miR-802 regulates human angiotensin II type 1 receptor expression in intestinal epithelial C2BBe1 cells

Abstract

Studies have demonstrated that angiotensin II (Ang II) can regulate intestinal fluid and electrolyte transport and control intestinal wall muscular activity. Ang II is also a proinflammatory mediator that participates in inflammatory responses such as apoptosis, angiogenesis, and vascular remodeling; accumulating evidence suggests that this hormone may be involved in gastrointestinal (GI) inflammation and carcinogenesis. Ang II binds to two distinct G protein-coupled receptor subtypes, the AT(1)R and AT(2)R, which are widely expressed in the GI system. Together these studies suggest that Ang II-AT(1)R/-AT(2)R actions may play an important role in GI tract physiology and pathophysiology. Currently it is not known whether miRNAs can regulate the expression of the human AT(1)R (hAT(1)R) in the GI system. PCR and in situ hybridization experiments demonstrated that miR-802 was abundantly expressed in human colon and intestine. Luciferase reporter assays demonstrated that miR-802 could directly interact with the bioinformatics-predicted target site harbored within the 3'-untranslated region of the hAT(1)R mRNA. To validate that the levels of miR-802 were physiologically relevant in the GI system, we demonstrated that miR-802 "loss-of-function" experiments resulted in augmented hAT(1)R levels and enhanced Ang II-induced signaling in a human intestinal epithelial cell line. These results suggest that miR-802 can modulate the expression of the hAT(1)R in the GI tract and ultimately play a role in regulating the biological efficacy of Ang II in this system.

Fig. 1.

A: schematic representation of the location of the prioritized putative miRNA binding sites harbored in the human AT₁R (hAT₁R) 3′-untranslated region (3′-UTR). TargetScan bioinformatic analysis predicted that a total of 58 miRNA binding sites were harbored in the hAT₁R 3′-UTR. Only those predicted miRNAs with a context score greater than −0.30 are shown. The miRNAs shown in bold print denote those predicted miRNAs with an 8-mer complementary “seed” sequence site-type (18, 21, 38). The UGA represents the beginning of the 3′-UTR, which is 883 bp in length. B: relative expression of mature miR-802 in human tissues. Total RNA samples isolated from a number of human tissues were quantified by utilizing miR-802 and RNU48 TaqMan microRNA assays (Applied Biosystems, Foster City, CA) as previously described (, , –42). The relative expression of the mature miR-802 gene was normalized to RNU48 expression in each tissue. Relative gene expression was calculated as 2^{−(Ct miR-802−Ct RNU48)}. Relative gene expression was multiplied by 10⁵ to simplify data presentation. The mean activities ± SE from 3 independent quantitative PCR (qPCR) experiments are shown.

Fig. 2.

In situ detection of miR-802 in paraffin-embedded, formalin-fixed human fetal colon. Representative example of the distribution of miR-802 after in situ hybridization analysis with a locked nucleic acid (LNA) miR-802-specific probe in a section of unremarkable human fetal colon (n = 3) at ×100 (A), ×200 (B), ×400 (C), and ×400 (D). The probe-target complex (blue signal) was visualized by utilizing a streptavidin-alkaline phosphatase conjugate acting on the chromogen nitroblue tetrazdium and bromochloroindolyl phosphate (NBT/BCIP). Nuclear fast red served as counterstain. miR-802 signal was evident in the epithelial (blue arrows) and stromal cells of the lamina propria (orange arrows) and in the submucosa (yellow arrows). A miR-802 signal was also visualized in the outer muscularis layer (green arrows). The signal was not evident in the serial section (4 μm away) when the scrambled LNA miRNA probe was utilized (data not shown). Representative example of the distribution of miR-802 after in situ hybridization analysis with a LNA miR-802-specific probe in a section of unremarkable human adult colon at ×400 (E) and ×1,000 (F). miR-802 signal was evident in the epithelial cells (blue arrows).

