Function and mechanism of long non-coding RNA Gm21284 in the development of hippocampal cholinergic neurons

Abstract

Background: Increasing evidence has revealed that long non-coding RNAs (lncRNAs) play a pivotal role in the development of nervous system. Our previous studies have demonstrated that enhanced cholinergic neurogenesis occurs in the subgranular zone (SGZ) of the hippocampal dentate gyrus (DG) after cholinergic denervation, which is closely associated with the core transcription factor Lhx8. This study aimed to identify novel lncRNAs in a denervated hippocampal niche, which may affect cholinergic neurogenesis, and to explore the molecular mechanisms underlying cholinergic neurogenesis.

Methods: The gene expression profiles of the denervated hippocampus were examined by microarray analysis, and targeted lncRNAs were filtered using bioinformatics analysis. The lncRNA Gm21284 was predicted to be associated with Lhx8. RT-PCR and FISH were used to observe the expression and localization of Gm21284 in vitro and in vivo. The interaction between Gm21284 and Lhx8 and miR-30e-3P was verified using the luciferase reporter gene assay. Cell proliferation and differentiation was observed to reveal the effects of Gm21284 in cholinergic neurogenesis.

Results: Microarray analysis demonstrated 482 up-regulated and 135 down-regulated mRNAs, 125 up-regulated and 55 down-regulated lncRNAs, and 10 up-regulated and 3 down-regulated miRNAs in the denervated hippocampal niche. Overall, 32 lncRNAs were differentially expressed in the denervated hippocampal niche, which could interact with miR-30e-3p, miR-431, and miR-147. Among these 32 lncRNAs, Gm21284 and Adarb1 were identified after interleaving with lncRNAs in a co-expression network and WGCNA. Gm21284 was mainly located in the hippocampal DG. Furthermore, Gm21284-positive cells were considerably increased in the denervated hippocampus than in the normal side. EdU proliferation assay revealed that the proliferation of neural stem cells was repressed after the overexpression of Gm21284. Compared with the control group, the proportion of ChAT-positive cells increased at 7 days of differentiation of NSCs overexpressing Gm21284.

Conclusion: Thus, Gm21284 functions as a competing endogenous RNA, which inhibits the proliferation of hippocampal NSCs and promotes their differentiation toward cholinergic neurons by inhibiting miR-30e-3P competitively.

Keywords: Cholinergic neuron; Development; Hippocampus; NSCs; lncRNA.

Fig. 1

Differentially expressed transcripts in hippocampal niche at 7 days after fimbria–fornix transection. a The volcano map shows differently expressed mRNAs in hippocampal niche after fimbria–fornix transection, the screening threshold of differentially expressed genes (fold change) is ≧ 1.5, P < 0.05. b The volcano map shows differently expressed lncRNAs in hippocampal niche after fimbria–fornix transection, the screening threshold of differentially expressed genes (fold change) is ≧ 1.5, P < 0.05. c The volcano map shows differently expressed miRNAs in hippocampal niche after fimbria–fornix transection, the screening threshold of differentially expressed genes (fold change) is ≧ 1.5, P < 0.05. d Co-expression networks of differentially expressed genes in the transected group. Lhx8 is found in the network core and is associated with 111 mRNA and 26 lncRNAs. e Weighted co-expression network for differential genes in microarray detection. Lhx8 is present in the brown module

Fig. 2

Identification of differential lncRNAs associated with Lhx8 by ceRNA strategy. a Prediction of top 10 miRNAs targeting on 3′UTR of Lhx8 by miRanda. b, c Western blots showed lower expression of Lhx8 in miRNA cells and mimics transfected LV-Lhx8 PC12 cells compared to empty vector infected cells. The difference in Lhx8 expression level between the two groups was significant (P < 0.05). d Selection and determination of Lhx8 associated lncRNAs. Colored circles represent various lncRNAs: red circle represents the 125 lncRNAs up-regulated in the Gene Chip; yellow circle represents the co-expression of 26 lncRNAs associated with Lhx8 in the co-expression analysis network; brown circle represents the 23 lncRNAs with the same expression module of Lhx8 in WGCNA; blue circle represents 32 lncRNAs predicted by miRanda, which is associated with miR-30e-3P, miR-431, and miR-503-5p, 2 lncRNAs were confirmed after intersection

