Construction of a lncRNA-associated competing endogenous RNA regulatory network after traumatic brain injury in mouse

Abstract

Traumatic brain injury (TBI) is a major public health problem worldwide which causes high mortality and disability. Functioning as microRNA (miRNA) sponges, long non-coding RNA (lncRNA) regulates the expression of protein-coding genes in a competing endogenous RNA (ceRNA) network. However, the lncRNA-associated ceRNA in TBI remains unclear. In this study, we processed the raw SRR files of mice cortex samples of sham injury (n = 3) and TBI groups (n = 3) to count files. Then, the expression profiles of lncRNAs and mRNAs were identified, and 86 differentially expressed (DE) lncRNAs and 1201 DEmRNAs between sham and TBI groups were identified. The DEmRNAs were used to perform enrichment analyses. Next, a lncRNA-miRNA-mRNA regulatory ceRNA network was constructed. The network consisted of 23 mRNAs, 5 miRNAs and 2 lncRNAs. The expression alternations of the 5 miRNAs were validated via qRT-PCR. The subnetwork of hub lncRNA Neat1 was extracted. We identified a potential inflammatory associated regulatory axis: Neat1/miR-31-5p/Myd88 axis. The PPI network based on DEmRNA involved in ceRNA network was constructed PPI networks to identify the hub genes. Finally, DElncRNAs and DEmRNAs were selected randomly and validated by qRT-PCR. In conclusion, with the lncRNA-miRNA-mRNA ceRNA network provided above, we can improve our understanding of the regulatory mechanisms and interaction among lncRNAs, miRNAs and mRNAs in TBI process.

Keywords: Bioinformatics analysis; Myd88; Neat1; Traumatic brain injury; ceRNA; lncRNA.

Fig. 1

LncRNA profile based on RNA-seq data. A Distribution of all identified lncRNAs in TBI samples. B Volcano plot of DElncRNAs in mice cortex 24 h post-TBI compared with control cortex. Red points represent upregulated DElncRNAs and blue points represent downregulated DElncRNAs in TBI group. The genesymbols of ten upregulated lncRNAs and ten downregulated lncRNAs with the most significant expression differences are shown in the plot. C Hierarchical cluster heatmaps of DElncRNAs. Each row represents an RNA, and each column represents a sample. Red indicates relatively high expression, and blue indicates relatively low expression. D Distribution of DElncRNAs in TBI, showing upregulated (gray) and downregulated (black) lncRNAs in each chromosome (chr)

Fig. 2

mRNA profile based on RNA-seq data. A Distribution of all identified mRNA in TBI samples. B Volcano plot of DEmRNAs in mice cortex 24 h post-TBI compared with control cortex. Red points represent upregulated DEmRNAs and blue points represent downregulated DEmRNAs in TBI group. The genesymbols of ten upregulated mRNAs and ten downregulated mRNAs with the most significant expression differences are shown in the plot. C Hierarchical cluster heatmaps of DEmRNAs. Each row represents an RNA, and each column represents a sample. Red indicates relatively high expression, and blue indicates relatively low expression. D Distribution of DEmRNAs in TBI, showing upregulated (gray) and downregulated (black) mRNAs in each chromosome (chr)

Fig. 3

A The top 20 enriched terms in the GO biological process analysis. B The top 20 enriched pathways in the KEGG pathway analysis. C, D ceRNA network and subnetwork related to TBI. C lncRNA-associated ceRNA regulatory network related to TBI. D Subnetwork of lncRNA Neat1. Blue squares represent lncRNAs, tan triangles represent miRNAs, and red circles represent mRNAs. E–I Validation of expression alternations of miRNAs after TBI via qRT-PCR. All the experiments were performed and analyzed in triplicate. (*P < 0.05)

Fig. 4

A The top 20 enriched terms in the GO biological process analysis. B PPI network of mRNAs in ceRNA network. Circles represent protein-coding genes, and edges represent the interactions between two proteins. The purple circles represent top 10 hub genes with highest degrees. C–J Validation of expression differences of DElncRNAs and DEmRNAs via qRT-PCR. C–D P2ry12 and Hes5 expression significantly decrease in injury cortex 24 h post-TBI compared to control cortex. E–F Cxcr2 and Mmp12 expression significantly increase in injury cortex 24 h post-TBI compared to control cortex. G–H lncRNA C030018K13Rik and Gm36823 expression significantly decrease in injury cortex 24 h post-TBI compared to control cortex. I–J lncRNA H19 and Mir155hg expression significantly increase in injury cortex 24 h post-TBI compared to control cortex. All the experiments were performed and analyzed in triplicate. (*P < 0.05, **P < 0.01, ***P < 0.001)