Genome-wide profiling of host-encoded circular RNAs highlights their potential role during the Japanese encephalitis virus-induced neuroinflammatory response

- Background: Japanese encephalitis virus (JEV) is one of the common causes of acute encephalitis in humans..
- In recent years, with the advancement of high-throughput sequencing technology, studies have shown that circRNAs, by competing with endogenous miRNAs, play a vital role in the pathology of CNS diseases.
- Our established competing endogenous RNA (ceRNA) interaction network suggested the correlation of several circRNAs, miRNAs, and mRNAs in regulating the inflammatory response during JEV infection.
- Among the predicted interactions, the correlation between circ_0000220, miR-326-3p, and BCL3/MK2/TRIM25 mRNAs was experimentally validated by knockdown or overexpression of the non-coding RNA entities in cultured mouse microglia.
- The knockdown of circ_0000220 or overexpression of miR-326-3p caused a lower production of JEV-induced inflammatory cytokines..
- Conclusions: Conclusively, our study provides new insights into the host response to JEV infection and proposes the circRNA-targeting therapeutic interventions to rein in Japanese encephalitis..
- According to an estimation cases of JE are reported annually in these regions with 10,000 death, and ~ 50% of the recovered patients undergo permanent neurological sequelae [3].
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- Full list of author information is available at the end of the article Li et al.
- Albeit several mo- lecular mechanisms of JEV pathogenesis have been un- veiled in the last few years, further studies are needed to advance the understanding of JEV pathogenesis..
- Circular RNAs (circRNAs) are identified as a new class of endogenous non-coding RNAs, produced as a result of the back-splicing process [7].
- Anomalous regulation of circRNAs has also been as- sociated with a variety of pathologies, especially cancers and autoimmunity [12, 13].
- Moreover, recent RNA-seq studies showed that the expression of a large number of circRNAs undergoes alteration during the replication of the herpes virus, human immunodeficiency virus, and avian leukosis virus [13–15].
- Albeit miRNAs are known to participate in the JEV pathogenesis, the role of host-encoded circRNAs in me- diating the JEV-induced neuroinflammation is undeter- mined [17, 18]..
- In this study, the genome-wide transcriptomic profil- ing of circRNAs and miRNAs from JEV-infected mice brain tissues was performed using the Illumina sequen- cing.
- A large number of circRNAs and miRNAs were found to be differentially expressed upon JEV infection with their potential role in regulating the JEV-induced neuroinflammation.
- JEV infection alters the expression profile of circRNAs in mice brain.
- To investigate the expression profile of circRNAs in mice brain infected with JEV, the rRNA- and linear RNA-depleted total RNAs extracted from JEV-infected or mock-infected brain tissue samples were employed for genome-wide sequencing.
- 0.05) circRNAs were identified in JEV-infected mice brain samples compared to mock-infected samples..
- 1a) and the volcano plot (Fig.
- 1b) showed the global regulatory pattern of circRNAs upon JEV infection..
- Properties of the circRNAs detected in JEV-infected and mock-infected mice brain.
- Among the 49,632 detected circRNAs, 25,039 were observed to be common between both experimental groups, whereas 11,143 and 12,893 circRNAs were uniquely expressed in the JEV-infected and the mock- infected group, respectively (Fig.
- The distribution of circRNAs length was analyzed and displayed in Fig.
- Most of the circRNAs were observed shorter than 2000 nt, and the number of circRNAs was decreased with the increase in their sequence length.
- As it is reported that most of the circRNAs are generated from the exons [7], we next analyzed the genomic origin of the circRNAs identified in two experimental groups.
- 90% of circRNAs were originated from exons, whereas only a minute fraction of them derived from introns or intergenic regions (Fig.
- 6000 circRNAs detected in each sample were annotated in the circBase (Fig.
- 2e), and the remaining ones.
- The cellular distribution analysis predicted their localization pri- marily in the synapses (Additional file 3: Figure S1A and Additional file 4: Table S3).
- Construction of the circRNA-miRNA-mRNA competing endogenous RNA (ceRNA) network.
- 0.05) upon JEV infection when compared to mock-infected mice sam- ples (Additional file 5: Table S4), as assessed through the R package limma [24].
- The overall expression pattern of miRNAs induced by JEV infection is displayed by the.
