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. 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).