Transcriptome perturbations within NIH3T3 cell lines expressing rhodopsin and its retinitis pigmentosa mutant and implication for drug screening

Mammalian cells are commonly employed to identify active compounds that could affect progression of many human diseases including retinitis pigmentosa. Here, using transcriptome analysis to compare NIH3T3 cells expressing rod opsin with the disease-causing a single point P23H mutation, we differentiated between genes affected by heterologous opsin expression and those influenced by the P23H opsin mutant gain- or loss-of-function. Surprisingly, heterologous expression of normal opsin causes changes in 783 out of 16888 protein coding transcripts more than 1 fragments per kilobase of transcript per million mapped reads (FPKM) in NIH3T3 cells despite that opsin is exogenous to this cell. The perturbed genes are involved in cell adhesion, morphology and migration and encode extracellular matrix proteins, growth factors, cytoskeleton proteins, glycoproteins or metalloproteases. Not fully overlapping 347 differentially expressed genes were affected when the P23H mutant opsin was expressed. Transcriptome perturbation by individual drug candidates also revealed that different active compounds can target distinct molecular pathways that result in a similar phenotype selected by a cell-based high throughput screen. This transcriptome approach is capable of detecting minute changes in the transcriptome and can be a key to therapeutic success of a candidate drug to restore the normal gene expression landscape in affected tissue. Overall design: Five cell assay conditions were arranged: 1) NIH3T3(GFP) cells were treated with 0.05% DMSO, labeled as WT; 2) NIH3T3(WT-opsin/GFP) cells were treated with 0.05% DMSO, labeled as OPSIN; 3) NIH3T3(P23H-opsin/GFP) cells were treated with 0.05% DMSO, labeled as P23H; 4) NIH3T3(P23H-opsin/GFP) cells were treated with 5 µM of compound 1 (an isoquinoline-2(3H)-hexanamide), labeled as T1; 5) NIH3T3(P23H-opsin/GFP) cells were treated with 10 µM of compound 2 (4-(5-chlorothiophen-2-yl)furan-2(5H)-one), labeled as T2. Each treatment condition was repeated in three 50 mm dishes as biological replicates. The medium was aspirated, and cells were washed with PBS after 24 h of each treatment before being lysed. Cells were lysed with TRIzol and total RNA was extracted from each sample according to the TRIzol reagent manual. High quality total RNA was subjected to sequencing library construction using 100 ng of total RNA as input. Transcript libraries were made with TruSeq Stranded mRNA Sample Prep Kit (Illumina, San Diego, CA, USA). The 100 base paired-end sequenced samples were run on 2 lanes of a HiSeq 2500. Fastq files were generated from reads passing chastity filter.

哺乳动物细胞常被用于筛选可影响包括色素性视网膜炎在内的多种人类疾病进展的活性化合物。本研究通过转录组分析，对比表达野生型视杆视紫红质（rod opsin）与携带致病单点P23H突变视紫红质的NIH3T3细胞，区分了受异源视紫红质表达影响的基因，以及受P23H视紫红质突变体功能获得或功能缺失调控的基因。令人意外的是，尽管视紫红质对于NIH3T3细胞属于外源性蛋白，野生型视紫红质的异源表达仍使该细胞中16888个蛋白编码转录本里的783个发生了超过1个每百万映射读段每千碱基转录本（FPKM, fragments per kilobase of transcript per million mapped reads）的表达变化。受表达扰动的基因参与细胞黏附、形态维持与迁移过程，编码细胞外基质蛋白、生长因子、细胞骨架蛋白、糖蛋白或金属蛋白酶。当表达P23H突变视紫红质时，另有347个差异表达基因受到调控，且该基因集与异源表达野生型视紫红质所影响的基因集并未完全重叠。对单个候选药物引发的转录组扰动分析还显示，不同活性化合物可靶向不同的分子通路，最终产生基于细胞的高通量筛选所筛选到的相似表型。该转录组分析方法可检测转录组中细微的表达变化，有望成为候选药物恢复受累组织正常基因表达谱、实现治疗成功的关键突破口。整体实验设计：共设置5组细胞培养条件：1）用0.05%二甲基亚砜（DMSO）处理NIH3T3(GFP)细胞，标记为WT组；2）用0.05% DMSO处理NIH3T3(野生型视紫红质/GFP)细胞，标记为OPSIN组；3）用0.05% DMSO处理NIH3T3(P23H突变视紫红质/GFP)细胞，标记为P23H组；4）用5 μM化合物1（异喹啉-2(3H)-己酰胺）处理NIH3T3(P23H突变视紫红质/GFP)细胞，标记为T1组；5）用10 μM化合物2（4-(5-氯噻吩-2-基)呋喃-2(5H)-酮）处理NIH3T3(P23H突变视紫红质/GFP)细胞，标记为T2组。每组处理设置3个50 mm培养皿作为生物学重复。每组处理24小时后，吸去培养基，用磷酸盐缓冲液（PBS）洗涤细胞后进行裂解。使用TRIzol试剂裂解细胞，并按照TRIzol试剂说明书从每个样本中提取总RNA。取100 ng高质量总RNA作为起始材料，构建测序文库。转录组文库采用TruSeq Stranded mRNA Sample Prep Kit（Illumina, San Diego, CA, USA）构建。将样本进行100 bp双端测序，上机运行于HiSeq 2500的2个测序泳道。从通过纯度过滤（chastity filter）的读段中生成Fastq格式文件。