Altered long noncoding RNA profile after intracerebral hemorrhage

Abstract

Objective: We investigated the expression pattern of long noncoding RNAs (lncRNA) and messenger RNAs (mRNA) from two different intracerebral hemorrhage (ICH) rat models, and performed gene ontology and gene/protein interaction analyses.

Methods: We harvested hemorrhagic brain 1, 3, and 7 days after ICH induction by stereotactic collagenase injection. We performed microarray analyses with Agilent array platform to compare the expression of lncRNA and mRNAs from hemorrhagic and normal brains. The RNA expression patterns were also examined from the autologous blood injection ICH model at days 1 and 3, and significantly altered lncRNAs from two ICH models were validated by quantitative reverse transcriptase-polymerase chain reaction. Gene ontology analysis and pathway analysis were performed with differentially expressed mRNAs after ICH. Gene and protein interaction analysis was performed to elucidate the functional role of upregulated lncRNA in neuronal damage.

Results: Among the 13,661 lncRNAs studied, 83, 289, and 401 lncRNAs were significantly elevated after 1, 3, and 7 days after collagenase-induced ICH, respectively. NR_027324, or H19, was the most upregulated lncRNA after 1 day from the two ICH models and its elevation persisted until the 7th day. Gene ontology analysis revealed that immune-related biological processes such as immune response, immune system process, and defense response were upregulated from both ICH models. Gene and protein interaction study demonstrated that NR_027324 was closely related to the type I interferon signaling pathway.

Interpretation: This study illustrates the dynamic expression pattern of the lncRNA profile following ICH, and that H19 is the most consistently upregulated lncRNA after ICH.

Figure 1

Overall study design and the expression pattern of RNAs in a collagenase‐induced intracerebral hemorrhage model. (A) The flow diagram summarizes the present study scheme, which analyzes the expression patterns of lncRNA and mRNA from two different ICH models followed by gene ontology analysis, pathway analysis, and gene/protein interaction analysis. (B) Temporal trends of RNA expression reveal that both lncRNAs and mRNAs showed robust expression change as time passes from collagenase induced model. Comparison with blood injection model revealed that 53 lncRNAs and 370 mRNAs were concordantly upregulated at day 1, and 7 lncRNAs and 6 mRNAs concordantly downregulated (black number = the number of lncRNAs, white number in bracket = the number of mRNAs). (C) Volcano plots show an increasing trend in significantly altered lncRNAs with more than a twofold change and P < 0.05 from collagenase‐induced hemorrhage model.

Figure 2

The expression levels of lncRNAs after intracerebral hemorrhage. (A) Heatmap analysis of significantly altered lncRNAs expression patterns from the collagenase‐induced hemorrhagic model showed a distinct temporal pattern of lncRNA. (B) The intergenic lncRNA subfamily is the most up/downregulated group among the six lncRNA subfamilies throughout the three time points. (C) The most elevated lncRNA in the ICH model was NR_027324, which remained elevated until the 7th day after ICH. Among several downregulated lncRNAs, only ENSRNOT00000076904 was consistently found to be decreased by qRT‐PCR. The black bar represents fold changes after collagenase induced ICH on the reference of normal healthy brain derived from microarray analysis; the white bar represents those fold changes derived from qRT‐PCR. Relative fold changes at each time point compared to normal brain from microarray analysis are illustrated in Tables 1 and 2. Relative fold changes (P values) from qRT‐PCR are illustrated in Table S4. Each group consists of four individual rat brains; * stands for P < 0.05 by Mann–Whitney U‐test.

Figure 3

Pathway analysis from two different intracerebral hemorrhage models. (A) Pathway analysis with increased RNAs in the collagenase‐induced ICH model after 1 day showed Herpes simplex infection as the most significantly involved pathway, which includes 34 genes. From the blood injection ICH model, the most significantly involved pathway was antigen processing and presentation which included 27 genes. (B) Three days after ICH, phagosome was the most enriched pathway in the collagenase‐induced model and Herpes simplex infection in the blood‐injected model. The black bars depict enrichment scores from the collagenase‐induced ICH model, and the white bars represent those values from the blood injection ICH model. The right‐sided numbers are gene numbers directly associated with the listed pathway.

Figure 4

H19 related gene/protein interaction study. (A) Three‐dimensional gene interaction viewer and database (3DIV) analysis revealed that H19 is located in chromosome 11 and related to many protein coding sequences with six topologically associating domains. Those protein sequences which were significantly elevated in the intracerebral hemorrhage model were marked with a red box. (B) STRING analysis showed that the elevated proteins related to H19 after ICH consist of two main groups; one harboring interferon regulatory factor 7 and interferon‐induced transmembrane protein 1–3, which are associated with the type I interferon response pathway, and the other group loosely related to cell survival and proliferation.