基于RNA-seq的炎症小鼠肺和脑组织的转录组分析
发布时间：2020-11-13 10:39
病毒感染通常具有器官特异性,而一些病毒可同时感染多个器官并导致炎症性疾病。呼吸和中枢神经系统(CNS)的病毒感染是公共卫生中最关键的问题,包括麻疹病毒和疱疹病毒等在内的一些已知的嗜神经病毒,通常与中枢神经系统疾病有关。然而,包括人类呼吸道合胞体病毒、流感病毒、冠状病毒和人化生病毒在内的一些呼吸道病毒已成为中枢神经系统疾病的相关媒介。对于病毒感染多个组织器官,不同组织器官针对同一病毒感染的不同炎症反应的作用或机制是免疫学的关键问题,有待进一步研究。因此,本研究旨在探寻病毒模拟物处理后小鼠大脑和肺组织炎症差异的机制。本研究利用病毒dsRNA模拟物poly(I:C)(polyinosinic acid-polycytidylic acid)刺激小鼠,阐述了病毒感染器官后炎症或免疫的分子机制。本研究通过检测细胞因子Il-1β、Tnf-α的表达,发现了大脑和肺组织中不同的炎症状态。本研究对小鼠肺和脑组织炎症进行转录组、蛋白相互作用和干扰素分析等后续实验,阐述了不同炎症状态背后的原因。本研究在肺中鉴定了629个差异表达基因(DEGs),在脑组织中鉴定了137个DEGs,其中有一些基因重叠。这些DEGs大部分是干扰素刺激基因(ISGs)。转录组GO分析显示,大脑和肺组织的DEGs在细胞粘附、炎症、病毒防御反应和先天免疫等方面起着关键作用。就DEGs和ISGs的数量而言,病毒模拟物在肺中的炎症反应较脑组织剧烈。本研究通过蛋白相互作用和信号通路关联分析,分别在肺组织和脑组织中鉴定出29个和14个免疫相关的hub基因。肺组织中与免疫相关的前五大hub基因为Itgam、Il-1b、Il-7、Cdkn1a、Myc,脑组织中与免疫相关的hub基因为Irf7、Oas2、Ifit1、Isg15、Rsad2。除这些hub基因外,包括病毒核酸受体2'-5'-寡腺苷酸合成酶(OAS)在内的不同ISGs在病毒感染时调节炎症和免疫反应,其表达具有一定的组织特异性。此外,序列特异性病毒和细菌核酸受体Tlr13在poly(I:C)处理组中上调,刺激小鼠肺及其与许多其他炎症调节蛋白的相互作用,反映出其在调节肺炎症反应中的关键作用。有趣的是,核糖体蛋白Rpl29和细胞表面肝素结合蛋白在大脑中上调,而在肺中下调。siRNA抑制RPL29,导致炎性细胞因子IL-1β、TNF-α高表达。本研究首次证实Rpl29参与炎症反应,其组织特异性和不同表达水平造成了调控病毒感染时肺和脑组织炎症反应的差异。MiR22可以在翻译后水平上调控许多生理事件,其中包括炎症。在病毒模拟物处理的组织和Rpl29阳性共表达的不同细胞系中,miR22的表达也存在差异。本研究还发现,Rpl29通过调节Fos-B和c-Fos,以在病毒感染后调节不同组织中miR22的表达。综上所述,本研究表明,Rpl29与不同的hub基因Tlr13、mi R22和ISGs一起,是机体特异性炎症或免疫反应的关键协调因子,借此可以调节不同组织中mi R22的表达差异。总之,该项研究的发现将有助于为探索与炎症、免疫相关多种严重病毒疾病的治疗策略提供新见解。
【学位单位】：华中农业大学
【学位级别】：博士
【学位年份】：2019
【中图分类】：R373
【文章目录】：
摘要
Abstract
Table of Abbreviations
Chapter1:Introduction
1.1 Inflammation and its mechanisms
1.1.1 PRR activation
1.1.2 Inflammatory pathways activation
1.1.3 Inflammatory markers
1.1.4 Cell types associated with inflammatory responses
1.2 Inflammatory and immune responses against virus infection
1.2.1 Production of inflammatory cytokines,IFNs,and ISGs
1.2.2 The inflammatory cytokines mediated antiviral response
1.2.3 IFNs and ISGs meditated antiviral responses
1.2.4 IFNs desensitization
1.2.5 The antiviral effects of other ISGs
1.3 Poly（I:C）,the viral mimic
1.4 RNA-seq based transcriptome profiling
1.4.1 Next-generation sequencing
1.4.2 Gene ontology and pathway analysis
1.5 Biological network
1.5.1 Transcriptomic integration in protein-protein interaction network
1.6 Interferome analysis
1.7 Toll-like receptors
1.7.1 Structure of TLRs
1.7.2 Signaling pathways of TLRs
1.7.3 Nucleic acid-TLR complexes
1.7.4 Toll-like receptor 13
1.8 Ribosome protein L29
1.9 MicroRNA22 in inflammation and immunity
1.10 Study proposal
Chapter2:Materials and Methods
2.1 Experimental animal
2.2 Experimental cells
2.3 Reagents
2.4 Solution preparation
2.4.1 Preparation of phosphate buffer（1XPBS）
2.4.2 Preparation of SDS-PAGE related solutions
2.5 Animal experiment
2.6 Cell culture and treatment
2.7 RNA isolation
2.8 Removal of genomic DNA and cDNA synthesis for qRT-PCR
2.9 Quantitative real-time polymerase chain reaction
2.10 RNA library preparation and sequencing
2.10.1 Removal of DNA contamination and detection of total RNA quality
2.10.2 mRNA purification and fragmentation
2.10.3 First strand cDNA synthesis
2.10.4 Second strand cDNA synthesis
2.10.5 Purification the double-stranded cDNA
2.10.6 End prep of cDNA library/3`adenylation
2.10.7 Adaptor ligation
2.10.8 Purification of the library
2.10.9 PCR enrichment of adaptor ligated cDNA
2.11 Validation of library
2.12 Differential expression analysis
2.13 KOG& GO enrichment analysis
2.14 Interferome analysis
2.15 KEGG pathway enrichment analysis
2.16 Functional protein association networks construction
2.17 Western blotting
2.17.1 Preparation of lysate from tissues
2.17.2 Determination of protein concentration
2.17.3 Sample preparation
