title: miR-20a regulates proliferation, differentiation and apoptosis in P19 cell model of cardiac differentiation by targeting Smoothened.
abstract: MicroRNA (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.
authors.affiliation: Department of Cardiovascular Surgery, Henan Provincial People's Hospital and People's Hospital of Zhengzhou University, Zhengzhou 450000, People's republic of China.
authors.forename: Feng
authors.initials: F
authors.lastname: Ai
authors.affiliation: Department of Cardiovascular Surgery, Henan Provincial People's Hospital and People's Hospital of Zhengzhou University, Zhengzhou 450000, People's republic of China.
authors.forename: Yanwei
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Department of Cardiovascular Surgery, Henan Provincial People's Hospital and People's Hospital of Zhengzhou University, Zhengzhou 450000, People's republic of China pbangtian@163.com.
authors.forename: Bangtian
authors.identifier: http://orcid.org/0000-0003-0110-607X
authors.initials: B
authors.lastname: Peng
keywords: Apoptosis; Differentiation; P19 cell; Proliferation; Smoothened; miR-20a