- 3a) and the volcano plot (Fig.
- To determine the role of circRNAs in JEV-induced neuroinflammatory response, we chose those differen- tially expressed circRNAs that showed the potential to be involved in the inflammatory response, and subse- quently, constructed the inflammation-related circRNA- miRNA-mRNA ceRNA network (Fig.
- The list of the degree of interaction of circRNA-miRNA-mRNA in the ceRNA network is presented in Table 1..
- Validation of the differentially expressed circRNAs and miRNAs.
- To validate the expression levels of JEV-induced cir- cRNAs and miRNAs found in our sequencing data, we randomly selected six differentially expressed circRNAs and five differentially expressed miRNAs, and validated their expression in JEV-infected or mock-infected mice.
- 1 The expression profile of circRNAs in mice brain during JEV infection.
- The up-regulated circRNAs are presented as red dots and the down-regulated circRNAs are presented as blue dots.
- CircRNA_0000220 positively regulates the production of JEV-mediated inflammatory cytokines and acts as a ceRNA of miR-326-3p.
- To interrogate the role of cirRNAs in the JEV-induced inflammatory response, we selected one of the differen- tially expressed circRNAs, the circ_0000220, predicted to be involved in inflammation.
- 6a), and subsequently, used these cells to evaluate the inflammatory response produced upon JEV infection.
- It was observed that the silencing of circ_0000220 followed by JEV infection re- sulted in a significantly reduced production of inflamma- tory cytokines as assessed by quantitative real-time PCR.
- Considering the circRNAs as miRNAs sponges [16], the up- and down-regulation of circ_0000220 and miR-326-3p, respectively, in our data, the functional en- richment of circ_0000220 and miR-326-3p in the inflam- mation, we speculated that circ_0000220 may positively regulate JEV-mediated inflammatory response via miR- 326-3p.
- To this end, cultured wild-type BV2 cells were transfected with synthetic miR-326-3p mimics or mimics control and then infected with JEV.
- Overexpression of miR-326-3p caused a significantly diminished expression of inflammatory cytokines (Fig.
- Furthermore, ec- topic expression of miR-326-3p led to a markedly down- regulated expression of circ_0000220 and miR-326-3p- target mRNAs (BCL3, MK2, and TRIM25), determined through quantitative real-time PCR and dual-luciferase assay (Fig.
- Taken together, these data suggest that circ_0000220 positively regulates the JEV-induced in- flammatory response, and this observed effect may occur via sponging the miR-326-3p and by subsequently affect- ing the expression of miR-326-3p-target mRNAs..
- 2 The genomic features of circRNAs detected in mice brain during JEV infection.
- b The length distribution statistics of circRNAs.
- c The genomic origin distribution of circRNAs.
- d The distribution of circRNAs on mouse chromosomes.
- e The number of detected circRNAs matched with circRNAs in the circBase database.
- f The proportion of detected circRNAs matched with circRNAs in the circBase database.
- 3 The expression profile of miRNAs in mice brain during JEV infection.
- Thus, it is intriguing to examine the expression profile and interaction of circRNAs with other endogenous RNAs.
- genome-wide RNA-sequencing of circRNAs and miR- NAs from JEV-infected mice brain tissues was per- formed.
- The findings obtained from this study indicated for the first time the intricate regulation of circRNAs during JEV infection and suggests their potential role in JEV neuropathogenesis..
- The expression of circRNAs is also evolutionary conserved from mouse to human, highlighting their functions during brain development [28].
- Given the role of circRNAs in brain physiology and the tendency of JEV to infect the brain, we surmised that circRNAs may participate in regulating the neuroinflammatory response triggered upon JEV infection.
- Our RNA-sequencing data analyses identified 180 circRNAs and 58 miRNAs differ- entially regulated in JEV-infected mice brain tissues.
- The CRISPR-Cas9-mediated knocking down of a selected circRNA, the circ_0000220, in cultured mouse microglial cells validated its function in regulating the production of inflammatory cytokines Table 1 The top 10 degrees of ncRNAs in the inflammation-.
- miR-326-3p 58 Arid5a 9 circ_0000082 12.
- 5 Validation of the sequencing data using the quantitative real-time PCR.
- The relative expression levels of circRNAs (a) and miRNAs (b) measured in JEV-infected or mock-infected mice brains.
