Microarray Analysis Identifies the Potential Role of Long Non-Coding RNA in Regulating Neuroinflammation during Japanese Encephalitis Virus Infection

Abstract

Japanese encephalitis virus (JEV) is the leading cause of epidemic encephalitis worldwide. JEV-induced neuroinflammation is characterized by profound neuronal cells damage accompanied by activation of glial cells. Albeit long non-coding RNAs (lncRNAs) have been emerged as important regulatory RNAs with profound effects on various biological processes, it is unknown how lncRNAs regulate JEV-induced inflammation. Here, using microarray approach, we identified 618 lncRNAs and 1,007 mRNAs differentially expressed in JEV-infected mice brain. The functional annotation analysis revealed that differentially regulated transcripts were predominantly involved in various signaling pathways related to host immune and inflammatory responses. The lncRNAs with their potential to regulate JEV-induced inflammatory response were identified by constructing the lncRNA-mRNA coexpression network. Furthermore, silencing of the two selected lncRNAs (E52329 and N54010) resulted in reducing the phosphorylation of JNK and MKK4, which are known to be involved during inflammatory response. Collectively, we first demonstrated the transcriptomic landscape of lncRNAs in mice brain infected with JEV and analyzed the coexpression network of differentially regulated lncRNAs and mRNAs during JEV infection. Our results provide a better understanding of the host response to JEV infection and suggest that the identified lncRNAs may be used as potential therapeutic targets for the management of Japanese encephalitis.

Keywords: Japanese encephalitis virus; brain; long non-coding RNAs; microarray; neuroinflammation.

Figure 1

Validation of successful Japanese encephalitis virus (JEV) infection in mice brain. For the infection group, mice were inoculated intracranially with 200 PFU JEV in 20 µl Dulbecco’s modified Eagle’s medium (DMEM), whereas mice belonging to the control group were injected intracranially with equal volume of DMEM. On day 5 postinfection, mice from both groups were euthanized, and brain samples were collected. (A) Quantification of infectious virus titer in mice brain by plaque assay. (B) Expression analysis of JEV NS5 by Western blot. (C) Hematoxylin–eosin (H&E) staining of brain sections to examine the histopathological changes. Scale bar = 200 µm. (D) Immunohistochemistry (IHC) analysis of brain sections to determine the activation of microglia and astrocytes using anti-ionized calcium-binding adapter molecule (anti-IBA) and antiglial fibrillary acidic protein (anti-GFAP) antibodies, respectively. Cell death was examined by terminal deoxynucleotidyl transferased UTP nick end labeling (TUNEL) assay. Scale bar = 50 μm. Integrated option density (IOD) analysis was performed to quantify the results of staining. Data represent the ranges observed from three sections from three mice in each group.

Figure 2

Expression profiles of mRNAs and long non-coding RNAs (lncRNAs) in Japanese encephalitis virus (JEV)-infected and mock-infected mouse brain. (A,B) Scatter plot to show the variation in mRNA (A) and lncRNA (B) expressions. The values of X and Y axes are the mean normalized signal values in each group (log 2-scaled). (C,D) Hierarchical clustering of differently expressed mRNAs (C) and lncRNAs (D) between the two groups. Red color indicates relatively higher expression, whereas green color indicates relatively lower expression.

Figure 3

The coexpression network of significantly regulated long non-coding RNAs (lncRNAs) and mRNAs. (A) lncRNA-mRNA network. (B) lncRNA-lncRNA network. Red color specifies selected upregulated lncRNAs, and blue color indicates downregualted lncRNAs. Yellow color denotes mRNAs, whereas pink designates other lncRNAs. (C) Detection of indicated mRNAs using quantitative real-time PCR in the sample pulled down by biotin-labeled E52329. (D) Detection of indicated mRNAs using quantitative real-time PCR in the sample pulled down by biotin-labeled N54010.

