RNA-Seq Transcriptome Profiling Identifies CRISPLD2 as a Glucocorticoid Responsive Gene that Modulat

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

RNA-Seq转录组分析鉴定CRISPLD2是一种糖皮质激素应答基因，可调节气道平滑肌细胞中的细胞因子功能

Asthma is a chronic inflammatory respiratory disease that affects over 300 million people worldwide. Glucocorticoids are a mainstay therapy for asthma because they exert anti-inflammatory effects in multiple lung tissues, including the airway smooth muscle (ASM). However, the mechanism by which glucocorticoids suppress inflammation in ASM remains poorly understood. Using RNA-Seq, a high-throughput sequencing method, we characterized transcriptomic changes in four primary human ASM cell lines that were treated with dexamethasone a potent synthetic glucocorticoid (1 mM for 18 hours). Based on a Benjamini-Hochberg corrected p-value ,0.05, we identified 316 differentially expressed genes, including both well known (DUSP1, KLF15, PER1, TSC22D3) and less investigated (C7, CCDC69, CRISPLD2) glucocorticoidresponsive genes. CRISPLD2, which encodes a secreted protein previously implicated in lung development and endotoxin regulation, was found to have SNPs that were moderately associated with inhaled corticosteroid resistance and bronchodilator response among asthma patients in two previously conducted genome-wide association studies. Quantitative RT-PCR and Western blotting showed that dexamethasone treatment significantly increased CRISPLD2 mRNA and protein expression in ASM cells. CRISPLD2 expression was also induced by the inflammatory cytokine IL1b, and small interfering RNA-mediated knockdown of CRISPLD2 further increased IL1b-induced expression of IL6 and IL8. Our findings offer a comprehensive view of the effect of a glucocorticoid on the ASM transcriptome and identify CRISPLD2 as an asthma pharmacogenetics candidate gene that regulates anti-inflammatory effects of glucocorticoids in the ASM.

哮喘是一种慢性呼吸道炎症性疾病，影响着全世界超过3亿人。
糖皮质激素是治疗哮喘的主要药物，因为它们在包括气道平滑肌(ASM)在内的多个肺组织中发挥抗炎作用。
然而，糖皮质激素抑制ASM炎症的机制仍不清楚。
利用RNA-Seq(一种高通量测序方法)，我们分析了四种主要的人类ASM细胞系在使用地塞米松(一种强效合成糖皮质激素，1mm持续18小时)处理后的转录组变化。
基于benjamin - hochberg校正的p值，0.05，我们鉴定了316个差异表达基因，包括已知的(DUSP1, KLF15, PER1, TSC22D3)和较少研究的(C7, CCDC69, CRISPLD2)糖皮质激素应答基因。
CRISPLD2编码一种以前与肺发育和内毒素调节有关的分泌蛋白，在之前进行的两项全基因组关联研究中发现，它具有与哮喘患者吸入皮质类固醇抵抗和支气管扩张剂反应中度相关的snp。
定量RT-PCR和Western blotting结果显示，地塞米松处理显著提高了ASM细胞CRISPLD2 mRNA和蛋白的表达。
炎性细胞因子IL1b也可诱导CRISPLD2的表达，小干扰rna介导的CRISPLD2的敲除进一步增加了IL1b诱导的IL6和IL8的表达。
我们的发现为糖皮质激素对ASM转录组的影响提供了一个全面的观点，并确定CRISPLD2作为哮喘药物遗传学候选基因，调节糖皮质激素在ASM中的抗炎作用。

Asthma, a chronic inflammatory respiratory disease that affects over 25 million Americans and 300 million people world-wide, is characterized by variable airflow limitation and airway hyperresponsiveness [1,2]. Glucocorticoids (GCs) are common medications used to treat various inflammatory diseases, including asthma [3]. Inhaled corticosteroids, GC medications that act directly in the lung, are among the most common asthma controller medications and treatment of asthma patients with them leads to improved clinical outcomes, including decreased asthma symptoms and exacerbations [4]. At a cellular level, GCs act by binding to GC receptors (GRs), causing them to translocate to cell nuclei where they modulate transcription of various genes in a tissuedependent fashion [5]. The anti-inflammatory action of GCs is partly a result of 1) GC-GR complexes stimulating antiinflammatory genes by directly binding to DNA at glucocorticoid receptor enhancer elements, and of 2) GC-GR complexes inhibiting proinflammatory transcription factors such as nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB) [6]. In addition to directly reducing inflammation, GCs have been shown to affect other asthma-related phenotypes, including bronchodilation [7], airway hyperresponsiveness [8], and airway smooth muscle (ASM) contractility [9]. Many cells and tissues are involved in asthma and are targeted by GCs, including inflammatory [10,11], airway epithelium [12], and ASM [13]. Of these, the ASM is involved in altered airway contractility [14], a major asthma-specific trait that is assessed clinically and for research studies by measures such as bronchodilator response [15] and airway hyperresponsiveness [16].

