Transcriptome profiling of Transforming Growth Factor B1-Induced Mesangial Gene Expression

TGF-beta1 is a pleiotropic cytokine that regulates multiple cellular functions including cell proliferation, differentiation, and apoptosis. It has been identified as one of the mediators of renal hypertrophy and accumulation of extracellular matrix proteins in the diabetic glomerular mesangium. Previous results from our and other laboratories indicate that TGF-beta1 mediates some of the effects of high glucose and glucosamine on extracellular matrix gene expression in mesangial cells. The global regulatory mechanism(s), however, still remains to be understood. In this study, we have utilized an in vitro model of mouse mesangial cells (MES-13) to investigate the genetic effects of TGF-beta1 on global patterns of gene expression, transcriptome and signaling networks. A total number of 179 genes are regulated by TGF-beta1 in MES-13 cells at 2.0-fold, which cover across 44 physiological processes, 31 metabolic functions, and 11 major signaling pathways. We observed that several genes regulated by high glucose and glucosamine are also targeted by TGF-beta1 in MES-13 cells such as thioredoxin interacting protein, glutathione S-transferase theta 1, selenium binding protein 1, inhibitor of DNA binding 2, and plasminogen activator inhibitor-1. The results of this study further support the hypothesis that some effects of high glucose and glucosamine on mesangial gene expression are mediated by TGF-beta1. The identification of these common genes will help us to investigate further their roles in the development and progression of diabetic kidney disease. Cell culture: Stable murine mesangial (MES-13) cells transformed with non-capsid-forming SV-40 virus were obtained from the ATCC, Manassas, VA. These cells display a differentiated mesangial cell phenotype including the typical spindle-like appearance, positive staining for vimentin and desmin, and contraction in response to ANG II and expression of AT1 receptor. The cells were maintained in DMEM and F-12 Nutrient Mixture (Ham's) (4:1 ratio) (GIBCO BRL, Gaithersburg, MD) containing a normal D-glucose concentration of 5.5 mmol/L, 2% FCS, 100 µg/ml streptomycin, 100 U/ml penicillin, and 2 mmol/L glutamine (26). The cells were incubated in a humidified incubator of 5% CO2 at 37 °C and routinely passaged at confluence every 3 days by trypsinization using 10-cm culture dishes. Approximately 50% confluent monolayers were starved in the above medium without FCS for 1 day and then incubated in the starvation medium with 100 ng/ml TGF-beta1 for 24hrs. Total RNA isolation, cDNA and cRNA synthesis and genechip hybridization: Total RNA was isolated using Trizol reagent (Life Technologies, Inc., Massachusetts). cDNA and cRNA synthesis, genechip hybridization, and scanning of the Affymetrix murine expression U430 2.0 chips (the Affymetrix M430 2.0 chip contains 39,000 transcripts targeted at 34,000 well characterized genes) were performed according to the manufacturer's protocol (Affymetrix, Santa Clara, CA). Briefly, 5 mg of RNA was converted into double-stranded cDNA by reverse transcription using a cDNA synthesis kit (SuperScript Choice, Life Technologies, Inc., MA) with an oligo(dT)24 primer containing a T7 RNA polymerase promoter site added 3' of the poly(T) (Genset, La Jolla, CA). After second-strand synthesis, labeled cRNA was generated from the cDNA sample by an in vitro transcription reaction supplemented with biotin-11-CTP and biotin-16-UTP (Enzo, Farmingdale, NY). The labeled cRNA was purified by using RNeasy spin columns (Qiagen, Valencia, CA). 15 mg of each cRNA was fragmented at 94 °C for 35 min in fragmentation buffer (40 mM Tris acetate, pH 8.1, 100 mM potassium acetate, 30 mM magnesium acetate) and then used to prepare 300 ml of hybridization mixture (100 mM MES, 0.1 mg/ml herring sperm DNA (Promega), 1M sodium chloride, 10 mM Tris, pH 7.6, 0.005% Triton X-100) containing a mixture of control cRNAs samples were heated at 94 °C for 5 min, equilibrated at 45 °C for 5 min, and clarified by centrifugation (14,000 x g) at room temperature for 5 min. Aliquots of each sample (10 mg of cRNA in 200 ml of the master mix) were hybridized to GeneChip Mouse Genome 430 2.0 Array at 45 °C for 16 h in a rotisserie oven set at 60 rpm, then washed with non stringent wash buffer (6x saline/sodium phosphate/EDTA) at 25 °C, followed by stringent wash buffer (100 mM MES (pH 6.7), 0.1 M NaCl, 0.01% Tween 20) at 50 °C, stained with streptavidin-phycoerythrin (Molecular Probe), washed again with 6x saline/sodium phosphate/EDTA, stained with biotinylated anti-streptavidin lgG, followed by a second staining with streptavidin-phycoerythrin, and a third washing with 6x saline/sodium phosphate/EDTA using the GeneChip Fluidics Station 450. The arrays were then scanned using a GeneChip Scanner 3000 (Affymetrix). Each experiment was repeated twice. Microarray Data analysis: The chips were read with Affymetrix GCOS v1.2, and the probe intensity files were modeled with DChip 1.3 (Harvard School of Public Health) in both PM-only and PM-MM modes. Consensus differentially regulated genes were initially derived from repeat experiments on the bases of a 90% CI of greater than 2-fold change in expression and with p<0.05 of error in paired t-test across repeats. This was further validated through modeling of variance between sample groups (one-way ANOVA) conducted in GeneSpring 6.0 (Silicon Genetix). The consensus gene-set was clustered in DChip and GeneSpring 6.0, with OntoExpress (Wayne State University) to explore ontological associations. Further ontological and pathway analyses were conducted with the GeneGo software and NIH's DAVID bioinformatics programs (heep://appls.niaid.nih.gov/david). Keywords = TGFBeta1 Keywords = mesangial cells Keywords = diabetic glomerulopathy Keywords: repeat sample