Figure 4

Validation of the microarray data using quantitative real-time PCR. (A,B) Mice were infected with Japanese encephalitis virus (JEV) or mock infected with Dulbecco’s modified Eagle’s medium (DMEM), and brain samples were collected at 5 days postinfection for analysis of selected long non-coding RNAs (lncRNAs). The levels of upregulated lncRNAs (A) and downregulated lncRNAs (B) were detected by quantitative real-time PCR. All data are representative of three independent experiments.

Figure 5

Detection of selected long non-coding RNAs (lncRNAs) in BV2 cells. (A) BV2 cells were infected with Japanese encephalitis virus (JEV) at multiplicity of infection (MOI) of 5 for the indicated times, and titers of infectious virus in the culture supernatants were determined by plaque assay. (B) BV2 cells were infected with JEV at MOI of 5 for 2 h. At 12, 24, and 36 h postinfection, cells were fixed and subjected to indirect immunofluorescence to detect JEV NS5 protein (green). Nuclei are shown by 6-diamidino-2-phenylindole dihydrochloride (DAPI, blue) staining. The images of the cells were acquired with a fluorescence microscope (Zeiss) with 20× magnification. Scale bar = 50 µm. (C) BV2 cells were infected with JEV at MOI of 5 for 2 h. The expression levels of lncRNAs NONMMUT054010 and ENSMUST00000152329 were detected by quantitative real-time PCR. All data are representative of three independent experiments.

Figure 6

Selected long non-coding RNAs (lncRNAs) regulate Japanese encephalitis virus (JEV)-mediated production of inflammatory cytokines. (A) BV2 cells were transfected with siE52329, siN54010, or their non-specific control small-interfering RNA (siRNA, final concentration, 50 nM) for 24 h, and then expression levels of lncRNA NONMMUT054010 and ENSMUST00000152329 were detected by quantitative real-time PCR. (B,C) BV2 cells were transfected with siE52329, siN54010, or their non-specific control siRNA (final concentration, 50 nM) for 24 h, and then infected with JEV at multiplicity of infection (MOI) of 5 for 24 h. The mRNA (B) and protein (C) levels of tumor necrosis factor alpha (TNF-α), interleukin (IL)-6, and IL-1β were analyzed by quantitative real-time PCR and ELISA, respectively. IFN-β mRNA level was determined by quantitative real-time PCR. (D) BV2 cells were transfected with siE52329, siN54010, or their non-specific control siRNA (final concentration, 50 nM) for 24 h, and then infected with JEV at MOI of 5 for the indicated times. The titers of infectious virus in the culture supernatants were determined by plaque assay. Data were expressed as means ± SEM from three independent experiments. One-way ANOVA with subsequent Bonferroni’s multiple comparison. All data are representative of three independent experiments.

Figure 7

Selected long non-coding RNAs (lncRNAs) regulate MKK4 phosphorylation and mediate Japanese encephalitis virus (JEV)-induced inflammatory response. Immunoblot analysis of MKK4, JNK, and IκBα phosphorylation in JEV-infected cells and mice brain. BV2 cells were transfected with siE52329, siN54010, or their non-specific control small-interfering RNA (siRNA, final concentration, 50 nM) for 24 h, and then infected with JEV at multiplicity of infection (MOI) of 5 for 6 h (A) or 12 h (B). BALB/c mice were inoculated intracranially with 20 µl of phosphate-buffered saline (PBS) or 50 plaque-forming units (PFU) of JEV in 20 µl of PBS, and the brain tissues were collected at 5 days postinfection. The extracts from BV2 cells and mouse brain homogenate were subjected to Western blot analysis with antibodies recognizing the indicated proteins. Levels of phosphorylated MKK4, IκBα, and JNK of BV2 cells were quantified by ImageJ software (NIH, Bethesda, MD, USA) and normalized to the amount of β-tubulin. After the quantification, one-way ANOVA analysis was performed (*P < 0.05, **P < 0.01, ***P < 0.001). Data are representative of three independent experiments.