哮喘是一种慢性炎症性呼吸道疾病，影响了超过2500万美国人和全世界3亿人，其特征是可变气流受限和气道高反应性[1,2]。
糖皮质激素(GCs)是治疗各种炎症性疾病的常用药物，包括哮喘[3]。
吸入糖皮质激素，一种直接作用于肺的GC药物，是最常见的哮喘控制药物之一，对哮喘患者的治疗可改善临床结果，包括减轻哮喘症状和加重[4]。
在细胞水平上，GCs通过与GC受体(GRs)结合而起作用，使它们转移到细胞核上，并以一种组织依赖的方式[5]调控各种基因的转录。
gc的抗炎作用在一定程度上是由于1)GC-GR复合物刺激抗炎在糖皮质激素受体基因通过直接绑定到DNA增强器元素,和2)GC-GR复合体抑制促炎核转录因子kappa-light-chain-enhancer等转录因子激活B细胞(NFkB)[6]。
除了直接减少炎症，GCs已经被证明影响其他哮喘相关表型，包括支气管扩张性[7]，气道高反应性[8]，气道平滑肌(ASM)收缩性[9]。
许多细胞和组织参与哮喘并被GCs靶向，包括炎症[10,11]、气道上皮[12]和ASM[13]。
其中，ASM与气道收缩性[14]改变有关，这是哮喘特有的主要特征，可通过支气管扩张剂反应[15]和气道高反应性[16]等指标进行临床和研究评估。

However, compared to the other airway cells, much less is known about how GCs work specifically in the ASM to alleviate asthma. 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. Several studies have been conducted to identify GCs-induced transcript changes in the ASM. For example, two microarray-based gene expression studies have measured the effect of GCs on ASM cells using in vitro models where human ASM cells were stimulated with dexamethasone or fluticasone [17,18]. Although both were limited by the inherent biases of microarrays, these studies identified some genes involved in the ASM GC response, with one focusing on validating the function of the KLF15 gene in airway hyperresponsiveness [17] and the other on the overlap between GC and beta-agonist response of the ASM [18]. Recent advances in sequencing technologies have made possible the comprehensive and in-depth characterization of transcriptomes via a technique known as RNA-Seq [19 21]. 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, and (3) cover a wider dynamic range of signal [22]. In this study, we used RNA-Seq to comprehensively characterize changes of the ASM transcriptome in response to GCs using an in vitro model. We identified 316 significantly differentially expressed genes representing various functional categories such as glycoprotein/extracellular matrix, vasculature and lung development, regulation of cell migration, and extracellular matrix organization. One of these genes, cysteine-rich secretory protein LCCL domain-containing, 2 (CRISPLD2; OMIM 612434), had single nucleotide polymorphisms (SNPs) that were nominally associated with two asthma drug response phenotypes (i.e., inhaled corticosteroid response and short-acting bronchodilator response). Functional experiments showed that in ASM cells, CRISPLD2 mRNA and protein levels changed in response to treatment with a glucocorticoid or proinflammatory cytokine, and that knockdown of CRISPLD2 resulted in increased levels of IL1b-induced IL6 and IL8 mRNA expression.

然而，与其他气道细胞相比，我们对GCs在ASM中具体如何工作以减轻哮喘的了解要少得多。
由于GCs通过激活GR直接调节转录基因表达来发挥作用，因此需要更好地理解ASM转录组如何响应GCs，从而为改善哮喘治疗提供机制上的见解。
已经进行了几项研究来鉴定gcs诱导的ASM转录变化。
例如，两项基于微阵列的基因表达研究使用地塞米松或氟替卡松刺激人类ASM细胞的体外模型，测量了GCs对ASM细胞的影响[17,18]。
虽然都是有限的微阵列的固有偏见,这些研究发现了一些基因参与了ASM GC响应,一个专注于验证KLF15基因的功能[17]气道高反应,另一个GC之间的重叠和ASM beta-agonist响应[18]。
近年来，测序技术的发展使得RNA-Seq技术能够对转录组进行全面深入的表征[19 21]。
与使用微阵列相比，RNA- seq能够

