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LncRNA Neat1 Mediates miR-124-induced Activation of Wnt/β-catenin Signaling in Spinal Cord Neural Progenitor Cells

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LncRNA Neat1 Mediates miR-124-induced Activation of Wnt/β-catenin Signaling in Spinal Cord Neural Progenitor Cells

Yi Cui et al. Stem Cell Res Ther.

Abstract

Background: Emerging evidence suggests that miR-124 performs important biological functions in neural stem cells (NSCs); it regulates NSC behavior and promotes the differentiation of NSCs into neurons, but the exact molecular mechanism remains unknown. And also, the role of miR-124 during spinal cord injury regeneration is unclear.

Materials and methods: In order to explore the function of miR-124 in neural differentiation, the molecular markers (Tuj1, Map2, and GFAP) correlated with the differentiation of NSCs were detected by immunofluorescence staining both in cultured mouse spinal cord progenitor cells (SC-NPCs) and in spinal cord injury (SCI) animal models. The migration ability and apoptosis of cultured SC-NPCs were also evaluated by Transwell migration assay and TUNEL assay. In addition, the relative expression of lnRNA Neat1- and Wnt/β-catenin signaling-related genes were detected by quantitative real-time PCR.

Results: In this study, we revealed that lncRNA Neat1 is involved in regulating Wnt/β-catenin signaling that is activated by miR-124 in SC-NPCs. LncRNA Neat1 was also found to play an important role in regulating neuronal differentiation, apoptosis, and migration of SC-NPCs. Furthermore, we demonstrated that overexpression of miR-124 resulted in elevated Neat1 expression, accompanied with the functional recovery of locomotion in a mouse model of spinal cord injury.

Conclusions: Our results confirm the therapeutic effectiveness of miR-124 on the functional recovery of injured spinal cord, supporting the rationale and feasibility of miR-124 for spinal cord injury treatment in future clinical therapy. Furthermore, we concluded that the miR-124-Neat1-Wnt/β-catenin signaling axis is involved in regulating the cell function of SC-NPCs, and this may offer novel therapeutic avenues for future treatment of SCI.

Keywords: Neat1; Spinal cord injury (SCI); Spinal cord neural stem cells (SC-NPCs); miR-124.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Relative expression of Neat1 gene in the spinal cord neural progenitor cells (SC-NPCs) and spinal cord injury (SCI) animals. Moreover, the relative expression of miR-124 and RXRα were also detected by quantitative real-time PCR (q-PCR). GAPDH expression served as a loading control. a The expression of Neat1 in SC-NPCs during differentiation when miR-124 was overexpressed or knocked down. b The relative expression of Neat1, miR-124, and RXRα at the injury site after SCI. Expression levels of each gene were normalized to GAPDH. The data are shown as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001
Fig. 2
Fig. 2
Relative expression of Wnt/β-catenin signaling-related genes (Wisp1, Wnt5a, DKK1, and Wnt2) in each group of SC-NPCs (a) and SCI animals (b). Expression levels of each gene were normalized to GAPDH. The data are shown as the mean ± SD *p < 0.05, **p < 0.01, ***p < 0.001
Fig. 3
Fig. 3
Mir-124/Neat1 promotes neuronal differentiation in cultured SC-NPCs. Representative pictures of immunofluorescence staining of Tuj1+ (a), Map2+ (b), and GFAP+ (c) from each group in cultured SC-NPCs. The graph shows the relative percentages of Tuj1+ (a, left graph), Map2+ (b), and GFAP+ (c) cells in SC-NPCs when cultured in the differentiation medium for 4 days. In addition, we measured the length of neurite extensions from Tuj1-positive cells and then quantified by using Image-Pro Plus software (a, right graph). The data are shown as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001
Fig. 4
Fig. 4
The effect of miR-124 on SCI animals. a The surgical procedure used to create the SCI mouse model. Mir-124 mimics were given by multiple injections around the injury site. b Overview immunofluorescence staining images of Tuj1+ (Left) and Map2+ (Right) at the injury site in SCI animal models at 1 and 2 weeks post-injury were shown. c The enlarged immunostaining images of GFAP+ at the glial scar located in the two sides of broken ends of all groups in SCI animals. The graph shows the relative percentages of GFAP+ cells quantified by using Image-Pro Plus software. d The enlarged immunostaining images of Tuj1+ at the injury site of all groups in SCI animals. The graph shows the neurite length calculated by using Image-Pro Plus software. The data are shown as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001
Fig. 5
Fig. 5
Mir-124/Neat1 inhibits apoptosis in cultured SC-NPCs. Quantitative analysis showing the percentage of apoptotic SC-NPCs in each group when cultured in the differentiation medium for 4 days. The data are shown as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001
Fig. 6
Fig. 6
Mir-124/Neat1 facilitates migration in cultured SC-NPCs. Quantitative analysis showing the percentage of migratory SC-NPCs in each group when cultured in the differentiation medium for 4 days. The numbers of migratory cells were normalized to the number of migratory cells in the control group. The data are shown as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001
Fig. 7
Fig. 7
The motor function evaluation of SCI animals and the regulatory mechanism of miR-124 on SC-NPCs. a BMS scores were determined at the particular point in time (1, 2, and 3 weeks) after miR-124 treatment. Mir-124-treated animals had significantly higher locomotor scale scores than control mice at 1 and 2 weeks. Error bars represent the mean ± SD of (at least) triplicate experiments. **p < 0.01. b Proposed regulatory mechanism of miR-124-NEAT1-Wnt/β-catenin signaling in SC-NPCs. The diagram shows that Neat1 acts as a key mediator between miR-124 and Wnt/β-catenin signaling in regulating SC-NPC behavior

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