MicroRNA 214 Is a Potential Regulator of Thyroid Hormone Levels in the Mouse Heart Following Myocardial Infarction, by Targeting the Thyroid-Hormone-Inactivating Enzyme Deiodinase Type III

Abstract

Cardiac thyroid-hormone signaling is a critical determinant of cellular metabolism and function in health and disease. A local hypothyroid condition within the failing heart in rodents has been associated with the re-expression of the fetally expressed thyroid-hormone-inactivating enzyme deiodinase type III (Dio3). While this enzyme emerges as a common denominator in the development of heart failure, the mechanism underlying its regulation remains largely unclear. In the present study, we investigated the involvement of microRNAs (miRNAs) in the regulation of Dio3 mRNA expression in the remodeling left ventricle (LV) of the mouse heart following myocardial infarction (MI). In silico analysis indicated that of the miRNAs that are differentially expressed in the post-MI heart, miR-214 has the highest potential to target Dio3 mRNA. In accordance, a luciferase reporter assay, including the full-length 3'UTR of mouse Dio3 mRNA, showed a 30% suppression of luciferase activity by miR-214. In the post-MI mouse heart, miR-214 and Dio3 protein were shown to be co-expressed in cardiomyocytes, while time-course analysis revealed that Dio3 mRNA expression precedes miR-214 expression in the post-MI LV. This suggests that a Dio3-induced decrease of T3 levels is involved in the induction of miR-214, which was supported by the finding that cardiac miR-214 expression is down regulated by T3 in mice. In vitro analysis of human DIO3 mRNA furthermore showed that miR-214 is able to suppress both mRNA and protein expression. Dio3 mRNA is a target of miR-214 and the Dio3-dependent stimulation of miR-214 expression in post-MI cardiomyocytes supports the involvement of a negative feedback mechanism regulating Dio3 expression.

Keywords: Dio3; SECIS; microRNA; myocardial infarction; thyroid hormone metabolism.

Figure 1

Conserved miR-214 target site in the 3′-UTR of Dio3. In silico analysis predicts a target sequence for miR-214 in the stem of SECIS element of the 3′UTR of Dio3 in mice, which is highly conserved among species. (Source: Targetscan and microRNA.org).

Figure 2

Increased miR-214 expression in the post-MI LV. Mir-214 expression levels were determined by quantitative RT PCR analysis in remote LV tissue from sham and MI animals isolated at day 7 post surgery. U6 snRNA expression levels were used as correction factor. Values are means ± SEM, n = 6, normalized to the sham level, *p < 0.05.

Figure 3

Dio3 is targeted by miR-214. A dual-luciferase construct containing the complete Dio3 3′UTR downstream of the Renilla luciferase gene was used to analyze the interaction between miR-214 and the 3′UTR of Dio3. Renilla luciferase activity (RLuc) is expressed relative to firefly luciferase activity (FLuc), correcting for differences in transfection efficiency. Four independent transfections were performed in duplicate. No-miR: control without co-transfection; ctrl-miR: + negative control/scrambled miR-1; P214: + Pre-miR-214; PA214: + Pre-miR-214 and Anti-miR-214. Values are means ± SEM, n = 4, normalized to the RLuc/Fluc ratio in the no-miR group, *p < 0.05.

Figure 4

Western blot analysis of DIO3 expression in vitro. (A) Transfection of COS-7 cells with increasing amounts of wtD3 plasmid confirmed the specificity of Ab 677 for full-length DIO3 (36 kDa) and of Ab 675 for truncated DIO3 protein (18 kDa). Equal amounts of protein were loaded. (B) COS-7 cells transfected with the CysD3 plasmid confirmed the expression of the truncated isoform of DIO3 in COS-7 cells transfected with the wtD3 plasmid, which was detected with Ab 675 (18 kDa) (lower blot; the upper blot is stained using Ab 677). (C) COS-7 cells were transfected with 100 ng of the wtD3 plasmid containing full-length human DIO3 together with either mimic-214 (DIO3 – 214) or Negative control miR-1 (DIO3-nc#1) at a final concentration of 500 nM. Addition of the negative control miR-1 did not significantly affect transfection efficiency (results not shown). Analysis of COS-7 cells transfected with empty pCDNA vector using Ab 677 and Ab 675 did not show bands at 36 kDa and 18 kDa, respectively, confirming antibody specificity (Figure S2 in Supplementary Material). Expression levels of the neomycin-resistance gene were analyzed using qPCR and used to normalize transfection efficiency. Values are means ± SEM, normalized to the value of the parameter in the DIO3-nc#1 group, *p < 0.05.

Figure 5

Dio3 protein and miR-214 are co-expressed. Representative images of immunohistochemical staining for Dio3 (A) and miR-214 in situ hybridization (B) in sequential sections of LV tissue 7 days post-MI. The indicated area shows a group of cardiomyocytes expressing both Dio3 protein and miR-214, with adjoining cells negative for both. Bar = 100 μM.

Figure 6

Time course of miR-214 expression, Dio3 mRNA and Dio3 activity after MI. For the sake of clarity, the miR-214 data are shown in separate panels in combination with Dio3 mRNA expression (A) and Dio3 activity (B). The different parameters are expressed relative to their basal expression level determined in sham-operated mice at day 7. Dio3 mRNA and Dio3 activity data from this set of samples were published in part before (8). Values are means ± SEM, miR-214 (n = 5), Dio3 mRNA (n_3d = 7, n_5d = 6, n_7d = 16, n_28d = 10, n_56d = 16) and Dio3 activity (n_3d = 8, n_5d = 9, n_7d = 16, n_28d = 6, n_56d = 6).

Figure 7

Effect of T3 treatment on the levels of miR-214 and Dio3 mRNA. Total RNA isolated from the LV of hypothyroid (−T3) and hypothyroid mice treated with T3 for three days (+T3) was analyzed by qPCR for miR-214 expression using U6 snRNA as correction factor (A), and for Dio3 mRNA expression using Hprt as correction factor (B). Values are means ± SEM, n = 6, normalized to the −T3 values, *p < 0.05.

Figure 8

Proposed mechanism of Dio3 regulation by miR-214. MI-induced cardiac stress results in upregulation of Dio3, leading to a reduced cardiac level of T3. The decreased level of T3 stimulates the expression of miR-214, which adds to the effects of other MI-induced regulatory pathways on miR-214 expression (the dotted arrow indicates that the relationship between T3 and miR-214 is inferred from indirect evidence). Increased miR-214 expression in turn decreases Dio3 expression, thereby dampening the reduction of T3 levels by Dio3. In this way, a negative feedback loop is created, possibly aimed at protecting the adult cardiomyocyte from the adverse effects of excessively low levels of T3.