(1)量化更多的RNA物种，包括非编码和新剪接变异，

(2)量化基线RNA，而不仅仅是测量不同条件下的折叠变化，以及

(3)覆盖更广泛的动态信号[22]范围。
在这项研究中，我们使用RNA-Seq在体外模型中全面描述了ASM转录组在GCs反应中的变化。
我们鉴定了316个显著差异表达基因，它们代表了不同的功能类别，如糖蛋白/细胞外基质、血管系统和肺发育、细胞迁移的调节和细胞外基质组织。
其中一个基因，富含半胱氨酸的分泌蛋白LCCL结构域，2 (CRISPLD2;OMIM 612434)的单核苷酸多态性(SNPs)与两种哮喘药物反应表型(即吸入皮质类固醇反应和短效支气管扩张剂反应)有关。
功能实验表明，在ASM细胞中，CRISPLD2的mRNA和蛋白水平在糖皮质激素或促炎细胞因子的作用下发生变化，抑制CRISPLD2导致il1b诱导的IL6和IL8 mRNA表达水平升高。

Results
RNA-Seq Transcriptome Profiling of GC-treated Primary Human ASM Cells
To identify GC-responsive genes in ASM, we performed RNASeq expression profiling of primary ASM cells from four white male donors treated with 1 mM dexamethasone (DEX) or control vehicle for 18 h, a treatment protocol that captures a large set of genes regulated by the GR [17]. We obtained an average of 58.9 million raw sequencing reads per sample (range 44.2 71.3 million reads per sample). Of these reads, an average of 83.36% were aligned to hg19 genome reference files downloaded from Illumina s iGenomes project (range 81.94% 84.34%) [Table S1]. An average of 26.43% of the mapped reads spanned junctions. Most bases in mapped reads corresponded to mRNA (.98%) [Table S2]. Plots of normalized read coverage of transcripts vs. normalized position, reveals that there was even coverage of transcripts by reads [Figure S1]. Based on these and various quality control (QC) summary metrics, including ERCC spike-in dose response plots, the sequencing and alignment results for each sample were deemed of sufficiently high quality to include in differential expression analyses. Quantification of transcript and gene expression levels was performed using Cufflinks according to hg19 RefSeq annotation files from Illumina s iGenomes Project. Overall, 316 genes were significantly differentially expressed after correcting for false discovery rate by the Benjamini- Hochberg [23] approach [Figure 1A, Table S3]. Table 1 contains the genes with Q-value ,1E-10 that were considered for further study. Some of these top genes have been previously related to steroid responsiveness and inflammation (i.e., DUSP1 [24], FKBP5 [25], KLF15 [17], PER1 [12,26], and TSC22D3 [25,27]), and their upregulation by 1 mM for 18 DEX was confirmed by quantitative real time PCR (qRT-PCR) in ASM cells from three donors [Figure 1B]. qRT-PCR results for the fourth donor used in the RNA-Seq experiment were also consistent [Figure S2]. Other genes identified via the RNA-Seq experiment were considered potentially novel GC-responsive genes as they have little published evidence regarding a relationship with steroid responsiveness and/ or inflammation. Gene set enrichment analysis using the NIH DAVID tool [28] identified various Gene Ontology and other annotation categories that were overrepresented by the 316 genes. The top six gene functional annotation clusters (enrichment scores .3) had terms related to: glycoprotein/extracellular matrix, vasculature development, circulatory system process, response to nutrients, thrombospondin type-1, and response to hormone stimulus terms [Table S4]. Other clusters among the 19 with enrichment scores .1.5 that may be relevant to lung disease included lung development, regulation of cell migration, and extracellular matrix organization.

Verification of GC-responsive Genes by q-PCR

A subset of the top differentially expressed genes (i.e., CRISPLD2, C13orf15, KCTD12, SERPINA3) was selected for follow-up based on each gene s potential to be a novel steroid responsiveness gene. Differential expression for these four genes and one additional gene selected from the top 316 differentially expressed ones (i.e., PTX3) was verified via qRT-PCR by treating with 1 mM DEX for 18 h three of the ASM cell lines used for RNA-Seq [Figure 2] to compare biological sample variability and effect sizes obtained via RNA-Seq vs. qRT-PCR. qRT-PCR results for the fourth donor were consistent with those for the other three cell lines [Figure S2]. Gene expression levels varied among the primary cell lines, suggesting an inherent heterogeneity in individual GC responsiveness. Nevertheless, the qRT-PCR data for each of the genes was consistent in direction of fold-change with the RNA-Seq results.

