The human angiotensin II type 1 receptor +1166 A/C polymorphism attenuates microRNA-155 binding

Abstract

The adverse effects of angiotensin II (Ang II) are primarily mediated through the Ang II type 1 receptor (AT1R). A silent polymorphism (+1166 A/C) in the human AT1R gene has been associated with cardiovascular disease, possibly as a result of enhanced AT(1)R activity. Because this polymorphism occurs in the 3'-untranslated region of the human AT1R gene, the biological importance of this mutation has always been questionable. Computer alignment demonstrated that the +1166 A/C polymorphism occurred in a cis-regulatory site, which is recognized by a specific microRNA (miRNA), miR-155. miRNAs are noncoding RNAs that silence gene expression by base-pairing with complementary sequences in the 3'-untranslated region of target RNAs. When the +1166 C-allele is present, base-pairing complementarity is interrupted, and the ability of miR-155 to interact with the cis-regulatory site is decreased. As a result, miR-155 no longer attenuates translation as efficiently as demonstrated by luciferase reporter and Ang II radioreceptor binding assays. In situ hybridization experiments demonstrated that mature miR-155 is abundantly expressed in the same cell types as the AT1R (e.g. endothelial and vascular smooth muscle). Finally, when human primary vascular smooth muscle cells were transfected with an antisense miR-155 inhibitor, endogenous human AT1R expression and Ang II-induced ERK1/2 activation were significantly increased. Taken together, our study demonstrates that the AT1R and miR-155 are co-expressed and that miR-155 translationally represses the expression of AT1R in vivo. Therefore, our study provides the first feasible biochemical mechanism by which the +1166 A/C polymorphism can lead to increased AT1R densities and possibly cardiovascular disease.

FIGURE 1. The hAT₁R + 1166 A/C SNP occurs in the miR-155-binding site

A, complementarity between miR-155 and the hAT₁R 3′-UTR site targeted (70 –90 bp downstream from the hAT₁R stop codon). The +1166 A/C SNP corresponds to the nucleotide 86 bp downstream from the hAT₁R stop codon (in boldface). The binding of miR-155 to the hAT₁R 3′-UTR target site fulfills the requirement of a 7-bp seed sequence of complementarity at the miRNA 5′ end (21, 22) when the +1166 A-allele is expressed. B, complementarity between miR-155 and the hAT₁R 3′-UTR harboring the +1166 C-allele. If the +1166 C-allele is expressed then the seed sequence requirement would not be met. C, computational modeling of the interaction between miR-155 and the hAT₁R 3′-UTR cis-response element +1166 A/C SNP was performed utilizing the RNAHYBRID software (35). Hybridization of miR-155 and hAT₁R mRNA harboring the A-allele. D, hybridization of miR-155 and hAT₁R mRNA harboring the C-allele. Arrows highlight the polymorphic site. Calculated mfe, minimal free energy, is shown.

FIGURE 2. The hAT₁R + 1166 A/C SNP decreases miR-155 effectiveness

A, schematic representation of the firefly luciferase (f-luc) reporter constructs utilized. The p3′-UTR-A plasmid harbors the full-length hAT₁R 3′-UTR (i.e. 883 bp) in the 5′→3′ orientation with respect to the firefly luciferase open reading frame. The “A” represents the A-allele at position +1166. The p3′-UTR-C plasmid also contains the hAT₁R 3′-UTR however this plasmid harbors a point mutation to represent the +1166 C-allele. B, CHO cells were co-transfected with p3′-UTR-A or p3′-UTR-C, pRL-CMV, and either miR-Controls or miR-155 at the concentrations indicated. Forty eight hours after transfection, luciferase activities were measured. Firefly luciferase activity was normalized to Renilla luciferase expression, and mean activities ± S.E. from five independent experiments are shown (*, p < 0.01 versus CHO cells transfected with p3′-UTR-C at each concentration shown).

