Abstract
ABSTRACTMicroRNA (miR)-20a, a member of the miR-17-92 cluster related to cardiac development, was obviously downregulated in myocardially differentiated P19 cells compared with normal P19 cells. Smoothened (SMO) is a member of the Hh pathway. Hh signaling induces cardiac differentiation in P19 cells, and SMO mediates the Hh pathway during embryonic development. Using bioinformatic prediction software Targetscan (http://www.targetscan.org/), PicTar (http://pictar.bio.nyu.edu), and miRBase (http://microrna.sanger.ac.uk/), miR-20a and the 3′-untranslated region (3′-UTR) of SMO mRNA were predicted to have complementary binding regions. Accordingly, we inferred that miR-20a might act as a regulator of SMO, and regulate proliferation, differentiation and apoptosis in P19 cells. We determined the expression of miR-20a, SMO and marker proteins of cardiomyocytes (cTnT, GATA4 and desmin) by quantitative real-time PCR (qRT-PCR) and western blot assays, and found that P19 cells had differentiated into cardiomyocytes successfully at differentiation day 10, and downregulation of miR-20a and upregulation of SMO existed in myocardially differentiated P19 cells. Cell proliferation, differentiation and apoptosis detection showed that miR-20a upregulation inhibited proliferation and differentiation and enhanced apoptosis in P19 cells. Moreover, we verified that miR-20a directly targeted SMO and knockdown of SMO and miR-20a overexpression had similar effects on P19 cell proliferation, differentiation and apoptosis, which verified the speculation that miR-20a inhibits proliferation and differentiation and enhances apoptosis in P19 cells by directly targeting SMO. Our results suggest that miR-20a may be a potential target against congenital heart diseases.
Highlights
The vertebrate heart, derived from the mesodermal cells, is the first functional organ forming in vertebrate development (Zaffran and Frasch, 2002)
RESULTS miR-20a is underexpressed and SMO is overexpressed in P19 cells at differentiation day 10 To assess whether P19 cells showed cardiomyocyte differentiation, P19 cells were harvested at day 0 and 10 of differentiation, and western blot assay was conducted to detect the levels of cTnT, GATA4 and desmin
The expression levels of cTnT, GATA4 and desmin were obviously higher in P19 cells at differentiation day 10 than those in P19 cells at differentiation day 0, which confirmed that P19 cells had differentiated into cardiomyocytes successfully at differentiation day 10 (Fig. 1A)
Summary
The vertebrate heart, derived from the mesodermal cells, is the first functional organ forming in vertebrate development (Zaffran and Frasch, 2002). The formation of a mature normal heart is a complicated course which depends on the sequential expression of multiple genes and various pathways, including the Hedgehog (Hh), the BMP, the Notch, and the Wnt/β-Catenin pathway. The impaired process of embryonic stem cell differentiation into cardiomyocytes caused by gene deletions or mutations is the primary cause of CHD (Hoffman, 1995; Hoffman and Kaplan, 2002), better understanding of the molecular mechanism of embryonic stem cell differentiation into cardiomyocytes will contribute to finding effective approaches for CHD treatment
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