CRISPLD2 Variants Associated with Asthma

Pharmacogenetic Phenotypes Inhaled corticosteroid (ICS) responsiveness is a measure of improvement in pulmonary function after treatment with a glucocorticoid. To determine whether any of the differentially expressed genes were associated with this pharmacogenetic phenotype, defined as unchanged improvement in lung function among asthma patients after receiving ICS therapy for 4 8 weeks, we obtained previously conducted ICS GWAS results (unpublished) for SNPs within, or spanning 50 kb on either side, each of the genes in Table 1. Based on a threshold of 1E-03, the CRISPLD2 gene had SNPs that were nominally associated with ICS resistance [Table 2; Figure S3].

结果
gc处理的原代人类ASM细胞的RNA-Seq转录组分析
为了鉴定ASM中的gc应答基因，我们对4个白人男性供体的原代ASM细胞进行了RNASeq表达谱分析。
我们平均获得了5890万的原始测序reads每个样本(范围442,7130万reads每个样本)。
在这些reads中，平均有83.36%的reads与从Illumina s genomics project下载的hg19基因组参考文件(范围81.94% 84.34%)一致[表S1]。
平均26.43%的映射读码是跨接的。
大部分的碱基都与mRNA对应(0.98%)[表S2]。
规范化的转录本阅读覆盖率与规范化位置的图，揭示了甚至存在着通过reads对转录本的覆盖[图S1]。
基于这些和各种质量控制(QC)汇总指标，包括ERCC剂量响应曲线，每个样本的测序和比对结果被认为具有足够高的质量，可以纳入差异表达分析。
根据Illumina基因组项目的hg19 RefSeq注释文件，使用Cufflinks对转录本和基因表达水平进行定量。
总的来说，采用Benjamini- Hochberg[23]方法修正错误发现率后，有316个基因显著差异表达[图1A，表S3]。
表1包含q值为1E-10的基因，考虑进一步研究。
一些顶级基因已经被先前与类固醇反应和炎症(例如,DUSP1 [24], FKBP5 [25], KLF15 [17], PER1[12、26],和TSC22D3[25日27]),和他们证实了由1毫米upregulation 18敏捷实时定量PCR(存在)在ASM细胞从三个捐助者(图1 b)。
RNA-Seq实验中使用的第四个供体的qRT-PCR结果也一致[图S2]。
通过RNA-Seq实验确定的其他基因被认为是潜在的新型gc应答基因，因为它们很少有发表的证据表明与类固醇应答和/或炎症的关系。
使用NIH DAVID工具[28]进行基因集富集分析，识别出316个基因过度代表的各种基因本体和其他注释类别。
前六个基因功能注释簇(富集得分为3)的术语与:糖蛋白/细胞外基质、血管系统发育、循环系统过程、对营养物质的反应、血栓反应素-1型和对激素刺激的反应相关[表S4]。
在富集得分为.1.5的19个集群中，其他可能与肺疾病相关的集群包括肺发育、细胞迁移调节和细胞外基质组织。

用q-PCR方法验证gc应答基因

根据每个基因可能成为新的类固醇反应基因，我们选择顶部差异表达基因(如CRISPLD2, C13orf15, KCTD12, SERPINA3)中的一个亚组进行随访。
微分表达这四个基因和一个额外的选择前316个差异表达的基因(例如,PTX3)验证了通过治疗中存在的1毫米敏捷18 h三个ASM细胞株用于RNA-Seq(图2)比较生物样品的变化和尺度效应获得通过RNA-Seq与存在。
第四供体的qRT-PCR结果与其他三株细胞系一致[图S2]。
基因表达水平在原代细胞系之间存在差异，表明个体GC反应性存在固有的异质性。
然而，每个基因的qRT-PCR数据在fold-change方向上与RNA-Seq结果一致。

