and Cell Cycle Module (bottom) contains 155 genes. d Top 5 pathways identified by Ingenuity Pathway Analysis of the 2 modules (see Additional file 1: Tables S8, S9 for extended lists). e miR-146a directly targets DCX, GAD1, and PAK3 3′UTR. Due to their size, the 3′UTR of DCX and PAK3 were cloned into 2 and 3 fragments (frag), respectively. Each 3′UTR construct contains at least 3 predicted miR-146a binding sites. Ratio of Renilla/Firefly luciferase (±SD of 3 technical triplicates) was measured in cell lines co-transfected with either the synthetic miR-mimic (control) or the miR-146a. Graphs are representative of 3 independent assays showing the same results. *P < 0.05, **P < 0.01 by Student’s 2 tailed paired t test. f List of known and predicted miR-146a targets that are downregulated in each respective module
Fig. 3 miR-146a targets key genes to promote hNSC differentiation. a Distribution of DEGs identified by RNA-Seq in undifferentiated (Undiff) and differentiated (Diff) conditions. b Graph shows expression ratios of DEGs measured by RNA-Seq and RT-qPCR. Circle indicates only incompatible/non-validated DEGs. c Protein interaction network of all DEGs identified in differentiated cells mapped by STRING. Two modules are identified: Neuronal Module (top) contains 350 genes and Cell Cycle Module (bottom) contains 155 genes. d Top 5 pathways identified by Ingenuity Pathway Analysis of the 2 modules (see Additional file 1: Tables S8, S9 for extended lists). e miR-146a directly targets DCX, GAD1, and PAK3 3′UTR. Due to their size, the 3′UTR of DCX and PAK3 were cloned into 2 and 3 fragments (frag), respectively. Each 3′UTR construct contains at least 3 predicted miR-146a binding sites. Ratio of Renilla/Firefly luciferase (±SD of 3 technical triplicates) was measured in cell lines co-transfected with either the synthetic miR-mimic (control) or the miR-146a. Graphs are representative of 3 independent assays showing the same results. *P < 0.05, **P < 0.01 by Student’s 2 tailed paired t test. f List of known and predicted miR-146a targets that are downregulated in each respective moduleCC BY · PubMed Central
Fig. 4 miR-146a determines neuronal lineage identities. Heatmap of gradient of expression from the Cell Cycle (a) and Neuronal (b) Modules spanning human fetal development to late adulthood in distinct brain regions. A1C, auditory cortex; AMY, amygdala; CBC, cerebellar cortex; DFC, dorsolateral prefrontal cortex; HIP, hippocampus; IPC, posterior inferior parietal cortex; ITC, inferior temporal cortex; M1C, primary motor cortex; MD, mediodorsal nucleus of thalamus; MFC, medial prefrontal cortex; OFC, orbital prefrontal cortex; S1C, primary somatosensory cortex; STC, superior temporal cortex; STR, striatum; V1C, primary visual cortex; VFC, ventrolateral prefrontal cortex. Period labels: 3—Early fetal (10 ≤ Age ≤ 13 Post conception week, PCW); 4—Early midfetal (13 ≤ Age ≤ 16 PCW); 5—Early midfetal (16 ≤ Age ≤ 19 PCW); 6—Late midfetal (19 ≤ Age ≤ 24 PCW); 7—Late fetal (24 ≤ Age ≤ 38 PCW); 8—Neonatal and early infancy (Birth ≤ Age ≤ 6 Postnatal months, M); 9—Late infancy (6 ≤ Age ≤ 12 M); 10—Early childhood (1 ≤ Age ≤ 6 Postnatal years, Y); 11—Middle and late childhood (6 ≤ Age ≤ 12 Y); 12—Adolescence (12 ≤ Age ≤ 20 Y); 13—Young adulthood (20 ≤ Age ≤ 40 Y); 14—Middle adulthood (40 ≤ Age ≤ 60 Y); 15—Late adulthood (60 Y ≤ Age). c Expression difference of detectable neuronal specific markers in differentiated H9 hNSC. Dotted lines indicate expression difference threshold (fold change ≥ 1.5). *P < 0.05, **P < 0.01 and ***P < 0.001 by EdgeR analysis (as representative of 3 different RNA-Seq analyses)
Fig. 4 miR-146a determines neuronal lineage identities. Heatmap of gradient of expression from the Cell Cycle (a) and Neuronal (b) Modules spanning human fetal development to late adulthood in distinct brain regions. A1C, auditory cortex; AMY, amygdala; CBC, cerebellar cortex; DFC, dorsolateral prefrontal cortex; HIP, hippocampus; IPC, posterior inferior parietal cortex; ITC, inferior temporal cortex; M1C, primary motor cortex; MD, mediodorsal nucleus of thalamus; MFC, medial prefrontal cortex; OFC, orbital prefrontal cortex; S1C, primary somatosensory cortex; STC, superior temporal cortex; STR, striatum; V1C, primary visual cortex; VFC, ventrolateral prefrontal cortex. Period labels: 3—Early fetal (10 ≤ Age ≤ 13 Post conception week, PCW); 4—Early midfetal (13 ≤ Age ≤ 16 PCW); 5—Early midfetal (16 ≤ Age ≤ 19 PCW); 6—Late midfetal (19 ≤ Age ≤ 24 PCW); 7—Late fetal (24 ≤ Age ≤ 38 PCW); 8—Neonatal and early infancy (Birth ≤ Age ≤ 6 Postnatal months, M); 9—Late infancy (6 ≤ Age ≤ 12 M); 10—Early childhood (1 ≤ Age ≤ 6 Postnatal years, Y); 11—Middle and late childhood (6 ≤ Age ≤ 12 Y); 12—Adolescence (12 ≤ Age ≤ 20 Y); 13—Young adulthood (20 ≤ Age ≤ 40 Y); 14—Middle adulthood (40 ≤ Age ≤ 60 Y); 15—Late adulthood (60 Y ≤ Age). c Expression difference of detectable neuronal specific markers in differentiated H9 hNSC. Dotted lines indicate expression difference threshold (fold change ≥ 1.5). *P < 0.05, **P < 0.01 and ***P < 0.001 by EdgeR analysis (as representative of 3 different RNA-Seq analyses)CC BY · PubMed Central

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Research works printed but not linked: 31

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Metadata

Authors
Nguyen, L. S., Fregeac, J., Bole-Feysot, C., Cagnard, N., Iyer, A., Anink, J., Aronica, E., Alibeu, O., Nitschke, P., & Colleaux, L.
ORCID
Colleaux, Laurence
Venue
Molecular Autism
Publisher
Springer Science and Business Media LLC
Published
2018-06-19
Volume
9
Issue
1
ISSN
2040-2392
DOI
10.1186/s13229-018-0219-3
PMC
PMC6011198
Cited by
85
References
14
Language
en
Licence
creativecommons.org/licenses/by/4.0/ (tdm)
Funders
  • Fondation pour la Recherche Médicale (DEQ20160334938)
  • Crédit Agricole d'Ile-de-France Mécénat
  • Fondation de France
  • Agence Nationale de la Recherche (ANR-10-IHU-01)

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