转化生长因子-β1（TGF-beta1）是一种多效性细胞因子，可调控细胞增殖、分化与凋亡等多种细胞功能。研究已证实其为糖尿病肾小球系膜区肾肥大与细胞外基质蛋白蓄积的介导因子之一。本实验室及其他团队此前的研究结果表明，TGF-beta1可介导高糖与氨基葡萄糖对系膜细胞细胞外基质基因表达的部分影响，但其全局调控机制仍有待阐明。本研究利用小鼠系膜细胞（MES-13）体外模型，探究TGF-beta1对基因表达全局模式、转录组及信号网络的遗传调控效应。在MES-13细胞中，共有179个基因受TGF-beta1调控（表达变化达2.0倍），这些基因涉及44种生理过程、31项代谢功能以及11条主要信号通路。我们观察到，若干受高糖与氨基葡萄糖调控的基因同时也是TGF-beta1在MES-13细胞中的靶标，例如硫氧还蛋白相互作用蛋白、谷胱甘肽S-转移酶θ1、硒结合蛋白1、DNA结合抑制因子2以及纤溶酶原激活物抑制剂-1。本研究结果进一步支持了“高糖与氨基葡萄糖对系膜细胞基因表达的部分影响由TGF-beta1介导”这一假说。上述共同靶基因的鉴定将有助于我们进一步探究其在糖尿病肾病发生与进展中的作用。

细胞培养：将经非衣壳形成型SV-40病毒转化的稳定小鼠系膜细胞（MES-13）从美国典型培养物保藏中心（ATCC，弗吉尼亚州马纳萨斯）获取。该细胞呈现分化型系膜细胞表型，包括典型的梭形外观、波形蛋白与结蛋白染色阳性，以及对血管紧张素Ⅱ（ANG Ⅱ）的收缩反应和AT1受体的表达。细胞培养于达尔伯克改良伊格尔培养基（DMEM）与Ham's F-12营养混合物按4:1比例混合的培养基（GIBCO BRL，马里兰州盖瑟斯堡）中，该培养基含正常浓度D-葡萄糖（5.5 mmol/L）、2%胎牛血清（FCS）、100 µg/ml链霉素、100 U/ml青霉素以及2 mmol/L谷氨酰胺(26)。细胞置于含5% CO₂的湿润培养箱中37 ℃培养，常规每3天汇合后用胰蛋白酶消化传代，培养器皿为10 cm培养皿。将约50%汇合度的单层细胞在不含FCS的上述培养基中饥饿处理1天，随后在含100 ng/ml TGF-beta1的饥饿培养基中孵育24小时。

