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Circular RNA Transcriptomic Analysis of Primary Human Brain Microvascular Endothelial Cells Infected with Meningitic Escherichia coli.

ABSTRACT: With their essential regulatory roles in gene expression and high abundance in the brain, circular RNAs (circRNAs) have recently attracted considerable attention. Many studies have shown that circRNAs play important roles in the pathology of CNS diseases, but whether circRNAs participate in E. coli-induced bacterial meningitis is unclear. We used high-throughput sequencing to analyze the transcriptional profiles of host circRNAs in primary brain microvascular endothelial cells in response to meningitic E. coli. A total of 308 circRNAs were significantly altered, including 140 upregulated and 168 downregulated ones (p < 0.05). Kyoto Encyclopedia of Genes and Genomes (KEGG) and gene ontology enrichment of the parental genes of these altered circRNAs indicated that they are likely to be involved in diverse biological processes via influencing the expression of their parental genes. Coupled with our previous mRNA and microRNA sequencing data, a competing endogenous RNA analysis was performed, and the potential regulatory network was preliminarily constructed and validated. By revealing the transcriptional profiles of the host circRNAs involved in E. coli meningitis, it is envisaged that the novel insight gained into the regulatory mechanisms of circRNAs in the development of bacterial meningitis will lead to better understanding of how to prevent and treat bacterial CNS infections.

SUBMITTER: Yang R

PROVIDER: S-EPMC6258830 | biostudies-other | 2018 Dec

REPOSITORIES: biostudies-other

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Circular RNA Transcriptomic Analysis of Primary Human Brain Microvascular Endothelial Cells Infected with Meningitic Escherichia coli.

Yang Ruicheng R Xu Bojie B Yang Bo B Fu Jiyang J Liu Lu L Amjad Nouman N Cai Aoling A Tan Chen C Chen Huanchun H Wang Xiangru X

Molecular therapy. Nucleic acids 20181026

With their essential regulatory roles in gene expression and high abundance in the brain, circular RNAs (circRNAs) have recently attracted considerable attention. Many studies have shown that circRNAs play important roles in the pathology of CNS diseases, but whether circRNAs participate in E. coli-induced bacterial meningitis is unclear. We used high-throughput sequencing to analyze the transcriptional profiles of host circRNAs in primary brain microvascular endothelial cells in response to men ...[more]

PMID: 30497055

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Project description:BACKGROUND:Bacterial meningitis remains a big threat to the integrity of the central nervous system (CNS), despite the advancements in antimicrobial reagents. Escherichia coli is a bacterial pathogen that can disrupt the CNS function, especially in neonates. E. coli meningitis occurs after bacteria invade the brain microvascular endothelial cells (BMECs) that form a direct and essential barrier restricting the entry of circulating microbes and toxins to the brain. Previous studies have reported on several cellular proteins that function during meningitic E. coli infections; however, more comprehensive investigations to elucidate the potential targets involved in E. coli meningitis are essential to better understand this disease and discover new treatments for it. METHODS:The isobaric tags for relative and absolute quantification (iTRAQ) approach coupled with LC-MS/MS were applied to compare and characterize the different proteomic profiles of BMECs in response to meningitic or non-meningitic E. coli strains. KEGG and gene ontology annotations, ingenuity pathways analysis, and functional experiments were combined to identify the key host molecules involved in the meningitic E. coli-induced tight junction breakdown and neuroinflammatory responses. RESULTS:A total of 13 cellular proteins were found to be differentially expressed by meningitic E. coli strains PCN033 and RS218, including one that was also affected by HB101, a non-meningitic E. coli strain. Through bioinformatics analysis, we identified the macrophage migration inhibitory factor (MIF), granzyme A, NF-?B signaling, and mitogen-activated protein kinase (MAPK) pathways as being biologically involved in the meningitic E. coli-induced tight junction breakdown and neuroinflammation. Functionally, we showed that MIF facilitated meningitic E. coli-induced production of cytokines and chemokines and also helped to disrupt the blood-brain barrier by decreasing the expression of tight junction proteins like ZO-1, occludin. Moreover, we demonstrated the significant activation of NF-?B and MAPK signaling in BMECs in response to meningitic E. coli strains, which dominantly determined the generation of the proinflammatory cytokines including IL-6, IL-8, TNF-?, and IL-1?. CONCLUSIONS:Our work identified 12 host cellular targets that are affected by meningitic E. coli strains and revealed MIF to be an important contributor to meningitic E. coli-induced cytokine production and tight junction disruption, and also the NF-?B and MAPK signaling pathways that are mainly involved in the infection-induced cytokines production. Characterization of these distinct proteins and pathways in BMECs will facilitate further elucidation of meningitis-causing mechanisms in humans and animals, thereby enabling the development of novel preventative and therapeutic strategies against infection with meningitic E. coli.

