Human microRNA-155 on chromosome 21 differentially interacts with its polymorphic target in the AGTR1 3' untranslated region: a mechanism for functional single-nucleotide polymorphisms related to phenotypes

Abstract

Animal microRNAs (miRNAs) regulate gene expression through base pairing to their targets within the 3' untranslated region (UTR) of protein-coding genes. Single-nucleotide polymorphisms (SNPs) located within such target sites can affect miRNA regulation. We mapped annotated SNPs onto a collection of experimentally supported human miRNA targets. Of the 143 experimentally supported human target sites, 9 contain 12 SNPs. We further experimentally investigated one of these target sites for hsa-miR-155, within the 3' UTR of the human AGTR1 gene that contains SNP rs5186. Using reporter silencing assays, we show that hsa-miR-155 down-regulates the expression of only the 1166A, and not the 1166C, allele of rs5186. Remarkably, the 1166C allele has been associated with hypertension in many studies. Thus, the 1166C allele may be functionally associated with hypertension by abrogating regulation by hsa-miR-155, thereby elevating AGTR1 levels. Since hsa-miR-155 is on chromosome 21, we hypothesize that the observed lower blood pressure in trisomy 21 is partially caused by the overexpression of hsa-miR-155 leading to allele-specific underexpression of AGTR1. Indeed, we have shown in fibroblasts from monozygotic twins discordant for trisomy 21 that levels of AGTR1 protein are lower in trisomy 21.

Figure 1.

Nine experimentally supported miRNA target sites harbor 12 SNPs. The studies that reported the nine miRNA-target interactions shown here used one or more of the several publicly available computational target-prediction programs to compute the putative binding diagrams. Seven of the nine SNPs (rs8179014, rs17500932, rs41269629, rs17860720, rs11416, rs5185, and rs12721277) occur in a region of a 5′-dominant target site that does not affect 5′-dominant base pairing. Three SNPs (rs11541536, rs9579381, and rs9341070) occur in a region of a 3′-compensatory target site that can affect 3′-compensatory base pairing. Two SNPs (rs5186 and rs8829) occur in a region of a 5′-dominant target site that does affect 5′-dominant base pairing.

Figure 2.

Effects of miR-155 on the luciferase reporter genes bearing 3′ UTR segments from hAGTR1 3′-UTR. 293T cells were cotransfected with pTAL-Luc hAGTR1 3′-UTR 1166A or pTAL-Luc hAGTR1 3′ UTR 1166C, pTAL-Luc hAGTR1 3′ UTR deletion 1163–1169, and different concentrations (20, 10, or 2.5 nM) of miR-155 (white bars) or no miRNA (black bars) or 20 nM of Let-7c (unrelated miRNA [dashed lines]). Values are means of firefly/Renilla ratio from three independent experiments (each with three culture replicates). Error bars indicate 1 SD of three independent experiments. P values were calculated from a two-sided, two-sample t test.

Figure 3.

Real-time quantitative PCR for mature miR-155 expression in fibroblast cells from MZ twins discordant for trisomy 21. Data are means (±SD) from three independent experiments analyzed in six replicates. Data are normalized with reference microRNAs, as mentioned in the text. P values were calculated from a two-sided, one-sample t test.

Figure 4.

AGTR1 radioligand binding studies in fibroblasts from MZ twins discordant for trisomy 21. There is a lower amount of AGTR1 protein in trisomy 21 fibroblasts than in the unaffected euploid twin fibroblasts.

Figure 5.

Model for molecular mechanism of 1166C association with hypertension. The 1166C allele in the 3′ UTR of AGTR1 abrogates miR-155 binding, which induces elevated levels of AGTR1.