Fig. 3.

hAT₁R is expressed in human fetal colon specimens. Representative photomicrographs of hAT₁R expression in fetal colon (A, ×20; B, ×40; C, ×1,000; D, ×1,000). Immunoreactivity was visualized with fast red chromogen (positive staining deep pink color). hAT₁R is expressed in colon epithelial (orange arrows) and endothelial cells (yellow arrows) and in the muscularis (green arrows). Hematoxylin (blue) was the counterstain. All hAT₁R staining was lost if the primary antibody was omitted (data not shown).

Fig. 4.

miR-802-mediated inhibition of luciferase/hAT₁R 3′-UTR activity. A: complementarity between miR-802 and the putative hAT₁R 3′-UTR binding site (616 base pairs downstream from the hAT₁R stop codon). B: the miR-802 “seed sequence” was mutated by using PCR and the resulting construct was designated psiCHECK/hAT₁R/mut-802. C: CHO cells were transfected with psiCHECK, psiCHECK/hAT₁R, or psiCHECK/mut-802 luciferase reporter constructs and miR-802 or scrambled miRNA at the concentrations indicated. Twenty-four hours following transfection, luciferase activities were measured. Renilla luciferase activity was normalized to firefly luciferase activity, and mean activities ± SE from 5 independent experiments are shown (*P < 0.01 psiCHECK/hAT₁R + miR-802 vs. psiCHECK/hAT₁R alone).

Fig. 5.

miR-802 gain- and loss-of-function experiments inversely regulates hAT₁R expression. A: human intestinal epithelial C2BBe1 cells were either mock transfected or transfected with miR-802 mimic or anti-miRNA-802 and 72 h after transfection the cells were utilized as follows. AT₁R radioreceptor binding assays were performed as described in materials and methods. Data have been normalized for protein and transfection differences and represent specific binding. Values are shown as percent of maximal specific binding of mock-transfected C2BBe1 cells and represent means ± SE from 4 independent experiments (*P < 0.01 vs. mock-transfected cells). B: qPCR experiments were performed as described in materials and methods utilizing total RNA isolated from transfected C2BBe1 cells. The relative gene expression of hAT₁R mRNA was normalized to 18S rRNA expression and is expressed in arbitrary units. The mean of hAT₁R steady-state mRNA levels from 4 independent transfection experiments are shown. C: Ang II-induced phospho-ERK1/2 experiments were performed utilizing serum-starved, transiently transfected C2BBe1 cells as described in materials and methods. A representative immunoblot is shown. Results are representative of 4 independent experiments. D: 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 means ± SE from 4 independent transfection experiments (*P < 0.01).

Fig. 6.

Antisense miR-802-mediated increase of hAT₁R expression levels attenuates Ang II-induced paracellular flux of macromolecules across intestinal epithelial cell monolayers. A: C2BBel cells were cultured as monolayer in complete medium on gold electrodes at 37°C and they were subsequently treated with culture media alone or culture media containing Ang II (1, 5, and 10 μM) up to 6 h. and transepithelial electrical resistance of the epithelial cell monolayer was measured on electric cell substrate impedance sensing system as described in materials and methods. The normalized resistance values obtained from 3 independent measurements at selected time points of treatment of C2BBel cell monolayers treated as described above are shown. Each histogram represents means ± SD from 3 independent determinations (*P < 0.05 culture media-treated vs. Ang II-treated cells). B: C2BBel cells (untransfected or transfected with anti-miR-802) were cultured as monolayers on 12-well sterile inserts (0.4 μM size) and FITC-dextran (molecular weight 70,000) paracellular flux (leak) across the epithelial cell monolayers 60 min following the treatment with culture media alone or culture media containing Ang II (5 μM) for 30 min, was assayed as described in materials and methods. Data are expressed as % transport of FITC-dextran normalized to the Ang II-untreated control cell monolayers set at 100%. Each histogram represents means ± SD from 3 independent determinations (*P < 0.05 culture media-treated vs. Ang II-treated cells or #P < 0.01 nontransfected Ang II-treated cells vs. anti-miR-802-transfected Ang II-treated cells).