Skip to main content
. 2017 Mar 23;8(4):1032–1045. doi: 10.1016/j.stemcr.2017.02.017

Figure 6.

Figure 6

miR-342-5p Targeted GFAP and Regulated Astrocyte Differentiation

(A) The sequence of the 3′ UTR of Gfap was aligned with the seed sequence of miR-342-5p. The recognized sequences (nucleotides 202–207 and 1,246–1,251) are marked in red.

(B) Adherently cultured NS/PCs were transfected with miR-342-5p mimics or the control (five independent transfections performed), and 4 hr later 5% FBS medium was applied to induce the differentiation for another 48 hr. Cells were collected and cell lysates were analyzed by western blot using anti-GFAP, anti-TUJ1, and anti-β-actin.

(C) Reporter assays. Cos7 cells were transfected with different reporter constructs, pcDNA6.2-GW/EmGFP-miR-342-5p and pcDNA6.2-GW/EmGFP-miR-negative control, and phRL-TK-expressing Renilla luciferase as an internal control. Cells were lysed 48 hr after the transfection, and luciferase activities in the cell lysates were determined. Reporter assays were done by four independent transfections, and three wells of a 96-well-plate were prepared as replicate samples in each group.

(D) Primary neurospheres (NS/PCs), oligodendrocytes, astrocytes, and neurons were isolated from rats (each type of cells derived from three different rat samples). Total RNA was extracted from cells and the expression of miR-342-5p was determined by qRT-PCR, with U6 RNA as an endogenous reference.

(E and F) NS/PCs were transfected with miR-342-5p or the control (five independent transfections performed), and cells were induced to differentiate by 5% FBS for 48 hr, and then fixed and stained with anti-GFAP and anti-TUJ1. Nuclei were counterstained with a Hoechst stain. The percentages of GFAP+ cells and TUJ1+ cells were compared (eight fields counted in each group).

(G) After differentiation, cells were collected, and the expressions of genes related with progeny cells were analyzed by qRT-PCR. Transfections of miR-342-5p mimic and the negative control were performed with 50 and 100 nM dosages (three independent transfections performed).

Bars, means ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ns, not significant.