miR-145 is differentially regulated by TGF-β1 and ischaemia and targets Disabled-2 expression and wnt/β-catenin activity

Abstract

The effect of wnt/β-catenin signalling in the response to acute myocardial infarction (AMI) remains controversial. The membrane receptor adaptor protein Disabled-2 (Dab2) is a tumour suppressor protein and has a critical role in stem cell specification. We recently demonstrated that down-regulation of Dab2 regulates cardiac protein expression and wnt/β-catenin activity in mesenchymal stem cells (MSC) in response to transforming growth factor-β(1) (TGF-β(1)). Although Dab2 expression has been shown to have effects in stem cells and tumour suppression, the molecular mechanisms regulating this expression are still undefined. We identified putative binding sites for miR-145 in the 3'-UTR of Dab2. In MSC in culture, we observed that TGF-β(1) treatment led to rapid and sustained up-regulation of pri-miR-145. Through gain and loss of function studies we demonstrate that miR-145 up-regulation was required for the down-regulation of Dab2 and increased β-catenin activity in response to TGF-β(1). To begin to define how Dab2 might regulate wnt/β-catenin in the heart following AMI, we quantified myocardial Dab2 as a function of time after left anterior descending ligation. There was no significant Dab2 expression in sham-operated myocardium. Following AMI, Dab2 levels were rapidly up-regulated in cardiac myocytes in the infarct border zone. The increase in cardiac myocyte Dab2 expression correlated with the rapid and sustained down-regulation of myocardial pri-miR-145 expression following AMI. Our data demonstrate a novel and critical role for miR-145 expression as a regulator of Dab2 expression and β-catenin activity in response to TGF-β(1) and hypoxia.

Fig 1

(A) Alignment of miR-145 to three different regions of Dab2 3′UTR according to target scan. Notice the distance between target sites in a 3′UTR region of ∼1.7 Kb. (B) Alignment of conservation of Dab2 miR-145 target site at position 1213 across multiple species.

Fig 2

Regulation of the expression of Dab2 and miR-145 in mesenchymal stem cells. MSC pri-miR-145 expression is increased in response to TGF-β₁ (5 ng/ml, black bars) (A). Dab2 expression is significantly reduced in response to TGF-β₁ (5 ng/ml) both at the mRNA (B) and protein level (C). qPCR experiments are n = 3 ± S.D.; Western blot is a representative experiment of four separate studies performed under the same conditions.

Fig 3

Gain and loss of miR-145 function in MSC and the effect on Dab2 expression. Transfection of MSC with 50vpM miR-145 mimic (black bars) by electroporation resulted in a decrease of Dab2 expression at the mRNA and protein levels, as determined by qPCR (A) and Western blot (B), respectively. No difference relative to control in Dab2 expression was observed when MSC where transfected with miR-145 inhibitors (grey bars). The presence of miR-145 did not alter the MSC response to TGF-β₁ (24 hrs, 5 ng/ml). The presence of miR-145 inhibitor completely blocked the down-regulation of Dab2 by TGF-β₁. qPCR experiments are n = 3 ± S.D.; Western blot is a representative experiment of three performed in the same conditions.

Fig 4

miR-145 regulates wnt/β-catenin activity in MSC. (A) TGF-β₁ treatment induced a two-fold increase in luciferase activity of the TOPFLASH LIF promoter. MSC transduced with miR-145 mimic (black bars) similarly exhibited increased luciferase activity, whereas the presence of the miR-145 inhibitor (grey bars) inhibited wnt/β-catenin activity even in the presence of TGF-β₁ treatment. (B) The over-expression of Dab2 in the presence of miR-145 blocked the up-regulation of TOPFLASH LIF promoter. Data represent mean ± S.D. of three experiments performed in triplicates (**P < 0.01 and *P < 0.05 relative to CTRL).

Fig 5

Dab2 expression is increased in cardiomyocytes following myocardial infarction. (A) Western blot analysis of infarct and infarct-border zone from myocardial tissue at the specified times after LAD ligation. (B) Immunocytochemistry for nuclei (DAPI, blue), cardiac myosine (green), and Dab2 (red) on paraffin embedded cardiac tissue in sham (No MI) and 8, 24, and 48 hrs after myocardial infarction. Images demonstrate an increase in Dab2 expression in cardiomyocytes in the infarct border zone at 8 hrs post-AMI and maintained in surviving cells after 24 and 48 hrs.

Fig 6

miR-145 expression in infarcted rat myocardium. Total RNA was isolated from dissected infarct border zones from sham (No MI, open bar) and 1 hr (black bar) after AMI. The expression of miR-145 was analyzed by qPCR (n = 5 per group). A dramatic decrease in miR-145 expression was seen 1 hr after AMI. Results are presented as mean ± S.D. (**P < 0.01, *P < 0.01 Student's t-test).