MicroRNA-29a modulates axon branching by targeting doublecortin in primary neurons - PubMed (original) (raw)

MicroRNA-29a modulates axon branching by targeting doublecortin in primary neurons

Hanqin Li et al. Protein Cell. 2014 Feb.

Abstract

MicroRNAs (miRNAs) are endogenously expressed small, non-coding transcripts that regulate protein expression. Substantial evidences suggest that miRNAs are enriched in central nervous system, where they are hypothesized to play pivotal roles during neural development. In the present study, we analyzed miRNAs expression in mice cerebral cortex and hippocampus at different developmental stages and found miR-29a increased dramatically at postnatal stages. In addition, we provided strong evidences that miR-29a is enriched in mature neurons both in vitro and in vivo. Further investigation demonstrated that the activation of glutamate receptors induced endogenous miR-29a level in primary neurons. Moreover, we showed that miR-29a directly regulated its target protein Doublecortin (DCX) expression, which further modulated axon branching in primary culture. Together, our results suggested that miR-29a play an important role in neuronal development of mice cerebrum.

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Figures

Figure 1

Figure 1

Expression of miR-29a in mice cortex and hippocampus. (A) Relative expression level of miR-29a and miR-138 (U6 is used as internal control) in cortex of different developmental stages, *, P < 0.01 compared to the E12.5 stage in each group (n = 5); (B) relative expression level of miR-29a and miR-138 in hippocampus of different developmental stages, *, P < 0.01 compared to the E18.5 stage in each group (n = 5); (C) in situ hybridization of different members of miR-29 family in hippocampus (a–e) and cortex (a′–e′) (n = 3), miR-124 is used as positive control, arrows indicate neurons with positive signal in cortex or hippocampus, scale bar = 200 μm

Figure 2

Figure 2

Expression of miR-29a in neurons under different circumstances. (A) Expression pattern of miR-29a (purple) in DG area of hippocampus at different postnatal stages (a–c), expression pattern of NeuN (brown) in DG area of hippocampus at different postnatal stages (d–f), granule cell layers are indicated between two yellow lines (n = 3), scale bar = 50 μm; (B) relative expression level of miR-29a in primary neuron at different cultured stages, *, P < 0.01 compared to DIV 1 in each group (n = 5); (C) relative expression level of miR-29a in primary cortical neurons at different time points after the stimulation of 10 μmol/L GSM for 15 min, *, P < 0.01 compared to control (n = 3); (D) relative expression level of miR-29a in primary cortical neurons at 2 h after indicated treatments, *, P < 0.01 (n = 3)

Figure 3

Figure 3

DCX is a direct target of miR-29a. (A) Seed sequence and alignment of the miR-29a binding sites in the 3′UTRs of DCX mRNAs from different species, the predicted base-pairing of miR-29a with target recognition seed sequence is shown in colored; (B) Western-blot of DCX (β-tubulin is used as internal control) in cortex (left panel) or hippocampus (right panel) at different developmental stages indicated (n = 3); (C and D) quantification of DCX protein in cortex (C) or hippocampus (D) at different developmental stages indicated (n = 3); (E) expression pattern of DCX (brown, arrows) in DG area of hippocampus at different postnatal stages, granule cell layers are between two yellow lines (n = 3), scale bar = 50 μm; (F and G) Western-blot and quantification of DCX protein in primary neurons derived from cortex and hippocampus at different time points (n = 3); (H) relative luciferase activity measured in different groups of 293T cells, *, P < 0.01 compared to control (n = 3); (I) relative level of miR-29a in primary cortical neurons transfected with miR-29a mimics, *, P < 0.01 compared to scramble (n = 3); (J and K) Western-blot and quantification of DCX protein in primary cortical neurons treated as in G, *, P < 0.01 compared to scramble (n = 3); (L) relative level of miR-29a in primary cortical neurons transfected with antago-miR-29a, *, P < 0.01 compared to scramble (n = 3); (M, N) Western-blot and quantification of DCX protein in primary cortical neurons treated as in J, *, P < 0.01 compared to scramble (n = 3)

Figure 4

Figure 4

Effects of miR-29a on axon branching of primary cortical neurons. (A) Cortical neurons transfected with scramble or miR-29a mimics together with the eGFP expression plasmid were fixed 4 d after transfection and subjected to immunocytochemistry using the eGFP antibody. In all images of this type, arrowheads indicate primary axons, scale bars = 50 μm; (B) quantification of axon branching of cortical neurons treated in A, *, P < 0.01 compared to scramble (n = 20); (C) cortical neurons transfected with scramble or antago-miR-29a together with the eGFP expression plasmid were analyzed as in A, scale bars = 50 μm; (B) quantification of axon branching of cortical neurons treated in C, *, P < 0.01 compared to scramble (n = 20); (E, F) Western-blot and quantification of DCX protein in cortical neurons treated with scrambles or DCX RNAi (n = 3); (G) cortical neurons transfected with scrambles or DCX RNAi together with the eGFP expression plasmid were analyzed as in A, scale bars = 50 μm; (H) quantification of axon branching of cortical neurons treated in G, *, P < 0.01 compared to scramble (n = 20)

Figure 5

Figure 5

Over-expression of DCX rescues the axon branching phenotype induced by miR-29a. (A and B) Western-blot and quantification of DCX protein in cortical neurons under different treatment indicated (n = 3); (C) different groups of cortical neurons transfected together with the eGFP expression plasmid were analyzed as in Fig. 4A, scale bars = 50 μm; (D) quantification of axon branching of cortical neurons treated in C, *, P < 0.01 compared to scramble (n = 20)

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