Transcriptomic profiling of differentially expressed mRNA, lncRNA and circRNA in atorvastatin-treated human endothelial cells

Statins are competitive inhibitors of (3-hydroxy-3-methylglutaryl coenzyme A) HMG-CoA reductase. The primary mechanism of statins is the lowering of serum cholesterol through inhibiting hepatic cholesterol biosynthesis. Statins exert pleiotropic vasoprotective and atheroprotective effects in the vasculature, with its mechanism of action being incompletely understood.To depict the statin-regulated mRNA,long non-coding RNA (lncRNA) and circular RNA (circRNA) in human endothelial cells, we performed transcriptomic profiling of human umbilical vein endothelial cells (HUVECs) with atorvastatin treatment.Differentially expressed mRNA, lncRNA and circRNA may represent novel therapeutic targets against cardiovascular diseases. Overall design: Human umbilical vein endothelial cells (HUVECs) were seeded in 0.2% gelatin-coated dishes the day before experiment. The next day, cells were treated with DMSO or atorvastatin (10µM) for 24 hours. After treatment, total RNA was isolated using the RNA-Easy Mini Plus kit (QIAGEN). RNA libraries were prepared for sequencing using rRNA Removal Library Construction Protocol. Briefly, take a certain amount of total RNA samples, and remove rRNA by using RNase H or Ribo-Zero method, then fragment the RNA. Obtain the first and second strands of cDNA by reverse transcription. Then add adapters at both ends of the cDNA for PCR amplification to obtain the cDNA library. The library was amplified with phi29 to make DNA nanoball (DNB) which have more than 300 copies of one molecular. The DNBs were load into the patterned nanoarray and single end 50 (pair end 100) bases reads were generated in the way of combinatorial Probe-Anchor Synthesis (cPAS). The sequencing data was filtered with SOAPnuke (v1.5.2), afterwards clean reads were obtained and stored in FASTQ format. The clean reads were mapped to the reference genome using HISAT2 (v2.0.4). Bowtie2 (v2.2.5) was applied to align the clean reads to the gene set, a database built by BGI (Beijing Genomic Institute in ShenZhen), then expression level of gene was calculated by RSEM (v1.2.12).

他汀类药物为（3-羟基-3-甲基戊二酰辅酶A，即HMG-CoA）还原酶的竞争性抑制剂。其核心作用机制为通过抑制肝脏胆固醇生物合成，降低血清胆固醇水平。他汀类药物可在血管系统中发挥多效性血管保护与抗动脉粥样硬化作用，但其完整作用机制尚未完全阐明。

为解析人内皮细胞中他汀调控的信使RNA（mRNA）、长链非编码RNA（lncRNA）及环状RNA（circRNA）表达谱，我们对阿托伐他汀处理后的人脐静脉内皮细胞（HUVECs）开展了转录组表达谱分析。差异表达的mRNA、lncRNA与circRNA或可作为对抗心血管疾病的新型治疗靶点。

整体实验设计如下：实验前一日，将人脐静脉内皮细胞（HUVECs）接种于0.2%明胶包被的培养皿中。次日，采用二甲基亚砜（DMSO）或10μM阿托伐他汀处理细胞24小时。处理完成后，使用RNA-Easy Mini Plus试剂盒（QIAGEN）提取总RNA。采用核糖体RNA去除建库流程构建RNA测序文库：取适量总RNA样品，通过核糖核酸酶H（RNase H）或Ribo-Zero法去除核糖体RNA（rRNA），随后对RNA进行片段化；经逆转录合成互补DNA（cDNA）第一链与第二链；在cDNA两端添加接头后进行聚合酶链式反应（PCR）扩增，最终获得cDNA文库。将文库通过phi29酶扩增，得到包含单分子300余份拷贝的DNA纳米球（DNB）。将DNB加载至patterned纳米阵列，采用组合探针锚定合成（cPAS）技术生成单端50碱基（双端100碱基）的测序读段。

测序数据经SOAPnuke（v1.5.2）过滤后，得到干净读段（clean reads）并以FASTQ格式存储。使用HISAT2（v2.0.4）将干净读段比对至参考基因组；采用Bowtie2（v2.2.5）将干净读段比对至由深圳华大基因（BGI）构建的基因集数据库，随后通过RSEM（v1.2.12）计算基因表达水平。