156 Regulation of LDL-cholesterol Associated Gene Expression by Iron Responsive Elements and Iron

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Variants in 37 genes have been associated with low density lipoprotein cholesterol (LDL-C) levels, including HFE (High Fe) p.(C282Y), rs1800562. This variant is homozygous in ~90% patients with haemochromatosis. It may reduce signalling to hepcidin, increasing iron absorption. However it has an extended founder haplotype and the mechanism leading to association with LDL-C levels is not known. This study aimed to investigate the effects of predicted iron responsive elements (IREs) and iron on LDL-C associated gene expression. HepG2 cells were treated with 10 µM ferric ammonium citrate (FAC, “free” iron), 30 µM haemin (haem iron), 10 µM desferrioxamine (DFO, parenteral iron chelator) and 100 µM deferiprone (L1, oral iron chelator) for 24 h (> = 4 biological replicates). Total RNA was isolated from cells for real-time quantitative PCR TaqMan assays and relative quantification using the [INCREMENT]Ct method. The mean of ACTB and HPRT1 was used as the endogenous gene expression control. Eleven LDL-C mRNAs were predicted to contain IREs. SORT1 was predicted to contain a classical IRE in the 3’-untranslated region. SORT1 expression after FAC was 29% lower (p = 0.03) than control cells and was not significantly altered by haemin, DFO or L1. PCSK9 contained a predicted IRE but it was located within coding sequence. PCSK9 mRNA levels showed a trend towards 38% reduction after iron supplementation with FAC (p = 0.05) but were not significantly altered by haemin; they were 117 and 133% higher after iron reduction by chelation with DFO (p = 0.04) and L1 (p = 0.04) respectively compared to control cells. LDLR contained no predicted IRES. LDLR expression was not significantly altered by FAC or haemin but was 72% and 53% higher after treatment with DFO (p = 0.009) and L1 (p = 0.0001). These findings support a mechanism underlying the reported association of the HFE p.(C282Y) variant with lower LDL-C levels involving the regulation of LDL-C – associated gene expression by iron.

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