Mir-23a and mir-125b regulate neural stem/progenitor cell proliferation by targeting Musashi1

Abstract

Musashi1 is an RNA binding protein that controls the neural cell fate, being involved in maintaining neural progenitors in their proliferative state. In particular, its downregulation is needed for triggering early neural differentiation programs. In this study, we profiled microRNA expression during the transition from neural progenitors to differentiated astrocytes and underscored 2 upregulated microRNAs, miR-23a and miR-125b, that sinergically act to restrain Musashi1 expression, thus creating a regulatory module controlling neural progenitor proliferation.

Keywords: 3′UTR, 3′Untranslated Region; BrdU, 5-bromo-2′-deoxyuridine; Msi1, Musashi1; NSPCs, Neural Stem/Progenitor Cells; astrocyte differentiation; cell proliferation; miRNA, microRNA; microRNAs; musashi1; neural progenitors; post-transcriptional gene regulation.

Figure 1.

Analysis of Msi1 mRNA and protein levels during in vitro astrocyte differentiation. (A) Neural progenitors (NSPCs) propagated in expansion medium were virtually all positive for the neural stem/progenitor cell markers nestin and Musashi1; NSPCs differentiated in FBS-containing medium for 6 d (astrocytes) homogeneously expressed the glial marker GFAP. Note that very few differentiated astrocytic cells express Musashi1 (arrows). Nuclei are stained with Hoechst. Scale bar 20 μm. (B) RT-PCR of Msi1 mRNA in NSPCs treated with FBS for 3 and 6 d. The histogram shows the relative quantities of Msi1 mRNA versus the 0 time point, set to a value of 1. Gapdh mRNA was used as a loading control. Data are presented as the mean values ± SEM from 3 independent experiments. *p<0,05; **p<0,01; ***p<0,001. (C) Immunoblotting of Msi1 in NSPCs treated with FBS for 3 and 6 d. The densitometric analysis on the right shows the relative amount of Msi1 protein vs. untreated cells (0 time point), set to a value of 1. Legend details as in panel (B).

Figure 2.

miRNA profiling during in vitro astrocyte differentiation. (A) The relative expression of each miRNA upon treatment of NPSCs with FBS for 3 and 6 d was determined by high-throughput qRT-PCR. A green-red color scale (-3 to +3) depicts normalized miRNA expression level on a log scale. (B) Pie chart summarizing the data obtained by the qRT-PCR analysis.

Figure 3.

Validation of miRNA profiling. (A) Northern blot of a subset of miRNAs in NSPCs treated with FBS for the indicated times (days). (B) The histograms show the relative quantities of miRNAs versus the 0 time point, set to a value of 1. Data were normalized to 5S-rRNA hybridization signal.

Figure 4.

miR-23a and miR-125b may specifically interact with Msi1 3 ’UTR. (A) Schematic representation of the binding sites for miR-23a and miR-125b on Msi1 3 ’UTR. (B) Schematic representation of the constructs expressing the miRNAs. (C) Northern blot of the ectopically expressed miRNAs. 5S-rRNA was used as a loading control. Control cells (CTRL) were transfected with the empty vector. (D) The histogram reports the levels of luciferase activity in cells overexpressing the miRNAs indicated below and transfected with the wild type 3 ’UTR (gray bars) or with its mutant derivative (black bar) lacking the miRNA binding sites. Control cells (CTRL) were transfected with the empty vector. Data are presented as the mean values ± SEM from 3 independent experiments. *p < 0,05; **p < 0,01; ***p < 0,001.

Figure 5.

miR-23a and miR-125b control NSPC proliferation through regulation of Msi1 expression. (A) Immunoblotting of Msi1 in NSPCs ectopically expressing miR-23a, miR-125b, both of them, or the control vector (CTRL). Actin was used as a loading control. Data in the histogram on the right show the relative quantities of Msi1 vs. control cells. Data are presented as mean values ± SEM from at least 3 independent experiments. *p<0,05; **p<0,01; ***p < 0,001. (B) Analysis of Msi1 mRNA levels by qRT-PCR in NPSCs overexpressing the 2 miRNAs: the histogram shows the relative amount of Msi1 mRNA versus control cells (CTRL), set to a value of 1. Legend details as in (A). (C) BrdU incorporation assay in NSPCs ectopically expressing miR-23a and miR-125b or untransfected cells (CTRL). Legend details as in (A). (D) Immunoblotting of Msi1 in NPSCs knocked down for Msi1. Cells were transfected with anti-Msi1 siRNAs (siMsi1) or with a non-targeting siRNA as a control (CTRL). Actin was used as a loading control. The histogram on the right quantifies the BrdU incorporation upon Msi1 knockdown. *p<0,05; **p<0,01; ***p<0,001. (E) Rescue assay: BrdU incorporation was quantified in NSPCs after ectopic expression of miR-23a and miR-125b alone (bar miRNAs+/Msi1-) or upon further expression of exogenous Msi1, lacking 3 ’UTR (bar miRNAs+/Msi1+). Proliferation was fully recovered to levels of mock-electroporated control cells (bar miRNAs-/Msi1-). Data are presented as mean values ± SEM from 3 independent experiments. At least 300 cells were counted for each sample in each experiment. *p < 0,05; **p < 0,01; ***p<0,001.