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MiR-133 promotes cardiac reprogramming by directly repressing Snai1 and silencing fibroblast signatures.

ABSTRACT: Fibroblasts can be directly reprogrammed into cardiomyocyte-like cells (iCMs) by overexpression of cardiac transcription factors or microRNAs. However, induction of functional cardiomyocytes is inefficient, and molecular mechanisms of direct reprogramming remain undefined. Here, we demonstrate that addition of miR-133a (miR-133) to Gata4, Mef2c, and Tbx5 (GMT) or GMT plus Mesp1 and Myocd improved cardiac reprogramming from mouse or human fibroblasts by directly repressing Snai1, a master regulator of epithelial-to-mesenchymal transition. MiR-133 overexpression with GMT generated sevenfold more beating iCMs from mouse embryonic fibroblasts and shortened the duration to induce beating cells from 30 to 10 days, compared to GMT alone. Snai1 knockdown suppressed fibroblast genes, upregulated cardiac gene expression, and induced more contracting iCMs with GMT transduction, recapitulating the effects of miR-133 overexpression. In contrast, overexpression of Snai1 in GMT/miR-133-transduced cells maintained fibroblast signatures and inhibited generation of beating iCMs. MiR-133-mediated Snai1 repression was also critical for cardiac reprogramming in adult mouse and human cardiac fibroblasts. Thus, silencing fibroblast signatures, mediated by miR-133/Snai1, is a key molecular roadblock during cardiac reprogramming.

SUBMITTER: Muraoka N

PROVIDER: S-EPMC4198052 | biostudies-literature | 2014 Jul

REPOSITORIES: biostudies-literature

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MiR-133 promotes cardiac reprogramming by directly repressing Snai1 and silencing fibroblast signatures.

Muraoka Naoto N Yamakawa Hiroyuki H Miyamoto Kazutaka K Sadahiro Taketaro T Umei Tomohiko T Isomi Mari M Nakashima Hanae H Akiyama Mizuha M Wada Rie R Inagawa Kohei K Nishiyama Takahiko T Kaneda Ruri R Fukuda Toru T Takeda Shu S Tohyama Shugo S Hashimoto Hisayuki H Kawamura Yoshifumi Y Goshima Naoki N Aeba Ryo R Yamagishi Hiroyuki H Fukuda Keiichi K Ieda Masaki M

The EMBO journal 20140611 14

Fibroblasts can be directly reprogrammed into cardiomyocyte-like cells (iCMs) by overexpression of cardiac transcription factors or microRNAs. However, induction of functional cardiomyocytes is inefficient, and molecular mechanisms of direct reprogramming remain undefined. Here, we demonstrate that addition of miR-133a (miR-133) to Gata4, Mef2c, and Tbx5 (GMT) or GMT plus Mesp1 and Myocd improved cardiac reprogramming from mouse or human fibroblasts by directly repressing Snai1, a master regulat ...[more]

PMID: 24920580

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Project description:BackgroundMicroRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally regulate gene expression. Sequential cleavage of miRNA precursors results in a ~22 nucleotide duplex of which one strand, the mature miRNA, is typically loaded into the RNA-induced silencing complex (RISC) while the passenger strand is degraded. Very little is known about how genetic variation might affect miRNA biogenesis and function.ResultsWe re-sequenced the MIR1-1, MIR1-2, MIR133A1, MIR133A2, and MIR133B genes, that encode the cardiac-enriched miRNAs, miR-1 and miR-133, in 120 individuals with familial atrial fibrillation and identified 10 variants, including a novel 79T?>?C MIR133A2 substitution. This variant lies within the duplex at the 3' end of the mature strand, miR-133a-3p, and is predicted to prevent base-pairing and weaken thermostability at this site, favoring incorporation of the passenger strand, miR-133a-5p, into RISC. Genomic DNA fragments containing miR-133a-2 precursor sequences with 79T and 79C alleles were transfected into HeLa cells. On Northern blotting the 79T allele showed strong expression of miR-133a-3p with weak expression of miR-133a-5p. In contrast, the 79C allele had no effect on miR-133a-3p but there was a significant increase (mean 3.6-fold) in miR-133a-5p levels. Deep sequencing of small RNA libraries prepared from normal human and murine atria confirmed that nearly all the mature miR-133a was comprised of miR-133a-3p and that levels of miR-133a-5p were very low. A number of isomiRs with variations at 5' and 3' ends were identified for both miR-133a-3p and miR-133a-5p, with 2 predominant miR-133a-3p isomiRs and one predominant miR-133a-5p isomiR. Bioinformatics analyses indicate that the major miR-133a-3p and 5p isomiRs have numerous predicted target mRNAs, only a few of which are in common.ConclusionsMultiple miR-133a isomiRs with potential different mRNA target profiles are present in the atrium in humans and mice. We identified a human 79T?>?C MIR133A2 variant that alters miRNA processing and results in accumulation of the miR-133a-5p strand that is usually degraded.

Dataset Information

MiR-133 promotes cardiac reprogramming by directly repressing Snai1 and silencing fibroblast signatures.

Publications

MiR-133 promotes cardiac reprogramming by directly repressing Snai1 and silencing fibroblast signatures.

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