Fig. 4

MiR-184 directly targets SOX1, LINGO1, and BCL2L1. Schematic representation of the miR-184 binding sites in the 3′-UTR of predicted target sequences: SOX1 (a), LINGO1 (b), and BCL2L1 (c). d Right: SOX1, LINGO1, and BCL2L1 proteins in NPCs were measured by western blotting 96 h post-transduction. GAPDH was used as an internal loading control. Left: Average relative density of protein bands was obtained after densitometric analysis of the bands by ImageJ software followed by normalization to that of GAPDH as the internal loading control. One-way ANOVA method was used in each case to compare the amounts of results statistically. ns: non-significant (P value > 0.05), *P value < 0.05, **P value < 0.01, ***P value < 0.001. e To construct pCDH-Mut-miR-184, two mutations were generated in the seed region of miR-184 and pre-miR-184 mutated form was cloned into pCDH-GFP. The mir-184 mature sequence is designated in red and the mutated form of the nucleotides was indicated in underlined and black. f HEK293 cells were co-transfected with the pLenti-III-miR-184 or pCDH-Mut-miR-184 and 3′-UTR-pSICHEK2 reporter vectors. Activities of the SOX1-, LINGO1-, and BCL2L1-bearing pSICHEK2 vectors were significantly declined in pLenti-III-miR-184 transfected cells compared to pLenti-III-Ctrl and pCDH-Mut-miR-184 vectors used as negative controls. Luciferase activity was detected 48 h after transfection. Values are means ±SD of the ratio of the luciferase activity from three independent runs (*P < 0.001). g Schematic model of OL differentiation pathway induced by miR-184 through Inhibition of SOX1, LINGO1, and BCL2L1. During the NPC differentiation, miR-184 suppresses the expression of neural cell fate determination and differentiation gene (SOX1) leading to the neuron generation inhibition. It also suppresses the expression of BCL2L1 to block the astrocytic differentiation at the point of astrocyte/oligodendrocyte lineage divergence. Furthermore, inhibition of LINGO1 could facilitate OL lineage differentiation. Stimulatory impact on differentiation is shown by green lines with arrowheads and inhibitory roles in differentiation are indicated by the red line with an arrowhead