总RNA提取、cDNA与cRNA合成及基因芯片杂交：使用TRIzol试剂（Life Technologies公司，马萨诸塞州）分离总RNA。按照制造商方案（Affymetrix，加利福尼亚州圣克拉拉）完成cDNA与cRNA合成、基因芯片杂交以及Affymetrix小鼠表达谱U430 2.0芯片（Affymetrix M430 2.0芯片包含针对34000个已充分表征基因的39000条转录本）的扫描。简要步骤如下：取5 µg RNA，通过逆转录反应合成双链cDNA，所用试剂盒为SuperScript Choice（Life Technologies公司，马萨诸塞州），引物为带有T7 RNA聚合酶启动子位点的oligo(dT)₂₄，该位点添加于poly(T)的3'端（Genset，加利福尼亚州拉霍亚）。第二链合成完成后，通过体外转录反应从cDNA样本合成标记cRNA，反应体系补充生物素-11-CTP与生物素-16-UTP（Enzo，纽约州法明顿）。使用RNeasy离心柱（Qiagen，加利福尼亚州瓦伦西亚）纯化标记后的cRNA。取15 µg每份cRNA在裂解缓冲液（40 mM Tris 乙酸盐，pH 8.1，100 mM乙酸钾，30 mM乙酸镁）中94 ℃裂解35分钟，随后用于制备300 µl杂交混合液（100 mM MES，0.1 mg/ml鲑鱼精DNA（Promega），1 M氯化钠，10 mM Tris，pH 7.6，0.005% Triton X-100）。混合对照cRNA样本的杂交混合液先94 ℃加热5分钟，45 ℃平衡5分钟，室温下14000×g离心5分钟澄清。每份样本的分装液（200 µl主混合液中含10 µg cRNA）在45 ℃、60 rpm旋转烤箱中与GeneChip小鼠基因组430 2.0阵列杂交16小时。随后用非严谨洗涤缓冲液（6× 生理盐水/磷酸钠/EDTA）在25 ℃洗涤，再用严谨洗涤缓冲液（100 mM MES，pH 6.7，0.1 M氯化钠，0.01% Tween 20）在50 ℃洗涤。使用链霉亲和素-藻红蛋白（Molecular Probe）染色，再次用6× 生理盐水/磷酸钠/EDTA洗涤，随后用生物素化抗链霉亲和素IgG染色，再用链霉亲和素-藻红蛋白进行第二次染色，最后使用GeneChip Fluidics Station 450以6× 生理盐水/磷酸钠/EDTA完成第三次洗涤。使用GeneChip Scanner 3000（Affymetrix）扫描芯片。每个实验重复两次。

微阵列数据分析：使用Affymetrix GCOS v1.2读取芯片数据，探针强度文件通过DChip 1.3（哈佛大学公共卫生学院）以PM-only和PM-MM两种模式建模。基于重复实验中90%置信区间内表达变化大于2倍、配对t检验误差p<0.05，初步获得一致性差异表达基因。进一步通过GeneSpring 6.0（Silicon Genetix）中的样本组间方差建模（单因素方差分析）进行验证。使用DChip与GeneSpring 6.0对一致性基因集进行聚类，并通过OntoExpress（韦恩州立大学）探究其本体论关联。进一步的本体论与通路分析使用GeneGo软件及美国国立卫生研究院（NIH）的DAVID生物信息学程序（http://appls.niaid.nih.gov/david）完成。

关键词 = TGFBeta1；关键词 = 系膜细胞；关键词 = 糖尿病肾小球病；关键词：重复样本