- Cultured wild-type BV2 cells were infected with JEV at MOI of 5 for 24 h, and the relative expression level of circRNAs (c) and miRNAs (d) were measured.
- During the transcriptomic analysis of our data, we found that most of the identified circRNAs exhibit ex- onic origin, which is consistent with previous studies [7]..
- Upon comparison of our circRNAs sequences with the circBase database, we found that only ~ 50% of the de- tected circRNAs from each experimental replicate were pairable with the circBase sequences, indicating that half of the circRNAs identified in our study are non- annotated which could be novel or may reflect tissue/cell.
- Thus, our study suggests the need for enrich- ment of the circBase database to advance the research related to circRNAs..
- miR-7 axis controls the proliferation, apoptosis, and in- flammation of the chondrocytes [30].
- 6 CircRNA_0000220 positively regulates the production of JEV-mediated inflammatory cytokines and acts as a ceRNA of miR-326-3p.
- c and d The wild-type BV2 cells were transfected with miR-326-3p mimics or mimics control (miR-NC) for 24 h, followed by infection with JEV at 5 MOI.
- At 24 h post-infection, the relative mRNA expression levels of miR-326-3p (c) and TNF- α , IL-6, IL-1 β , and CCL5 (d) were measured by quantitative real-time PCR.
- e The wild- type BV2 cells were transfected with miR-326-3p mimics or mimics control (miR-NC) for 24 h.
- f HEK-293 T cells were transfected with miR-326-3p mimics or mimics control (miR-NC) together with luciferase reporter plasmid harboring the 3 ′ UTR of circ_0000220, BCL3, MK2, or TRIM25 for 24 h, and followed by measurement of the luciferase activity.
- 0000220 and miR-326-3p were confirmed to regulate the production of inflammatory cytokines during JEV infec- tion, possibly by controlling the expression of BCL3, MK2, and TRIM25 mRNAs.
- In summary, we first unveiled the signatures of circRNAs and related miRNAs in the JEV-infected mice brain using the high throughput RNA-sequencing method.
- The experimental val- idation of the involvement of selected circRNA during the JEV-induced inflammatory response confirmed our pre- dictions.
- This study may advance the understanding of the molecular aspects of the JEV neuropathogenesis.
- Since the roles of circRNAs during viral infections is still at its in- fancy, our study may serve as a resource for future studies..
- JEV infection.
- All animal experiments were performed following the National Institute of Health Guide for the Care and Use of Laboratory Animals, and the experimental protocols were approved by the Research Ethics Committee of College of Veterinary Medicine, Huazhong Agricultural.
- The expression of circRNAs.
- genome.ucsc.edu/cgi-bin/hgTables), and the spliced se- quences of circRNAs were extracted through the circPri- mer software [35].
- In total, the dataset contains 1978 ma- ture miRNA sequences of the mouse.
- analyses of differentially expressed circRNAs were car- ried out by using the R package clusterProfiler v .
- Construction of the circRNA–miRNA–mRNA network To investigate miRNAs interactions with mRNAs and circRNAs, the miRNA binding sites on these RNA en- tities were predicted by the miRanda v3.3a, an algorithm recognizing target sites based on sequence complemen- tarity between miRNAs and the free energy of RNA du- plex [43].
- Differentially expressed circRNAs between JEV-infected and mock-infected groups..
- Summary of the Gene Ontology (A) and KEGG pathway (B) analysis for the parental genes of the differentially expressed circRNA parent genes..
- Differentially expressed miRNAs between JEV-infected and mock-infected groups..
- JEV: Japanese encephalitis virus.
- The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript..
- All the RNA-seq data are available on the available in the NCBI Sequence Read Archive (SRA) repository with the accessions SRP254062 for circRNAs, SRP253986 for miRNAs..
- Review of climate, landscape, and viral genetics as drivers of the Japanese encephalitis virus ecology.
- Crosstalk in competing endogenous RNA networks reveals new circular RNAs involved in the pathogenesis of early HIV infection.
- CircRNAs in the brain.
- Circular RNAs in the mammalian brain are highly abundant, conserved, and dynamically expressed.
- Role of the ciRS- 7/miR-7 axis in the regulation of proliferation, apoptosis and inflammation of chondrocytes induced by IL-1 β

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