Project description:Escherichia coli K1 is the most common gram-negative organism causing neonatal meningitis, but the mechanism by which E. coli K1 crosses the blood-brain barrier is incompletely understood. We have previously described the cloning and molecular characterization of a determinant, ibeA (also called ibe10), from the chromosome of an invasive cerebrospinal fluid isolate of E. coli K1 strain RS218 (O18:K1:H7). Here we report the identification of another chromosomal locus, ibeB, which allows RS218 to invade brain microvascular endothelial cells (BMEC). The noninvasive TnphoA mutant 7A-33 exhibited <1% the invasive ability of the parent strain in vitro in BMEC and was significantly less invasive in the central nervous system in the newborn rat model of hematogenous E. coli meningitis than the parent strain. The TnphoA insert with flanking sequences was cloned and sequenced. A 1,383-nucleotide open reading frame (ORF) encoding a 50-kDa protein was identified and termed ibeB. This ORF was found to be 97% identical to a gene encoding a 50-kDa hypothetical protein (p77211) and located in the 13-min region of the E. coli K-12 genome. However, no homology was observed between ibeB and other known invasion genes when DNA and protein databases in GenBank were searched. Like the TnphoA insertion mutant 7A-33, an isogenic ibeB deletion mutant (IB7D5) was unable to invade BMEC. A 7. 0-kb locus containing ibeB was isolated from a LambdaGEM-12 genomic library of E. coli RS218 and subcloned into a pBluescript KS vector (pKS7-7B). pKS7-7B was capable of completely restoring the BMEC invasion of the noninvasive TnphoA mutant 7A-33 and the ibeB deletion mutant IB7D5 to the level of the parent strain. More importantly, the ibeB deletion mutant IB7D5 was fully complemented by pFN476 carrying the ibeB ORF (pFN7C), indicating that ibeB is required for E. coli K1 invasion of BMEC. Taken together, these findings indicate that several E. coli determinants, including ibeA and ibeB, contribute to crossing of the blood-brain barrier.

Project description:Circular RNAs (circRNAs) are generated by head-to-tail splicing and are ubiquitously expressed in all multicellular organisms. Their important biological functions are increasingly recognized. Cerebral ischemia reperfusion injury-induced brain microvascular endothelial cell dysfunction is an initial stage of blood-brain barrier disruption. The expression profile and potential function of circRNAs in brain microvascular endothelial cells is unknown. Rat brain microvascular endothelial cells were extracted and cultured in glucose-free medium for 4 hours with 5% CO2 and 95% N2, and the medium was then replaced with complete growth medium for 6 hours. The RNA in these cells was then extracted. The circRNA was identified by Find_circ and CIRI2 software. Functional and pathway enrichment analysis of genes that were common to differentially expressed mRNAs and circRNA host genes was performed by the Database for Annotation, Visualization and Integrated Discovery Functional Annotation Tool. Miranda software was used to predict microRNAs that were potentially spong-ed by circRNAs. Furthermore, cytoscape depicted the circR-NA-microRNA interaction network. The results showed that there were 1288 circRNAs in normal and oxygen-glucose deprived/recovered primary brain microvascular endothelial cells. There are 211 upregulated and 326 downregulated differentially expressed circRNAs. The host genes of these differentially expressed circRNAs overlapped with those of differentially expressed mRNAs. The shared genes were further studied by functional enrichment analyses, which revealed that circRNAs may contribute to calcium ion function and the cyclic guanosine 3',5'-monophosphate (CAMP) dependent protein kinase (PKα) signaling pathway. Next, quantitative reverse transcription polymerase chain reaction assays were performed to detect circRNA levels transcribed from the overlapping host genes. Eight out of the ten circRNAs with the highest fold-change identified by sequencing were successfully verified. Subsequently, the circRNA-microRNA interaction networks of these eight circRNAs were explored by bioinformatic analysis. These results demonstrate that altered circRNAs may be important in the pathogenesis of cerebral ischemia reperfusion injury and consequently may also be potential therapeutic targets for cerebral ischemia diseases. All animal experiments were approved by the Chongqing Medical University Committee on Animal Research, China (approval No. CQMU20180086) on March 22, 2018.

Dataset Information

Circular RNA Transcriptomic Analysis of Primary Human Brain Microvascular Endothelial Cells Infected with Meningitic Escherichia coli.

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Circular RNA Transcriptomic Analysis of Primary Human Brain Microvascular Endothelial Cells Infected with Meningitic Escherichia coli.

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