文章为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差异表达倍数
ICS GWAS 全基因组关联性分析
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
splice junction剪切点
pro-inflammatory agents 促炎剂
preprocessed预处理
ligand-dependent配体依赖性
macrophages巨噬细胞
myeloid dendritic cell髓样树突状细胞
ERCC-spike in 表达量数据质量控制联盟（Variation in RNA expression data can be attributed to a variety of factors including the quality of the starting material, the level of cellularity and RNA yield, the platform employed, and the person performing the experiment. To control for these sources of variability, a common set of external RNA controls has been developed by the External RNA Controls Consortium (ERCC), an ad-hoc group of academic, private, and public organizations hosted by the National Institute of Standards and Technology (NIST). The controls consist of a set of unlabeled, polyadenylated transcripts designed to be added to an RNA analysis experiment after sample isolation, in order to measure against defined performance criteria. Up until the design of such universally accepted controls, it has been difficult to execute a thorough investigation of fundamental analytical performance metrics. From the trusted brand of quality RNA reagents, Ambion® ERCC Spike-In Control Mixes are commercially available, pre-formulated blends of 92 transcripts, derived and traceable from NIST-certified DNA plasmids. The transcripts are designed to be 250 to 2,000 nt in length, which mimic natural eukaryotic mRNAs.）
Cufflinks ：Cufflinks 利用Tophat比对的结果（alignments）来组装转录本，估计这些转录本的丰度，并且检测样本间的差异表达及可变剪接。这个软件其实是个套装，包括四个部分分别命名为：cufflinks、cuffcompare、cuffmerge及cuffdiff
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
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
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 。
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
显著P-value值
5.GC Induced CRISPLD2 mRNA and Protein Expression in Primary Human ASM Cells
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
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。
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。
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。
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。