CRISPLD2变异与哮喘相关

吸入皮质类固醇(ICS)反应性是糖皮质激素治疗后肺功能改善的一种措施。
来决定是否与这个相关的差异表达基因遗传表型,定义为不变改善哮喘患者肺功能在收到ICS治疗4 8周,我们之前获得了ICS GWAS结果(未发表)单核苷酸多态性,或跨越50 kb两侧,每个基因在表1。
根据1E-03的阈值，CRISPLD2基因具有与ICS抗性相关的SNPs[表2;
图S3)。

Because the beta-agonist and glucocorticoid pathways are known to overlap [29], we also examined the association of the differentially expressed genes with bronchodilator response, which measures the effect of betaagonists on lung function. Based on bronchodilator response GWAS results from a previous study where the phenotype was defined as change in FEV1 in response to administration of the beta-agonist albuterol [30], SNPs in CRISPLD2 and an additional gene CCDC69 were nominally associated with the bronchodilatorresponse [Table 2; Figure S3]. Additionally, replication results for
one SNP (rs8047416) from this bronchodilator response GWAS that had a primary P-value of 4.5E-04 had been obtained for 552 white subjects from the Severe Asthma Research Program (SARP) cohort and found to have a P-value of 0.038 (overall P-value 9.0E-05). Together these results suggest a role for CRISPLD2 in modulating two asthma pharmacogenetic phenotypes.

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

We analyzed publicly available data from two published gene expression microarray studies (GSE34313 [17] and GSE13168[18]) that measured the effect of GCs on human ASM cells to determine whether these previous studies supported our CRISPLD2 differential expression results. Although CRISPLD2 did not rank as one of the most highly differentially expressed genes in these studies, all comparisons available between ASM cells treated with a GC vs. baseline conditions demonstrate that CRISPLD2 had significant adjusted P-values [Table 3]. Specifically,the GSE34313 study found that CRISPLD2 was differentially expressed both 4 and 24 hours after ASM cells were treated with DEX, and the GSE13168 study found that the differential
CRISPLD2 expression was strongest when ASM cells were treated with a GC (i.e. fluticasone) vs. left untreated, than when cells were also stimulated with pro-inflammatory cytokines (i.e. EGF and IL1b).

因为已知-激动剂和糖皮质激素途径与[29]重叠，我们也检查了差异表达基因与支气管扩张剂反应的关系，这衡量了-激动剂对肺功能的影响。
基于先前研究的支气管扩张剂反应GWAS结果，该研究将表型定义为在使用β-激动剂沙丁胺醇[30]后FEV1反应的变化，CRISPLD2中的SNPs和另外一个基因CCDC69在理论上与支气管扩张剂反应相关[表2;
图S3)。
此外，复制结果为
在重症哮喘研究计划(SARP)队列中的552名白人受试者中，从这个支气管扩张剂反应GWAS中获得了一个初始p值为4.5E-04的SNP (rs8047416)，发现其p值为0.038(总体p值为9.0E-05)。
这些结果表明CRISPLD2在调节两种哮喘药物遗传表型中的作用。

CRISPLD2表达变化在之前的ASM GC反应的微阵列研究中

我们分析了两项公开发布的基因表达芯片研究(GSE34313[17]和GSE13168[18])的数据，它们测量了GCs对人类ASM细胞的影响，以确定这些先前的研究是否支持我们的CRISPLD2差异表达结果。
虽然在这些研究中CRISPLD2并不是表达差异最大的基因之一，但所有对经GC处理的ASM细胞与基线条件的比较表明，CRISPLD2具有显著的调整后的p值[表3]。
其中，GSE34313研究发现，CRISPLD2在DEX处理ASM细胞4小时和24小时后均有差异表达，GSE13168研究发现了差异
与未处理的细胞相比，用GC(即氟替卡松)处理ASM细胞时CRISPLD2表达最强，而用促炎细胞因子(即EGF和IL1b)刺激细胞时则最强。

GC Induced CRISPLD2 mRNA and Protein Expression in Primary Human ASM Cells
Because of its potential to modulate two important asthma drug response phenotypes vis-a`-vis these associations and published evidence of its involvement in lung development and endotoxin regulation [31], we focused our functional studies on the CRISPLD2 gene to investigate its potential role in steroid and immune response in ASM cells. We grew the most GC sensitive ASM cell line among those tested in Figure 2, treated those cells with DEX, and extracted RNA for qRT-PCR and protein for immune-blot analysis. Upon DEX treatment, CRISPLD2 mRNA increased 8.1-fold [Figure 3A]. Consistent with mRNA changes,protein levels of CRISPLD2 in ASM cells also increased upon DEX treatment by 1.7-fold at 24 hours [Figure 3B]. Using cells from a single donor, the effect of DEX on CRISPLD2 expression was found to be time [Figure S4A] and dose dependent [Figure S4B].
The induction of CRISPLD2 by DEX that was observed in ASM did not occur in A549 pulmonary epithelial cells derived from a lung carcinoma tissue, as analogous treatment of A549 cells with DEX caused a decrease of CRISPLD2 mRNA [Figure S5].

