β-Arrestin1/miR-326 Transcription Unit Is Epigenetically Regulated in Neural Stem Cells Where It Controls Stemness and Growth Arrest

Figure 3

CpG island methylation of miR-326/βarr-1 regulatory sequence controls βarr-1 and miR-326 expression. (a) Schematic representation of mouse miR-326/βarr-1 regulatory regions, highlighting the CpG islands regions (light blue) and the CpG islands sites (blue) derived from DBCAT software. (b) mRNA expression levels of βarr-1 and precursor forms of miR-326 and miR-326 expression levels in NSCs after 4 days of 5-azacytidine (5-AZA) treatment. Data are means ± SD from 3 independent experiments. . ((c)-(d)) mRNA expression levels (c) and Western blot analysis (d) of endogenous βarr-1, stemness markers (Nanog, Oct4, and Sox2), and differentiation markers (βIII-tub and Gfap) after 4 days of 5-azacytidine (5-AZA) treatment in NSCs. Actin as LC. Data are means ± SD from 3 independent experiments. . (e) Methylation specific PCR of the screened region in NSCs compared to the starting bulk population (T0). (f) Methylation specific PCR of the screened region in NSCs after 5-AZA treatment. ((e) and (f)) Data are representative images from 3 independent experiments.

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