MicroRNA-27a decreases the level and efficiency of the LDL receptor and contributes to the dysregulation of cholesterol homeostasis

Abstract

Rationale: A strong risk factor for atherosclerosis- the leading cause of heart attacks and strokes- is the elevation of low-density lipoprotein cholesterol (LDL-C) in blood. The LDL receptor (LDLR) is the primary pathway for LDL-C removal from circulation, and their levels are increased by statins -the main treatment for high blood LDL-C. However, statins have low efficiency because they also increase PCSK9 which targets LDLR for degradation. Since microRNAs have recently emerged as key regulators of cholesterol homeostasis, our aim was to identify potential microRNA-based therapeutics to decrease blood LDL-C and prevent atherosclerosis.

Methods and results: We over expressed and knocked down miR-27a in HepG2 cells to assess its effect on the expression of key players in the LDLR pathway using PCR Arrays, Elisas, and Western blots. We found that miR-27a decreases LDLR levels by 40% not only through a direct binding to its 3' untranslated region but also indirectly by inducing a 3-fold increase in PCSK9, which enhances LDLR degradation. Interestingly, miR-27a also directly decreases LRP6 and LDLRAP1, two other key players in the LDLR pathway that are required for efficient endocytosis of the LDLR-LDL-C complex in the liver. The inhibition of miR-27a using lock nucleic acids induced a 70% increase in LDLR levels and, therefore, it would be a more efficient treatment for hypercholesterolemia because of its desirable effects not only on LDLR but also on PCSK9.

Conclusion: The results presented here provide evidence supporting the potential of miR-27a as a novel therapeutic target for the prevention of atherosclerosis.

Keywords: Atherosclerosis; Cholesterol homeostasis; LDLR; LDLRAP1; LRP6; PCSK9; miR-27a.

Fig. 1. Effect of miR-27a on LRP6 expression in HepG2 cells

(A) Predicted annealing of human miR-27a to LRP6 3′UTR. HepG2 cells were transfected with either 50 nM of LNA anti-miR-27a, 50 nM LNA NC, 30 nM of miR-27a, or 30 nM NC mimic, in the absence (A) or presence (B) of plasmid pLRP6-3′UTR. Western blot was used to assess the expression of LRP6 protein in HepG2 cells transfected with either LNA anti-miR-27a or LNA NC (C) as well as with either miR-27a or NC mimic (E). The intensity of the protein bands was measured by densitometry (D and F). Data are mean of three independent experiments ± S.D. LNA, locked nucleic acids; NC, negative control; LRP6, LDLR-related protein 6. The significance is indicated only for samples that are significantly different from all the others. * P<.05; ** P<0.01; *** P<0.001.

Fig. 2. Regulation of LDLRAP1 by miR-27a

(A) Predicted annealing of human miR-27a to two sites on the LDLRAP1 3′UTR. (B and C) HepG2 cells were transfected with either 50 nM of LNA anti-miR-27a, 50 nM LNA NC, 30 nM of miR-27a mimic or 30 nM NC mimic, in the absence (B) or presence (C) of plasmid pLDLRAP1-3′UTR. The effect of miR-27a on the levels of LDLRAP1 mRNA was assessed by TaqMan qPCR (B), while luciferase activity was measured to determine the effect of miR-27a on protein levels (C). (D and E) The expression of LDLRAP1 protein in cells transfected with LNA anti-miR-27a or LNA NC was assessed by western-blot (D), and the intensity of the protein bands was measured by densitometry (E). Data are mean of three independent experiments ± S.D. LNA, locked nucleic acids; NC, negative control; LDLRAP1, LDLR-adapter protein 1. The significance is indicated only for samples that are significantly different from all the others. * P<.05; ** P<0.01; *** P<0.001.

