High-Throughput Sequencing and Co-Expression Network Analysis of lncRNAs and mRNAs in Early Brain Injury Following Experimental Subarachnoid Haemorrhage

Figures

• 

  Figure 1. Induced SAH model and quality assessment of the gene expression levels in the control group and SAH group. (A) Images of mouse brains between in the control and SAH groups. There were no blood clots in the sham-operated mouse brains, while a thick blood clot was found around the arterial circle of Willis after the SAH operation. (B) As shown in the box plot, almost all of the quality scores (−10*log10 (P-value)) were approximately 40 in all samples. (C) The Gaussian distribution curve indicates the average GC content in all reads of one sample, the red line represents actual content, and the blue line represents the theoretical content. The real GC content of all samples showed a Gaussian distribution, which nearly agreed with the theoretical content. SAH, subarachnoid haemorrhage; GC, guanine-cytosine.

• 

  Figure 2. Representative mapping of sequence reads in the Control and SAH groups. Overall coverage of the detected reads was mapped to mouse chromosomes.

• 

  Figure 3. Differentially expressed lncRNAs and mRNAs between mouse brains at 24 h after SAH and in control groups. (A) The volcano plot illustrates the differentially expressed genes (including lncRNAs and mRNAs) between the control and SAH groups, with red dots representing differentially expressed genes (log2fold change > 1; q < 0.05). (B, C) Chromosome distribution (B) and average length (C) of the differentially expressed lncRNAs and mRNAs. (D,E) The hierarchical cluster analysis generated heat maps of the top 100 significant differential expressed lncRNAs (D) and mRNAs (E) in the mouse brains at 24 h following SAH or in control groups (log2fold change >1.5; q < 0.05). Colour represents the log10 (RPKM + 1) value, with red indicating upregulated genes and green indicating downregulated genes. The legend represents the standard normal distribution of log10 (RPKM + 1) values, and the blue line in the legend indicates the gene amounts in this colour area. SAH, subarachnoid haemorrhage; lncRNAs, long non-coding RNAs; mRNAs, messenger RNAs; RPKM, expected number of Reads Per Kilobase of transcript sequence per Millions base pairs Sequenced.

• 

  Figure 4. GO enrichment and KEGG pathway analysis of differentially expressed genes in SAH brains. (A) BP, (B) CC and (C) MF presented the top 10 significance terms of GO enrichment analysis (P < 0.05 and FDR < 0.05). (D) The top 20 KEGG pathways of significantly differentially expressed genes between the control and SAH groups (P < 0.05 and FDR < 0.05). GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; BP, biological processes; CC, cellular components; MF, molecular functions; FDR, false discovery rate; DE, differentially expressed.

• 

  Figure 5. LncRNA/mRNA transcripts co-expression network after SAH. (A) The network represents the co-expression correlations between the significantly differentially expressed mRNA and lncRNA transcripts (|COR| > 0.95, P < 0.05). Circles indicate lncRNA transcripts and triangles indicate mRNA transcripts. Solid lines indicate positive correlations, and dashed lines indicate negative correlations. Red represents upregulated, and green represents downregulated. (B) KEGG analysis suggested that lncRNA-coexpressed mRNAs were mainly targeted to the Toll-like receptor signaling pathway (http://www.genome.jp/kegg)5455. Blue represents significant genes in this pathway of SAH, and pentagrams represent significant lncRNA-coexpressed genes.

• 

  Figure 6. Gene expression profile. (A,B) Subcellular localization pattern of fantom3_F730004F19 by RNA FISH staining. A significantly higher expression of fantom3_F730004F19 in treated BV-2 microglia cells was estimated, and fantom3_F730004F19 was predominantly expressed in the nucleus. (C) Cellular localization pattern of fantom3_F730004F19 by qRT-PCR. Although there was no statistical difference between BV2 and HT22 cells, the level of fantom3_F730004F19 present a higher in BV2 cell line, lesser extent in HT22 cells and MOPC cells, and not detected in MA cells. (D) Comparison between sequencing and qRT-PCR analysis of 5 randomly selected lncRNAs. Positive values refer to upregulation, and negative values refer to downregulation. β-actin was used to normalize the expression of samples. Red representes RNA-seq data, and blue representes qRT-PCR. The bars represent standard error of the mean (SEM). The qRT-PCR results were closely correlated with the sequencing data (P < 0.05). (E) The silencing efficiency of the lentivirused on fantom3_F730004F19 in BV-2 microglia cells. Lentivirus-50305 (KD1) and lentivirus-50307 (KD3) significantly inhibited fantom3_F730004F19 expression (P < 0.05 versus…

• 

  Figure 7. Silencing fantom3_F730004F19 attenuated the inflammatory response. (A–F) Inhibition of fantom3_F730004F19 reduced the expression of CD14 and TLR4 in BV-2 cells following LPS treatment. Protein expression was shown to be completely depleted by both western blot analysis (A–C) and immunofluorescence staining (D–F) (full-length blots/gels are presented in Supplementary Figure S5). Although the inhibitory effect was not obvious in the normal condition, silencing fantom3_F730004F19 significantly reduced the expression of CD14 and TLR4 protein levels after LPS treatment (#P < 0.05 and ##P < 0.01 versus the NC LPS-treated group). (G–I) Inhibition of fantom3_F730004F19 attenuated inflammatory cytokines release. Silence fantom3_F730004F19 significantly reduced the release of IL-1β (G), IL-6 (H) and TNF-α (I) after LPS treatment (#P < 0.05 and ##P < 0.01 versus the NC LPS-treated group). N: normal condition group; LPS: lipopolysaccharide treated; Blank: non-transfected cells; NC: negative control lentivirus transfected cells; KD: the lncRNA fantom3_F730004F19 lentivirus transfected cells.