MicroRNA-20a-5p Downregulation by Atorvastatin: A Potential Mechanism Involved in Lipid-Lowering Therapy

Abstract

The treatment of hypercholesterolemia is mainly based on statins. However, the response to pharmacological therapy shows high inter-individual variability, resulting in variable effects in both lipid lowering and risk reduction. Thus, a better understanding of the lipid-lowering mechanisms and response variability at the molecular level is required. Previously, we demonstrated a deregulation of the microRNA expression profile in HepG2 cells treated for 24 h with atorvastatin, using a microarray platform. In the present study, we evaluated the expression of hsa-miR-17-5p, hsa-miR-20a-5p and hsa-miR-106a-5p in hypercholesterolemic patients before and after atorvastatin treatment and in HepG2 cells treated for 24 h with atorvastatin The miRNA hsa-mir-20a-5p was repressed after atorvastatin treatment in hypercholesteremic subjects and in HepG2 cells in culture. Repression of hsa-mir-20a-5p increased LDLR gene and protein expression in HepG2 cells, while hsa-mir-20a-5p overexpression reduced LDLR gene and protein expression.

Keywords: atorvastatin; cardiovascular diseases; hypercholesterolemia; microRNA; statin.

Figure 1

Relative expression of miRNAs in hypercholesteremic patients treated with atorvastatin. (a) hsa-miR-17-5p, (b) hsa-miR-20a-5p and (c) hsa-miR-106a-5p. The relative expression was determined by RT-qPCR from enriched RNA extracted from leukocyte cells in the peripheral blood of hypercholesteremic patients before and after atorvastatin treatment (20 mg/day/4 weeks). Normalization was performed using hsa-miR-191 as a reference gene; p-value was obtained by paired t-test.

Figure 2

Relative expression of miRNAs in HepG2 cells treated with ATV 10 μM. Gene expression analysis of miRNAs hsa-miR-17-5p, hsa-miR-20a-5p and hsa-miR-106a-5p in control cells, vehicle-treated cells (methanol 0.1%) and cells treated with 10 μM ATV. * p = 0.0456 *** p < 0.0001 by unpaired t-test when compared with vehicle-treated cells (methanol 0.1%). The experiments were performed in technical and biological triplicates. ATV: atorvastatin.

Figure 3

Binding sites for the miRNA hsa-miR-20a-5p in the 3′UTR region of LDLR. The 3′UTR region of LDLR in humans has two predicted and conserved binding sites for hsa-miR-20a-5p. The seed sequence of hsa-miR-20a-5p is shown in red, while its respective complementary region in the 3′UTR is shown in blue. Conserved sites between species are shown below: hsa, human; ptr, chimpanzee; mmu, mouse; rno, rat. LDLR: low-density lipoprotein receptor; UTR: untranslated region.

Figure 4

Post-transcriptional regulation of LDLR expression by hsa-miR-20a-5p in HepG2 cells. (a) LDLR gene expression analysis by RT-qPCR in HepG2 cells transfected with a negative control mimic (NC) or an hsa-miR-20a-5p mimic. (b) Gene expression analysis of LDLR by RT-qPCR in HepG2 cells transfected with a negative control inhibitor (NC) or an hsa-miR-20a-5p inhibitor. (c) LDLR protein expression analysis by western blot (representative image) of LDLR in HepG2 cells transfected with a negative control mimic (NC) or an hsa-miR-20a-5p mimics. (d) LDLR protein expression analysis by western blot (representative image) of LDLR in HepG2 cells transfected with a negative control inhibitor (NC) or an hsa-miR-20a-5p inhibitor. * p < 0.05; ** p < 0.005, *** p < 0.0001, compared to cells treated with negative control mimics or the inhibitor as appropriate, by unpaired t-test. The experiments were performed in technical and biological triplicates.