In-depth transcriptomic analyses of LncRNA and mRNA expression in the hippocampus of APP/PS1 mice by Danggui-Shaoyao-San

Abstract

Alzheimer's disease (AD) is an age-related neurodegenerative disease with a high incidence worldwide, and with no medications currently able to prevent the progression of AD. Danggui-Shaoyao-San (DSS) is widely used in traditional Chinese medicine (TCM) and has been proven to be effective for memory and cognitive dysfunction, yet its precise mechanism remains to be delineated. The present study was designed to investigate the genome-wide expression profile of long non-coding RNAs (LncRNAs) and messenger RNAs (mRNAs) in the hippocampus of APP/PS1 mice after DSS treatment by RNA sequencing. A total of 285 differentially expressed LncRNAs and 137 differentially expressed mRNAs were identified (fold-change ≥2.0 and P < 0.05). Partial differentially expressed LncRNAs and mRNAs were selected to verify the RNA sequencing results by quantitative polymerase chain reaction (qPCR). A co-expression network was established to analyze co-expressed LncRNAs and genes. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were used to evaluate the biological functions related to the differentially co-expressed LncRNAs, and the results showed that the co-expressed LncRNAs were mainly involved in AD development from distinct origins, such as APP processing, neuron migration, and synaptic transmission. Our research describes the lncRNA and mRNA expression profiles and functional networks involved in the therapeutic effect of DSS in APP/PS1 mice model. The results suggest that the therapeutic effect of DSS on AD involves the expression of LncRNAs. Our findings provide a new perspective for research on the treatment of complex diseases using traditional Chinese medicine prescriptions.

Keywords: APP/PS1 mice; Alzheimer's disease; Dangui-Shaoyao-San; LncRNA; transcriptomic analyses.

Figure 1

LC-MS/MS chromatogram and mass spectrometry of Danggui-Shaoyao-San (DSS). (A–E) Retention time, chromatogram, and mass spectrogram of ferulic acid (A), paeoniflorin (B), ligustilide (C), atractylenolide I, (D) and alisol B 23-acetate (E) in DSS.

Figure 2

Danggui-Shaoyao-San rescued learning and memory deficits in APP/PS1 transgenic mice. (A) The genotype identification of APP/PS1 double transgenic mice; (B) Escape latency during the acquisition phase of the Morris water maze test; (C) The number of crossings over the previously hidden platform area in the Morris water maze test; (D) Escape latency during the visible platform phase of the Morris water maze test. N = 10 mice/group; Age = 7 months; Data are represented as mean ± standard deviation (SD), *, P< 0.05. (E) Immunohistochemistry of amyloid-β in the brain. N = 5 mice/group; Data are represented as mean ± SD, *, P< 0.05. (F) The protein expression of synaptic markers. N = 4 mice/group; Data are represented as mean ± SD, *, P< 0.05.

Figure 3

Differential expression analysis of messenger RNA (mRNAs) and long non-coding RNAs (LncRNAs) in Danggui-Shaoyao-San (DSS)-treated and untreated APP/PS1 double transgenic mice. (A) Heatmap analysis of differentially expressed mRNAs, DSS-treated VS. APP/PS1 mice; (B) Heatmap analysis of differentially expressed LncRNA, DSS-treated VS. APP/PS1 mice; (C) Principal component analysis (PCA), DSS-treated VS. APP/PS1 mice; (D) Differentially expressed LncRNAs and mRNAs; (E) qPCR for mRNAs; (F) qPCR for LncRNA. N = 5 mice/group; Data are represented as mean ± standard error of mean (SEM), *, P <0.05 vs. APP/PS1 mice group.

Figure 4

Co-expression network of differentially expressed long non-coding RNAs (LncRNAs) and differentially expressed messenger RNAs (mRNAs). The Pearson correlation coefficient between differentially expressed LncRNAs and mRNAs was calculated to construct the co-expression network, and the Pearson correlation coefficient ≥ 0.95 was selected. The circular nodes represent differentially expressed mRNAs (green: downregulated; red: upregulated). The triangular nodes represent differentially expressed LncRNAs. Connection line: co-expression between differentially expressed LncRNAs and mRNAs.

Figure 5

Co-expression network of differentially expressed long non-coding (LncRNAs) and messenger RNAs (mRNAs) related to Alzheimer’s disease. The construction method of the co-expression network is consistent with Figure 3.

Figure 6

The function and pathway analysis of co-expressed genes. The gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed on differentially expressed long non-coding RNA (LncRNA)-related genes to predict the potential biological processes and pathways affected by Danggui-Shaoyao-San-treated Alzheimer's disease mice. (A) The GO enrichment analysis (Biological Process) of differentially expressed genes co-expressed with differentially expressed LncRNAs; (B) The KEGG pathway enrichment analysis of differentially expressed genes co-expressed with differentially expressed LncRNAs.