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. 2024 Jan 5:14:1165378.
doi: 10.3389/fmicb.2023.1165378. eCollection 2023.

Circular RNA network plays a potential antiviral role in the early stage of JEV infection in mouse brain

Mengli Chen  1   2 , Lei Kang  1   2 , Tong Zhang  1 , Jiayang Zheng  1 , Dishi Chen  3 , Donghua Shao  1 , Zongjie Li  1 , Beibei Li  1 , Jianchao Wei  1 , Yafeng Qiu  1 , Xiuli Feng  2 , Zhiyong Ma  1 , Ke Liu  1
Affiliations

Circular RNA network plays a potential antiviral role in the early stage of JEV infection in mouse brain

Mengli Chen et al. Front Microbiol. .

Abstract

Japanese encephalitis is one of the most important insect-borne infectious disease with public health concern. The virus can break the blood-brain barrier and cause death or long-term sequela in infected humans or animals. Viral encephalitis is an important clinical feature of JEV infection. In recent studies, CircRNAs and related ceRNAs data illustrated the regulative role in many aspects of biological process and disease duration. It is believed that CircRNA regulates JEV infection in a ceRNA-dependent mechanism. In this study, brain tissues of experimental mice were sequenced and analysised. 61 differentially expressed circRNAs, 172 differentially expressed miRNAs and 706 differentially expressed mRNAs were identified by RNA-Sequencing and statistical analysis. CX3CR1 was determined as a key host factor impact JEV infection by microRNA interference measurement. CX3CR1 interaction network indicated circStrbp/miR709/CX3CR1 as a functional regulation axis. Further sequencing in BV2 cell shown CX3CR1 is a special target of miR-709 only during JEV infection. In summary, our study presented a new ceRNA pathway that impact JEV infection in vivo and in vitro, which could be a therapeutic target to fight against JEV.

Keywords: Japanese encephalitis; Japanese encephalitis virus; ceRNA; circular RNA; miR-709.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
The characteristics of circRNA during JEV infection in mouse brain. (A) Viral fold change in mouse brain. JEV (4 × 106 PFU) was injected intraperitoneally. The viral load was determined by detecting JEV envelope (E) gene and normalized with mouse GAPDH. All fold changes were measured by Real-time PCR. Data are shown as the mean ± standard error (SEM). Six mice for each group were detected, and the experiments were repeated three times. (B,C) Heat map (B) and Volcanic map (C) of differently expressed circRNAs enriched in mouse brain. Red color represents up-regulations and other colors represent down-regulatons. Difference were calculated as significant if a fold change >2 and a p value <0.05. (D) The genomic distribution of differently expressed circRNAs. Exonic, intron and unknown distribution were measured. (E) The length distribution of differently expressed circRNAs. Length range from 100 to more than 10,000 were analysed.
Figure 2
Figure 2
Network of differently expressed ceRNA. (A). circRNA (green)-miRNA (blue)-mRNA (purple) interaction network. CircRNA is inside the circle with green frame, miRNA is in the middle of the circle with blue frame, mRNA is outside the circle with purple frame. (B,C). Validation of circRNAs, miRNAs and mRNAs expression in infected mouse. Mice were infected or mock infected with JEV. Mouse brain was sampled in the indicated time points, then representative circRNAs, miRNAs and mRNAs were detected by qPCR.
Figure 3
Figure 3
Network of CX3CR1 related ceRNA. (A) circRNA (green)-miRNA (blue)-mRNA (purple) interaction network. (B) Validation of CX3CR1 related circRNAs, miRNAs and mRNAs expression in infected mouse. Each genes were measured by qPCR using 2−ΔΔCt method and then normalized by a mock sample at day 5 to calculate fold change. (C) RNA interference efficiency of circStrbp. CircStrbp gene was measured by qPCR with primers target to linkage sequences of circStrbp. (D) Detection of miR-709 levels in BV2 cell after circStrbp knock-down. (E) Detection of CX3CR1 mRNA levels in BV2 cell after circStrbp knock-down. (F) Detection of JEV mRNA levels in BV2 cell after circStrbp knock-down. All experiments were measured by qPCR using 2−ΔΔCt method and then normalized by a mock. All experiments were repeated three times. Data are shown as means ± standard errors of the means.
Figure 4
Figure 4
miR-709 targeted CX3CR1 during JEV infection. (A) JEV infection curve in BV2 cell. Three dose of JEV (MOI = 0.5, 1, 5) were inculated into BV2 cell, the infected or mock infected cells were collected at different time points. (B–D) miR-709 dynamic response after JEV infection in BV2 cell with different MOI. The infected or mock infected cells were collected at different time points, the relative expression of miR-709 was measured by qPCR. Data are shown as the mean ± standard error (SEM). The experiments were repeated three times. (E) JEV replication after miRNA transfection. miR-709, miR-6992-5p or NC were transfected in BV2 cell, JEV was inoculated at 24 h after transfection. 24 h post infection, cell samples were collected. (F) CX3CR1 mRNA changes after miRNA transfection. miR-709, miR-6992-5p or NC were transfected in BV2 cell, 24 h post transfection, cell samples were collected. *, p < 0.05 compared between groups. Data are shown as the mean ± standard error (SE). The experiments were repeated for three times. (G) Down-regulation of mRNAs of miR-709 transfected uninfected-BV2 cell. (H) Down-regulation of mRNAs of miR-709 transfected infected-BV2 cell. Red color represents up-regulations and blue colors represent down-regulatons. Difference were calculated as significant if a fold change >2 and a p value <0.05.
Figure 5
Figure 5
Mode pattern of the effect of CirStrbp-miR-709-CX3CR1 network on the JEV infection in mouse brain.
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Grants and funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This study was supported by the Shanghai Natural Science Fund (no. 21ZR1477100), Shanghai Veterinary Research Institute 2022 Fund.

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