2.17.4 Separation of protein and transferred to PVDF membrane
2.17.5 Blocking and hybridization
2.18 Enzyme-linked immunosorbent assay（ELISA）
2.18.1 Sample preparation
2.18.2 Reagent preparation
2.18.3 Standard preparation
2.18.4 Assay procedure
2.19 Statistical analysis
Chapter3:Results
3.1 The inflammatory cytokines Il-1βand Tnf-αexpression analysis
3.2 An overview of RNA-seq data
3.2.1 RNA extraction and quality
3.2.2 Library construction and quality
3.2.3 Quality control and mapping of sequencing reads
3.3 Differential gene expression analysis
3.4 Bioinformatics analysis
3.4.1 KOG enrichment analysis
3.4.2 GO enrichment analysis
3.4.3 Pathway enrichment analysis
3.5 Interferome analysis
3.6 Protein-protein interaction（PPI）
3.6.1 Construction of PPI networks of immune-related genes
3.6.2 Analysis of key DEGs related to immune responses
3.6.3 Inflammatory roles of Tlr13
3.7 qRT-PCR validation of RNA sequence data
3.8 Rpl29 is expressed differentially in the brain and lung in response to poly（I:C）
3.9 Knockdown of RPL29 stimulates inflammatory cytokines
3.10 MiR22 expression was different between the two tissues of poly（I:C）treated mouse and positively correlated with Rpl
3.11 RPL29 and miR22 positively correlated in different cells
3.12 RPL29 regulates the expression of miR22
3.13 RPL29 regulates miR22 expression through the transcriptional activities of Fos-B and c-Fos
Chapter4:Discussion
4.1 Tissue-specific inflammatory and immune responses to viral dsRNA
4.2 Speculation of hub proteins and hub genes
4.3 Functional analysis of hub genes and signaling pathways in the lung tissues
4.3.1 Immunoglobulin-like domain superfamily
4.3.2 OAS domain2 superfamily
4.3.3 SH2 domain superfamily
4.3.4 JAK-STAT signaling pathway
4.3.5 The top5 key genes on the nodes
4.3.6 Other signaling pathways and key genes
4.4 Functional analysis of hub genes and signaling pathways in brain tissues
4.4.1 Immunoglobulin-like domain superfamily
4.4.2 OAS domain2 superfamily
4.4.3 Ubiquitin domain
4.4.4 Herpes simplex infection pathways
4.4.5 The top5 key genes on the nodes
4.4.6 Other KEGG signaling pathways and hub genes in the brain tissues
4.5 ISGs orchestrate immune and inflammatory responses in viral diseases
4.6 Tlr13 involves in lung specific inflammation
4.7 Rpl29 is a novel regulator of tissue specific inflammation
4.8 Rpl29 regulates miR22 expression through Fos-B and c-Fos
4.9 Conclusion
Chapter5:Summary
References
Acknowledgements
Appendices
本文编号：2882097
【学位单位】：华中农业大学
【学位级别】：博士
【学位年份】：2019
【中图分类】：R373
【文章目录】：
摘要
Abstract
Table of Abbreviations
Chapter1:Introduction
1.1 Inflammation and its mechanisms
1.1.1 PRR activation
1.1.2 Inflammatory pathways activation
1.1.3 Inflammatory markers
1.1.4 Cell types associated with inflammatory responses
1.2 Inflammatory and immune responses against virus infection
1.2.1 Production of inflammatory cytokines,IFNs,and ISGs
1.2.2 The inflammatory cytokines mediated antiviral response
1.2.3 IFNs and ISGs meditated antiviral responses
1.2.4 IFNs desensitization
1.2.5 The antiviral effects of other ISGs
1.3 Poly（I:C）,the viral mimic
1.4 RNA-seq based transcriptome profiling
1.4.1 Next-generation sequencing
1.4.2 Gene ontology and pathway analysis
1.5 Biological network
1.5.1 Transcriptomic integration in protein-protein interaction network
1.6 Interferome analysis
1.7 Toll-like receptors
1.7.1 Structure of TLRs
1.7.2 Signaling pathways of TLRs
1.7.3 Nucleic acid-TLR complexes
1.7.4 Toll-like receptor 13
1.8 Ribosome protein L29
1.9 MicroRNA22 in inflammation and immunity
1.10 Study proposal
Chapter2:Materials and Methods
2.1 Experimental animal
2.2 Experimental cells