Fig. 3

Expression and location of Gm21284. a RT-PCR detected Gm21284 expression in hippocampal niche after fimbria–fornix transection at different time points, *: vs. control group, P < 0.05; **: vs. control group, P < 0.01. b RT-PCR detected Gm21284 in tissues originated from different germinal layer. c RT-PCR detected Gm21284 in forebrain cortices of E13, E15, E18, and P1 and in adult rats. d, e RT-PCR showed that the expression of Gm21284 in cytoplasm of PC12 cells was higher than that in nucleus, *: vs. nucleus, P < 0.05. f FISH assay showed Gm21284 mainly localized in cytoplasm of PC12 cells. g FISH assay showed Gm21284 localized mainly in the cytoplasm of hippocampal neurons. h FISH labeled Gm21284 positive cells in the dentate gyrus of the hippocampus. i FISH showed that the average fluorescence intensity of Gm21284 positive cells in transected side was higher than the control side in DG after fimbria–fornix transection at 7 days, *: vs. control group, P < 0.05. j Confocal microscopy (×40) showed that the number of Gm21284 positive cells on the transected side was higher than the control side in DG after fimbria–fornix transection at 7 days, *: vs. control group, P < 0.05. Scale bar = 50 μm

Fig. 4

Gm21284 acts as ceRNA to regulate expression of Lhx8 with miR-30e-3P. a The predicted target region of miRNAs on Lhx8 3′UTR. b Statistical representation of luciferase reporter gene assay for miR-30e-3P, *: vs. NC miRNA, P < 0.05; **: vs. GV268-lhx8 mut, P < 0.05. c Statistical graph of luciferase reporter gene assay for miR-431, *: vs. NC miRNA, P < 0.05; **: vs. GV268-lhx8 mut, P < 0.05. d Statistical graph of luciferase reporter gene assay for miR-147. e Relative Lhx8 expression in each group for rescue assay. f Statistical graph of rescue assay for miR-30e-3P, *: vs. NC LV-GM21284 + miR-30e-3P group, P < 0.05; **: vs. NC group, P < 0.05. g Statistical graph of rescue assay for miR-431, *: vs. NC LV-GM21284 + miR-431 group, P < 0.05; **: vs. NC group, P < 0.05. g RNAhybrid 2.2 predicted miRNA sponge sites of Gm21284. h Statistical graph of luciferase reporter gene assay for miR-30e-3P, *: vs. NC miRNA, P < 0.05; **: vs. GV268-lhx8 mut, P < 0.05. i Statistical graph of luciferase reporter gene assay for miR-431

Fig. 5

Gm21284 promotes differentiation of NSCs to cholinergic neurons by competitive inhibition of miR-30e-3P. a Immunofluorescence images of EdU-positive cells in each group. b Statistical graph representing percentage of EdU-positive cells, *: vs. NC group, P < 0.05, #: vs. miR30e-3P group, P < 0.05. Scale bar = 50 μm. c FCS image of cell cycle of hippocampal NSCs in each group. d Statistical graph of percentage of cells in the S and G2/M phases, *: vs. NC group, P < 0.05, #: vs. miR30e-3P group, P < 0.05. e Western blot detected the expression of LhX8 and ChAT at different time points in differentiating NSCs. f Statistical graph of expression of LhX8 and ChAT, *: vs. 1 day, P < 0.05; #: vs. 7 days, P < 0.05. g Western blot detected the expression of ChAT in each group at 7 days. h Statistical graph of expression of ChAT, *: vs. NC group, P < 0.05; #: vs. miR30e-3P group, P < 0.05. i Immunofluorescence images of cells at 7 days after differentiation of NSCs to cholinergic neurons. j Statistical graph of percentage of ChAT-positive cells, *: vs. NC group, P < 0.05; #: vs. miR30e-3P group, P < 0.01. Scale bar = 50 μm