FIGURE 3. A mutation in miR-155 can restore its ability to inhibit the hAT₁R +1166 C-allele

A, schematic representation of the location of the mutation introduced in miR-155 when it was chemically synthesized. This mutation eliminates a complementary bp in the miR-155 seed sequence when the hAT₁R +1166 A-allele is present. The mutation in mut-miR-155 restores the complementary bp in the miR-155 seed sequence when the hAT₁R + 1166 C-allele is present. B, CHO cells were co-transfected with p3′-UTR-A or p3′-UTR-C, pRL-CMV, and either miR-Controls or mutant miR-155 at the concentrations indicated. Forty eight hours after transfection, luciferase activities were measured. Firefly luciferase activity was normalized to Renilla luciferase expression, and mean activities ± S.E. from five independent experiments are shown (*, p < 0.01 versus CHO cells transfected with p3′-UTR-A at each concentration shown).

FIGURE 4. Mir-155 seed sequence complementarity is needed to inhibit hAT₁R expression

A, schematic representation of the hAT₁R expression constructs utilized in transfection experiments. The phAT₁R-A plasmid harbors a full-length hAT₁R cDNA in the 5′ → 3′ orientation with respect to the CMV promoter. The “A” represents the A-allele at position +1166. The phAT₁R-C plasmid also contains the full-length hAT₁R cDNA; however, this plasmid harbors a point mutation to represent the +1166 C-allele. B, CHO cells were co-transfected with phAT₁R-A, pRL-CMV, and either miR-Controls, miR-155, or mutant miR-155 at 50 nM final concentration. C, alternatively, identical experiments were performed utilizing the phAT₁R-C expression construct. Forty eight hours after transfection, luciferase activities were measured. Firefly luciferase activity was normalized to Renilla luciferase expression, and mean activities ± S.E. from five independent experiments are shown. B, *, p < 0.01 versus mut-miR-155; C, *, p < 0.01 versus miR-155. Identical experiments were performed as described above; however, total RNA was isolated from transfected CHO cells and subjected to real time PCR experiments to quantitate the expression of hAT₁R mRNA in cells transfected with either phAT₁R-A (D) or phAT₁R-C expression construct (E).

FIGURE 5. In situ detection of miR-155 in paraffin-embedded, formalin-fixed human tissues

A, representative example of the distribution of miR-155 after in situ hybridization analysis with an LNA miR-155-specific probe in a section of unremarkable human lung. Note that signal (blue due to the action of the alkaline phosphatase conjugate on the chromogen nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate) is evident in both the smooth muscle layer of the bronchiole wall (large arrow) as well as the epithelium that lines the lumen (small arrow). The signal was not evident in the serial section (4 μm away) when the scrambled LNA miRNA probe was utilized (B). C, high magnification near the bronchiole lumen where the signal in the epithelium (small arrow) and smooth muscle layer (large arrow) can be seen. Note that the signal is present in the cytoplasm and tends to concentrate around the nuclear membrane. D, arterioles in the lung also demonstrated signal (small arrow, endothelium; large arrow, smooth muscle cells). No signal was evident in the serial section when the scrambled probe was used (E). F, miR-155 detection was evident in an unremarkable human placenta in the endothelium; note the cytoplasmic localization and concentration around the nuclear membrane (arrow).

FIGURE 6. Anti-miR-155 enhances hAT₁R expression and Ang II-induced signaling in VSMCs

VSMCs were transfected with the anti-miRNA oligonucleotides as indicated; 48 h after transfection, the cells were utilized as follows. A, AT₁R radioreceptor binding assays were performed as described under “Experimental Procedures.” The data have been normalized for protein and transfection differences, and data represent specific binding. The values are shown as percent of maximal specific binding of mock-transfected VSMCs and represent the mean ± S.E. from four independent transfection experiments (*, p < 0.01 versus mock-transfected cells). B, Ang II-induced phospho-ERK1/2 experiments were performed utilizing serum-starved, transiently transfected cells as described under “Experimental Procedures.” A representative immunoblot is shown. Results are representative of four independent experiments. C, quantitation of Ang II-(1 μM for 5 min) induced ERK1/2 phosphorylation was determined by densitometry. Values are expressed as a percent of the maximal phosphorylation of ERK1/2 in response to Ang II in mock-transfected cells and represent the mean ± S.E. from four independent transfection experiments (*, p < 0.01).