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

Because GCs exert anti-inflammatory effects, we tested the role of GC-induced CRISPLD2 expression in regulating inflammatory responses in the ASM. Treatment of a single ASM cell line with the proinflammatory cytokine IL1b (5 ng/mL for 24 h) increased CRISPLD2 mRNA by 10.4-fold and protein levels by 1.9-fold[Figure 3C and 3D], suggesting that CRISPLD2 is not only GCinducible but also immuno-responsive. We next performed knockdown experiments to assess whether CRISPLD2 modulates IL1b-induced cytokine responses using a single ASM cell line.
Because IL1b acts as an important mediator of inflammatory responses by activating other cytokines, we investigated the role of
CRISPLD2 in IL1b-induced expression of other known immuneresponse genes (i.e. IL6 [32] and IL8 [33]). In ASM cells transfected with CRISPLD2-specific siRNA, CRISPLD2 mRNA expression was decreased by 74% and protein levels decreased by 60% [Figure 4A].
While expression levels of IL6 did not change in response to CRISPLD2 knockdown, treatment of ASM cells with IL1b induced significantly higher expression of IL6 in CRISPLD2-knockdown cells as compared to NT siRNA control cells [Figure 4B],suggesting that CRISPLD2 is an inhibitory modulator of immunoresponse in ASM cells. Consistent with this notion, another cytokine’s (i.e. IL8’s) induction by IL1b was also enhanced by CRISPLD2 knockdown [Figure S6]. To further characterize the effect of CRISPLD2 on immune response, we treated cells with 100 nM DEX alone or in combination with 5 ng/mL IL1b. IL6 expression was decreased with DEX treatment, but CRISPLD2 knockdown did not significantly change the IL6 response to DEX
[Figure 4C]. However, IL6 mRNA levels in CRISPLD2 knockdown cells were higher than that in NT siRNA control cells when IL1b and DEX were administered simultaneously [Figure 4D],again supporting a role for CRISPLD2 in modulating IL1b response.

CRISPLD2 is Not Required for the Expression of GC Target Genes

We performed siRNA-mediated CRISPLD2 knockdown experiments to assess whether CRISPLD2 affects the transcriptional expression of well-known GC-response genes (i.e. DUSP1 [24],FKBP5 [25] or TSC22D3 [25,27]). The expression levels of three GR target genes were not significantly altered in CRISPLD2-knockdown ASM cells relative to control non-targeting siRNAtransfected cells [Figure S7], suggesting that CRIPSLD2 does not modulate the direct transcriptional actions of GCs in the ASM.

GC诱导原代人ASM细胞CRISPLD2 mRNA和蛋白的表达
由于CRISPLD2基因具有调节两种重要的哮喘药物反应表型的潜力，并有已发表的证据表明其参与肺发育和内毒素调节[31]，我们将功能研究重点集中在CRISPLD2基因上，以研究其在类固醇和ASM细胞免疫反应中的潜在作用。
我们在图2中检测的细胞中培养了最敏感的GC ASM细胞系，用DEX处理这些细胞，提取RNA进行qRT-PCR，提取蛋白进行免疫印迹分析。
DEX处理后，CRISPLD2 mRNA增加了8.1倍[图3A]。
与mRNA变化一致的是，在DEX处理24小时后，ASM细胞中的CRISPLD2蛋白水平也增加了1.7倍[图3B]。
使用来自单个供体的细胞，发现DEX对CRISPLD2表达的影响是时间[图S4A]和剂量相关的[图S4B]。
在ASM中观察到的DEX诱导的CRISPLD2在来自肺癌组织的A549肺上皮细胞中没有发生，因为类似地用DEX处理A549细胞会导致CRISPLD2 mRNA的减少[图S5]。

