RNA-Seq Transcriptome Profiling

RNA-Seq Transcriptome Profiling Identifies CRISPLD2 as a Glucocorticoid Responsive Gene that Modulates Cytokine Function in Airway Smooth Muscle Cells

名词解释

CRISPLD2:cysteine-rich secretory protein LCCL domain-containing, 2

asthma 哮喘

inflammatory respiratory disease 炎性呼吸系统疾病

glucocorticoid （GC）糖皮质激素

anti-inflammatory 抗炎

airway smooth muscle（ASM）气道平滑肌

dexamethasone 地塞米松

airway contractility 气道收缩性

potent强效的

synthetic 合成物

inplicated 纠缠、牵连

endotoxin 内毒素

vasculature 脉管系统

inhaled 吸入性

corticosteroid 皮质类固醇

bronchodilator支气管扩张剂

pharmacogenetics遗传药理学

airway hyperresponsiveness 气道高反应性

airway smooth muscle contractility 气道平滑肌收缩

novel splice variants 新型剪切变异体

potent有效的

fold-change

ICS GWAS

cytokine细胞因子

soform同种型

splicing variation剪切变体

rank列为

serum血清

sepsis败血症

septic shock感染性休克

Inhaled corticosteroid (ICS) responsiveness 吸入皮质类固醇(ICS)反应性

quality control （QC）质控

Introduction

1.GCs act by binding to GC receptors (GRs)to treat various inflammatory diseases, including asthma.

2.Many cells and tissues are involved in asthma and are targeted by GCs, including inflammatory, airway epithelium ,and ASM.

3.Compared to the other airway cells, much less is known about how GCs work specifically in the ASM to alleviate asthma.

4.Because GCs function by activating GR to directly modulate transcriptional gene expression, a better understanding of how the ASM transcriptome responds to GCs is needed to provide mechanistic insights for improving asthma therapy.

5.Two microarray-based gene expression:

1)one focusing on validating the function of the KLF15 gene in airway hyperresponsiveness;

2)on the overlap between GC and beta-agonist response of the ASM

6.Compared to the use of microarrays, RNA-Seq is able to :

1)quantify more RNA species, including non-coding and novel splice variants,

2)quantify RNA at baseline, rather than only measure fold changes
across conditions

3)cover a wider dynamic range of signal

7.Used RNA-Seq to comprehensively characterize changes of the ASM transcriptome in response to GCs using an in vitro model

8.316 differentially expressed genes concludes glycoprotein/extracellular matrix, vasculatureand lung development, regulation of cell migration, and extracellular matrix organization.

9.CRISPLD2-SNP-inhaled corticosteroid response and short-acting bronchodilator response

10.Functional experiments showed that in ASM cells, CRISPLD2 mRNA and protein levels changed in response to treatment with a glucocorticoid or proinflammatory and that knockdown of CRISPLD2 resulted in increased
levels of IL1b-induced IL6 and IL8 mRNA expression.

Results

1.RNA-Seq Transcriptome Profiling of GC-treated Primary Human ASM Cells

1.四组-药物组和对照组，获得每个样本平均58.9亿raw sequencing reads，其中83.36%与hg19比对。质控的脚本根据hg19RefSeq的Cuffilinks。通过Benjamini-Hochberg approch校正后,得到316个差异表达基因.

2.其中table 1包括了Q-value <1E-10的基因将进一步研究

image-20181228204937552

先前报道过的：DUSP1, FKBP5, KLF15, PER1, and TSC22D3 与类固醇反应和炎症有关

Others:potentially novel GC-responsive genes

3.NIH DAVID tool用来Gene set enrichment analysis

4.功能注释集：6个enrichment score>3与...有关

​ enrichment score>1.5与...有关

2.Verification of GC-responsive Genes by q-PCR

高差异表达基因选了4个再加一个PTX3

image-20181228204730811

3.CRISPLD2 Variants Associated with Asthma Pharmacogenetic Phenotypes

CRISPLD2 in modulating two asthma pharmacogenetic phenotypes：1）Inhaled corticosteroid (ICS) responsiveness：2）bronchodilator response

吸入类固醇反应性ICS是评估糖皮质激素治疗后肺功能改善与否的一种方法

用先前ICS GWAS的结果， Based on a threshold of 1E-03, the CRISPLD2 gene had SNPs that were nominally associated with ICS resistance 。

image-20181228212914459

previous study：CRISPLD2 and an additional gene CCDC69 were nominally associated with the bronchodilatorresponse

4.CRISPLD2 Expression Changes in Previous Microarray Studies of the ASM GC Response

用先前两篇文献GSE34313\GSE13168来确定一下自己的结果lCRISPLD2是否差异表达的基因

image-20181228214856046

显著P-value值

5.GC Induced CRISPLD2 mRNA and Protein Expression in Primary Human ASM Cells

image-20181228220111871

A.CRISPLD2 mRNA increased 8.1-fold

B.protein levels of CRISPLD2 in ASM cells also increased upon DEX
treatment by 1.7-fold at 24 hours

并且是时间和计量依赖性的

image-20181228221342771 image-20181228220248237

A.ASM中CRISPLD2升，癌症细胞系A549中CRISPLD2降

B.在癌细胞中，DEX治疗后，CRISPLD2下降

6.CRISPLD2 is Induced by IL1b and Modulates the Expression of Two Immuno-Responsive Genes

由于GCs能触发免疫反应，因此检测在调节免疫反应中ASM中CRISPLD2的表达。应用免疫因子IL1后，CRISPLD2mRNA和蛋白水平均升高，证明CRISPLD2不仅可由GC作用后增多，也会因为免疫反应而增多。

7.CRISPLD2 is Induced by IL1b and Modulates the Expression of Two Immuno-Responsive Genes

敲低CRISPLD2，研究CRISPLD2在IL1诱导的其他免疫基因的表达（IL6 and IL8）。

A.干扰后，在ASM细胞中，CRISPLD2 mRNA
expression was decreased by 74% and protein levels decreased by
60%

B.用了IL1后，干扰CRISPLD2组相比较对照组中的IL6要增多很多，说明CRISPLD2是抑制IL6产生的，从而说明CRISPLD2在ASMs中是抑制免疫反应的。

image-20181228230621379

C.对照组，加了DEX，IL6就下降了，CRISPLD2敲低组，IL6的水平就比对照组高，说明DEX是通过CRISPLD2来抑制免疫反应。

image-20181228233758631

D.相比较对照组，把CRISPLD2敲低以后，同时应用DEX和IL1b是IL6的水平上升。其中DEX是使IL6下降的，IL1b是使IL6上升，按理两者一折中，应该是IL6的水平变化不大，这正好是对照组的水平，但是在CRISPLD2敲低以后，IL6的水平就上升了，说明CRISPLD2是参与到抗免疫反应的。

image-20181228234135127

8CRISPLD2 is Not Required for the Expression of GC Target Genes

CRISPLD2- knockdown的 ASM细胞中三个GR靶基因的表达水平相对于对照非靶向siRNA转染的细胞没有明显改变，说明CRISPLD2并没有调节GCs在ASM中的直接转录作用。

image-20181228235132964