Abstract
The cholesterol-lowering drug atorvastatin is among the most prescribed drug in the world. Alternative splicing in a number of genes has been reported to be associated with variable statin response. RNA-seq has proven to be a powerful technique for genome-wide splice variant analysis. In the present study, we sought to investigate atorvastatin responsive splice variants in HepG2 cells using RNA-seq analysis to identify novel candidate genes implicated in cholesterol homeostasis and in the statin response. HepG2 cells were treated with 10 µM atorvastatin for 24 hours. RNA-seq and exon array analyses were performed. The validation of selected genes was performed using Taqman gene expression assays. RNA-seq analysis identified 121 genes and 98 specific splice variants, of which four were minor splice variants to be differentially expressed, 11 were genes with potential changes in their splicing patterns (SYCP3, ZNF195, ZNF674, MYD88, WHSC1, KIF16B, ZNF92, AGER, FCHO1, SLC6A12 and AKAP9), and one was a gene (RAP1GAP) with differential promoter usage. The IL21R transcript was detected to be differentially expressed via RNA-seq and RT-qPCR, but not in the exon array. In conclusion, several novel candidate genes that are affected by atorvastatin treatment were identified in this study. Further studies are needed to determine the biological significance of the atorvastatin responsive splice variants that have been uniquely identified using RNA-seq.
Highlights
Atorvastatin is an efficient competitive inhibitor of 3-hydroxy-3methylglutaryl-Coenzyme A (HMG-CoA) reductase (HMGCR), the rate-limiting enzyme in cholesterol synthesis
Because we wanted to study the effects of statin that were due to the inhibition of cholesterol biosynthesis, we used the magnitude of increased HMGCR mRNA expression as a marker for statin response
The initial experiments showed that HepG2 cells grown in medium containing 10% FBS had low HMGCR mRNA levels, which remained unchanged when treated with atorvastatin
Summary
Atorvastatin is an efficient competitive inhibitor of 3-hydroxy-3methylglutaryl-Coenzyme A (HMG-CoA) reductase (HMGCR), the rate-limiting enzyme in cholesterol synthesis. Atorvastatin belongs to the statin class of drugs and is widely used to reduce cholesterol levels and the risk of cardiovascular disease. The cholesterol-lowering effect of statins is well documented. Statins block HMGCR and prevent the conversion of HMG-CoA to mevalonate and thereby decrease the level of sterol and non-sterol products derived from mevalonate, including cholesterol. The expression level of a minor splice variant of HMGCR lacking exon 13 has been shown to be associated with variations in the plasma LDL-cholesterol levels and statin response [5]. The exact molecular mechanisms underlying these LDL-cholesterol independent effects of statins are still unclear
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