2.3 Reagents
2.4 Solution preparation
2.4.1 Preparation of phosphate buffer（1XPBS）
2.4.2 Preparation of SDS-PAGE related solutions
2.5 Animal experiment
2.6 Cell culture and treatment
2.7 RNA isolation
2.8 Removal of genomic DNA and cDNA synthesis for qRT-PCR
2.9 Quantitative real-time polymerase chain reaction
2.10 RNA library preparation and sequencing
2.10.1 Removal of DNA contamination and detection of total RNA quality
2.10.2 mRNA purification and fragmentation
2.10.3 First strand cDNA synthesis
2.10.4 Second strand cDNA synthesis
2.10.5 Purification the double-stranded cDNA
2.10.6 End prep of cDNA library/3`adenylation
2.10.7 Adaptor ligation
2.10.8 Purification of the library
2.10.9 PCR enrichment of adaptor ligated cDNA
2.11 Validation of library
2.12 Differential expression analysis
2.13 KOG& GO enrichment analysis
2.14 Interferome analysis
2.15 KEGG pathway enrichment analysis
2.16 Functional protein association networks construction
2.17 Western blotting
2.17.1 Preparation of lysate from tissues
2.17.2 Determination of protein concentration
2.17.3 Sample preparation
2.17.4 Separation of protein and transferred to PVDF membrane
2.17.5 Blocking and hybridization
2.18 Enzyme-linked immunosorbent assay（ELISA）
2.18.1 Sample preparation
2.18.2 Reagent preparation
2.18.3 Standard preparation
2.18.4 Assay procedure
2.19 Statistical analysis
Chapter3:Results
3.1 The inflammatory cytokines Il-1βand Tnf-αexpression analysis
3.2 An overview of RNA-seq data
3.2.1 RNA extraction and quality
3.2.2 Library construction and quality
3.2.3 Quality control and mapping of sequencing reads
3.3 Differential gene expression analysis
3.4 Bioinformatics analysis
3.4.1 KOG enrichment analysis
3.4.2 GO enrichment analysis
3.4.3 Pathway enrichment analysis
3.5 Interferome analysis
3.6 Protein-protein interaction（PPI）
3.6.1 Construction of PPI networks of immune-related genes
3.6.2 Analysis of key DEGs related to immune responses
3.6.3 Inflammatory roles of Tlr13
3.7 qRT-PCR validation of RNA sequence data
3.8 Rpl29 is expressed differentially in the brain and lung in response to poly（I:C）
3.9 Knockdown of RPL29 stimulates inflammatory cytokines
3.10 MiR22 expression was different between the two tissues of poly（I:C）treated mouse and positively correlated with Rpl
3.11 RPL29 and miR22 positively correlated in different cells
3.12 RPL29 regulates the expression of miR22
3.13 RPL29 regulates miR22 expression through the transcriptional activities of Fos-B and c-Fos
Chapter4:Discussion
4.1 Tissue-specific inflammatory and immune responses to viral dsRNA
4.2 Speculation of hub proteins and hub genes
4.3 Functional analysis of hub genes and signaling pathways in the lung tissues
4.3.1 Immunoglobulin-like domain superfamily
4.3.2 OAS domain2 superfamily
4.3.3 SH2 domain superfamily
4.3.4 JAK-STAT signaling pathway
4.3.5 The top5 key genes on the nodes
4.3.6 Other signaling pathways and key genes
4.4 Functional analysis of hub genes and signaling pathways in brain tissues
4.4.1 Immunoglobulin-like domain superfamily
4.4.2 OAS domain2 superfamily
4.4.3 Ubiquitin domain
4.4.4 Herpes simplex infection pathways
4.4.5 The top5 key genes on the nodes
4.4.6 Other KEGG signaling pathways and hub genes in the brain tissues
4.5 ISGs orchestrate immune and inflammatory responses in viral diseases
4.6 Tlr13 involves in lung specific inflammation
4.7 Rpl29 is a novel regulator of tissue specific inflammation
4.8 Rpl29 regulates miR22 expression through Fos-B and c-Fos
4.9 Conclusion
Chapter5:Summary
References
Acknowledgements
Appendices
本文编号：2882097
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