CRISPLD2由IL1b诱导，调节两个免疫应答基因的表达

由于GCs发挥抗炎作用，我们测试了gc诱导的CRISPLD2表达在调节ASM炎症反应中的作用。
用促炎细胞因子IL1b (5 ng/mL)处理单个ASM细胞系24小时后，CRISPLD2的mRNA和蛋白水平分别提高了10.4倍和1.9倍[图3C和3D]，表明CRISPLD2不仅具有gcinible，而且具有免疫应答性。
接下来，我们使用单个ASM细胞系进行敲除实验，以评估CRISPLD2是否能调节il1b诱导的细胞因子反应。
由于IL1b通过激活其他细胞因子作为炎症反应的重要介质，我们研究了IL1b的作用
CRISPLD2在il1b诱导的其他已知免疫应答基因(即IL6[32]和IL8[33])表达中的作用。
在转染了CRISPLD2特异性siRNA的ASM细胞中，CRISPLD2 mRNA表达降低了74%，蛋白水平降低了60%[图4A]。
而白细胞介素6的表达水平并没有改变CRISPLD2击倒,治疗与IL1b ASM细胞诱导表达明显高于白细胞介素6的CRISPLD2-knockdown细胞相比NT siRNA控制细胞(图4 b),这表明CRISPLD2在ASM细胞是免疫反应的抑制调制器。
与这一观点相一致的是，CRISPLD2敲低也增强了IL1b对另一种细胞因子(即IL8)的诱导[图S6]。
为了进一步研究CRISPLD2对免疫应答的影响，我们用100 nM DEX单独或与5 ng/mL IL1b联合处理细胞。
DEX处理后，il - 6表达降低，但CRISPLD2敲除没有显著改变il - 6对DEX的反应
(图4 c)。
然而，当同时给予IL1b和DEX时，CRISPLD2敲除细胞中的IL6 mRNA水平高于NT siRNA对照细胞[图4D]，再次支持了CRISPLD2在调节IL1b反应中的作用。

CRISPLD2不是GC靶基因表达所必需的

我们通过sirna介导的CRISPLD2基因敲除实验来评估CRISPLD2是否影响已知的gc应答基因(即DUSP1 [24]，FKBP5[25]或TSC22D3[25,27])的转录表达。
与对照组非靶向sirna转染的细胞相比，crispld2敲除的ASM细胞中三个GR靶基因的表达水平没有明显改变[图S7]，这表明CRIPSLD2并没有调节GCs在ASM中的直接转录作用。

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

在这个RNA-Seq试验中，采用了4种呼吸道平滑肌肉细胞（airway smooth muscle cells），每种细胞均有 地塞米松治疗、非治疗两类。共计8个样本，储存在 airway 包中。

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.

1.GCs通过与GC受体(GRs)结合来治疗各种炎症性疾病，包括哮喘。

2.许多细胞和组织参与哮喘并被GCs靶向，包括炎症、气道上皮和ASM。

3.与其他气道细胞相比，我们对GCs在ASM中具体如何工作以减轻哮喘的了解要少得多。

4.由于GCs通过激活GR直接调节转录基因表达来发挥作用，因此需要更好地理解ASM转录组如何响应GCs，从而为改善哮喘治疗提供机制上的见解。

5.两种微阵列基因表达:

1)专注于验证KLF15基因在气道高反应性中的功能;

2)关于ASM的GC和beta激动剂反应的重叠

6.与使用微阵列相比，RNA-Seq能够:

1)量化更多的RNA物种，包括非编码RNA和新剪接变异RNA;

2)在基线时量化RNA，而不是仅仅测量fold的变化
在条件下

3)覆盖更大的信号动态范围

7.使用RNA-Seq在体外模型中全面描述ASM转录组在GCs反应中的变化

8.316差异表达基因包括糖蛋白/细胞外基质、血管和肺发育、细胞迁移调节和细胞外基质组织。

9.crispld2 - snp吸入皮质类固醇反应和短效支气管扩张剂反应

10.功能实验表明，在ASM细胞中，CRISPLD2的mRNA和蛋白水平在糖皮质激素或促炎治疗后发生变化，而抑制CRISPLD2导致il1b诱导的IL6和IL8 mRNA表达水平升高。

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

3.CRISPLD2 Variants Associated with Asthma Pharmacogenetic Phenotypes

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

CRISPLD2在调节两种哮喘药物遗传表型中的作用:1)吸入皮质类固醇(ICS)反应性:2)支气管扩张剂反应性

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

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