基于RNA-seq的炎癥小鼠肺和腦組織的轉(zhuǎn)錄組分析
發(fā)布時間：2020-11-13 10:39
病毒感染通常具有器官特異性,而一些病毒可同時感染多個器官并導致炎癥性疾病。呼吸和中樞神經(jīng)系統(tǒng)(CNS)的病毒感染是公共衛(wèi)生中最關(guān)鍵的問題,包括麻疹病毒和皰疹病毒等在內(nèi)的一些已知的嗜神經(jīng)病毒,通常與中樞神經(jīng)系統(tǒng)疾病有關(guān)。然而,包括人類呼吸道合胞體病毒、流感病毒、冠狀病毒和人化生病毒在內(nèi)的一些呼吸道病毒已成為中樞神經(jīng)系統(tǒng)疾病的相關(guān)媒介。對于病毒感染多個組織器官,不同組織器官針對同一病毒感染的不同炎癥反應的作用或機制是免疫學的關(guān)鍵問題,有待進一步研究。因此,本研究旨在探尋病毒模擬物處理后小鼠大腦和肺組織炎癥差異的機制。本研究利用病毒dsRNA模擬物poly(I:C)(polyinosinic acid-polycytidylic acid)刺激小鼠,闡述了病毒感染器官后炎癥或免疫的分子機制。本研究通過檢測細胞因子Il-1β、Tnf-α的表達,發(fā)現(xiàn)了大腦和肺組織中不同的炎癥狀態(tài)。本研究對小鼠肺和腦組織炎癥進行轉(zhuǎn)錄組、蛋白相互作用和干擾素分析等后續(xù)實驗,闡述了不同炎癥狀態(tài)背后的原因。本研究在肺中鑒定了629個差異表達基因(DEGs),在腦組織中鑒定了137個DEGs,其中有一些基因重疊。這些DEGs大部分是干擾素刺激基因(ISGs)。轉(zhuǎn)錄組GO分析顯示,大腦和肺組織的DEGs在細胞粘附、炎癥、病毒防御反應和先天免疫等方面起著關(guān)鍵作用。就DEGs和ISGs的數(shù)量而言,病毒模擬物在肺中的炎癥反應較腦組織劇烈。本研究通過蛋白相互作用和信號通路關(guān)聯(lián)分析,分別在肺組織和腦組織中鑒定出29個和14個免疫相關(guān)的hub基因。肺組織中與免疫相關(guān)的前五大hub基因為Itgam、Il-1b、Il-7、Cdkn1a、Myc,腦組織中與免疫相關(guān)的hub基因為Irf7、Oas2、Ifit1、Isg15、Rsad2。除這些hub基因外,包括病毒核酸受體2'-5'-寡腺苷酸合成酶(OAS)在內(nèi)的不同ISGs在病毒感染時調(diào)節(jié)炎癥和免疫反應,其表達具有一定的組織特異性。此外,序列特異性病毒和細菌核酸受體Tlr13在poly(I:C)處理組中上調(diào),刺激小鼠肺及其與許多其他炎癥調(diào)節(jié)蛋白的相互作用,反映出其在調(diào)節(jié)肺炎癥反應中的關(guān)鍵作用。有趣的是,核糖體蛋白Rpl29和細胞表面肝素結(jié)合蛋白在大腦中上調(diào),而在肺中下調(diào)。siRNA抑制RPL29,導致炎性細胞因子IL-1β、TNF-α高表達。本研究首次證實Rpl29參與炎癥反應,其組織特異性和不同表達水平造成了調(diào)控病毒感染時肺和腦組織炎癥反應的差異。MiR22可以在翻譯后水平上調(diào)控許多生理事件,其中包括炎癥。在病毒模擬物處理的組織和Rpl29陽性共表達的不同細胞系中,miR22的表達也存在差異。本研究還發(fā)現(xiàn),Rpl29通過調(diào)節(jié)Fos-B和c-Fos,以在病毒感染后調(diào)節(jié)不同組織中miR22的表達。綜上所述,本研究表明,Rpl29與不同的hub基因Tlr13、mi R22和ISGs一起,是機體特異性炎癥或免疫反應的關(guān)鍵協(xié)調(diào)因子,借此可以調(diào)節(jié)不同組織中mi R22的表達差異�？傊�,該項研究的發(fā)現(xiàn)將有助于為探索與炎癥、免疫相關(guān)多種嚴重病毒疾病的治療策略提供新見解。
【學位單位】：華中農(nóng)業(yè)大學
【學位級別】：博士
【學位年份】：2019
【中圖分類】：R373
【文章目錄】：
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
本文編號：2882097
【學位單位】：華中農(nóng)業(yè)大學
【學位級別】：博士
【學位年份】：2019
【中圖分類】：R373
【文章目錄】：
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
本文編號：2882097
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