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. 2019 Apr 21;10(4):313.
doi: 10.3390/genes10040313.

miR-194 Accelerates Apoptosis of Aβ1⁻42-Transduced Hippocampal Neurons by Inhibiting Nrn1 and Decreasing PI3K/Akt Signaling Pathway Activity

Tingting Wang  1 , Yaling Cheng  2 , Haibin Han  3 , Jie Liu  4 , Bo Tian  5 , Xiaocui Liu  6
Affiliations

miR-194 Accelerates Apoptosis of Aβ1⁻42-Transduced Hippocampal Neurons by Inhibiting Nrn1 and Decreasing PI3K/Akt Signaling Pathway Activity

Tingting Wang et al. Genes (Basel). .

Abstract

This article explores the mechanism of miR-194 on the proliferation and apoptosis of Aβ1-42-transduced hippocampal neurons. Aβ1-42-transduced hippocampal neuron model was established by inducing hippocampal neurons with Aβ1-42. MTT assay and flow cytometry were used to detect the viability and apoptosis of hippocampal neurons, respectively. qRT-PCR was used to detect changes in miR-194 and Nrn1 expression after Aβ1-42 induction. Aβ1-42-transduced hippocampal neurons were transfected with miR-194 mimics and/or Nrn1 overexpression vectors. Their viability and neurite length were detected by MTT assay and immunofluorescence, respectively. Western blot was used to detect protein expression. Aβ1-42 inhibited Aβ1-42-transduced hippocampal neuron activity and promoted their apoptosis in a dose-dependent manner. miR-194 was upregulated and Nrn1 was downregulated in Aβ1-42-transduced hippocampal neurons (p < 0.05). Compared with the model group, Aβ1-42-transduced hippocampal neurons of the miR-194 mimic group had much lower activity, average longest neurite length, Nrn1, p-AkT, and Bcl-2 protein expression and had much higher Bax, Caspase-3, and Cleaved Caspase-3 protein expression. Compared with the model group, Aβ1-42-transduced hippocampal neurons of the LV-Nrn1 group had much higher activity, average longest neurite length, Nrn1, p-AkT, and Bcl-2 protein expression and had much lower Bax, Caspase-3, and Cleaved Caspase-3 protein expression. Nrn1 is a target gene of miR-194. miR-194 inhibited apoptosis of Aβ1-42-transduced hippocampal neurons by inhibiting Nrn1 and decreasing PI3K/AkT signaling pathway activity.

Keywords: Alzheimer’s disease; Nrn1; PI3K/AkT signaling pathway; apoptosis; hippocampal neurons; miR-194; proliferation.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1
Culture and identification of hippocampal neurons (×200). Morphology of hippocampal neurons (A) immediately after inoculation, (B) 1 day, (C) 2 days, and (D) 7 days. (E,F) Immunofluorescence labeling of hippocampal neurons treated with mouse anti-rat β-III tubulin monoclonal antibody and fluorescein isothiocyanate (FITC)-labeled goat anti-mouse IgG secondary antibody. (G) Neuronal nuclei labeled by Hoechst 33258 labeling. (H) Merged picture of Figure (E,F).
Figure 2
Figure 2
Aβ1–42 inhibited hippocampal neuron viability and promoted its apoptosis in a dose-dependent manner. (A) MTT assay for hippocampal neuron activity after Aβ1–42 induction. (B) Detection of hippocampal neuron apoptosis rate by flow cytometry. * p < 0.05 when compared with the negative controls.
Figure 3
Figure 3
miR-194 was upregulated and Nrn1 mRNA was downregulated in hippocampal neurons after being induced by Aβ1–42. * p < 0.05 when compared with the blank group.
Figure 4
Figure 4
Upregulated miR-194 decreased viability of Aβ1–42-transduced hippocampal neurons (A) and increased the apoptosis of Aβ1–42-transduced hippocampal neurons (B). * p < 0.05 when compared with the model group.
Figure 5
Figure 5
Nrn1 is the target gene of miR-194. (A) Target Scan predicted the binding sites of Nrn1 and miR-194. (B) Luciferase reporter gene assay was used to verify the targeting relationship between miR-194 and Nrn1. * p < 0.05 when compared with the negative control (NC) + wild-type (Wt) group.
Figure 6
Figure 6
Nrn1 repaired the damage of miR-194 on Aβ1–42-transduced hippocampal neuron activity. * p < 0.05 when compared with the model group at the same time.
Figure 7
Figure 7
Nrn1 reversed the inhibitory effect of miR-194 on neurite growth of Aβ1–42-transduced hippocampal neurons. (A) Neurite of Aβ1–42-transduced hippocampal neurons were observed under a microscope after they were immunofluorescently stained. (B) Statistical data of the average longest neurite length. * p < 0.05 when compared with the model group at the same time.
Figure 8
Figure 8
miR-194 regulated apoptosis-related protein expression in Aβ1–42-transduced hippocampal neurons by targeting inhibition of Nrn1 expression and decreasing PI3K/AkT signaling pathway activity. Western blot analysis of the expression of (A) AkT and p-AkT, (B) Nrn1, (C) Bcl-2 and Bax, and (D) Caspase-3 and Cleaved Caspase-3. * p < 0.05 when compared with the model group.
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