Fig. 3. Effect of miR-27a on the levels of PCSK9

HepG2 cells were transfected with different concentrations of either LNA anti-miR-27a or LNA NC (A) or with either 30 nM miR-27a or NC mimic (B). The level of PCSK9 mRNA was quantified by TaqMan qPCR and the secreted PCSK9 protein by ELISA. Data are mean of three independent experiments ± S.D. LNA, locked nucleic acids; NC, negative control; PCSK9, proprotein convertase subtilisin/kexin type 9. The significance is indicated only for samples that are significantly different from all the others. * P<.05; ** P<0.01; *** P<0.001.

Fig. 4. Effect of miR-27a on the expression of LDLR in HepG2 cells

(A) Predicted annealing of human miR-27a to two sites on the LDLR 3′UTR. HepG2 cells were transfected with different concentrations of either LNA anti-miR-27a or LNA NC. (B) Cells were transfected with either 30 nM miR-27a, 30 nM NC mimic, 50 nM LNA anti-miR-27a or 50 nM LNA NC. The level of LDLR mRNA was quantified by TaqMan qPCR and the LDLR protein by ELISA. (C and D) Cells were co-transfected with 1 μg of reporter constructs pLDLR-3′UTR, pmiR-27a or mutated pmiR-27aM in the presence or absence of either 50 nM LNA anti-miR-27a, 50 nM LNA NC, 30 nM miR-27a or 30 nM NC mimic. (E) LDLR activity was assessed in HepG2 cells transfected with either 30 nM miR-27a or NC mimics and incubated overnight with LDL conjugated to DyLight^™ 549, a fluorescent probe for detection of LDL uptake. The degree of LDL uptake was examined in an Evos Digital Inverted Fluorescence Microscope (AMG, Fisher Scientific) with filters for excitation at 540 nm and emission at 570 nm. (F) The intensity of fluorescence was quantified at 540/570 nm excitation/emission using a Synergy MX plate reader (Biotek). Data are mean of three independent experiments ± S.D. LNA, locked nucleic acids; NC, negative control; LDLR, LDL receptor. The significance is indicated only for samples that are significantly different from all the others. * P<.05; ** P<0.01; *** P<0.001.

Fig. 5. Regulation of miR-27a in HepG2 cells

The level of miR-27a was assessed by TaqMan qPCR in HepG2 cells treated for 24 h with (A) Bay-11 (an inhibitor of NF-KB), LDL-C, simvastatin (commonly used statin that inhibits cholesterol synthesis), insulin and glucose; (B) 200 μM fatty acids (Sigma) conjugated with 0.2% BSA (30 mM) in 1% ethanol; different concentrations of simvastatin (C) or human LDL-C (D). Data are mean of three independent experiments ± S.D. The significance is indicated only for samples that are significantly different from all the others. * P<.05; ** P<0.01; *** P<0.001.

Fig. 6. Proposed mechanism for the effect of miR-27a and simvastatin on cholesterol homeostasis in the liver

(A) miR-27a directly downregulates LDLR, LDLRAP1 and LRP6 gene expression by binding to their 3′UTR region. LDLRAP1 and LRP6 proteins are essential for efficient endocytosis of LDLR in hepatocytes, a step required for the release of LDL-C inside the cell. miR-27a also upregulates the enzyme PCSK9, which induces LDLR degradation and, thus, further decreases the number of LDLRs on the surface of the cell. Therefore, miR-27a decreases the level and efficiency of the LDLR leading to a diminished LDL-C uptake and increased LDL-C levels in the blood that contributes to atherosclerosis. (B) Simvastatin (SIM) inhibits HMGCR (3-hydroxy-3-methylglutaryl-CoA reductase), the limiting rate enzyme of the cholesterol biosynthesis, which induces a decrease in intracellular (I. C.) cholesterol and subsequent increase in the expression of the LDLR. However, simvastatin not only increases the expression of PCSK9 --an enzyme that induces LDLR degradation-- but also miR-27a, which contribute to diminish the efficiency of the treatment with statins in patients with hypercholesterolemia.