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. 2016 Sep 15;5(9):1260-5.
doi: 10.1242/bio.019182.

miR-20a regulates proliferation, differentiation and apoptosis in P19 cell model of cardiac differentiation by targeting Smoothened

Feng Ai  1 , Yanwei Zhang  1 , Bangtian Peng  2
Affiliations

miR-20a regulates proliferation, differentiation and apoptosis in P19 cell model of cardiac differentiation by targeting Smoothened

Feng Ai et al. Biol Open. .

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.

Keywords: Apoptosis; Differentiation; P19 cell; Proliferation; Smoothened; miR-20a.

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Conflict of interest statement

The authors declare no competing or financial interests.

Figures

Fig. 1.
Fig. 1.
miR-20a is downregulated and SMO is upregulated in myocardially differentiated P19 cells. (A) Western blot assay shows that the expression levels of marker proteins of cardiomyocytes cTnT, GATA4 and desmin were obviously higher in P19 cells at differentiation day 10 than those in P19 cells at differentiation day 0. (B) qRT-PCR shows that compared with P19 cells at differentiation day 0, P19 cells at differentiation day 10 have an obvious decrease in miR-20a expression. (C) The expression level of SMO protein is significantly higher in P19 cells at differentiation day 10 than that in P19 cells at differentiation day 0. Error bars indicate s.d., n=3. *P<0.05 and **P<0.01 (Student's t-test).
Fig. 2.
Fig. 2.
miR-20a overexpression suppresses proliferation and differentiation and promotes apoptosis in P19 cells. (A) P19 cells transfected with miR-20a mimics proliferate significantly slower than those transfected with miR-control. Error bars indicate s.e.m. (n=3). (B) The expression level of Ki67 protein is obviously decreased in miR-20a-overexpressing P19 cells compared with that in control cells. Error bars indicate s.d. (n=3). (C) P19 cells transfected with miR-20a mimics have obvious decreases in cTnT, GATA4 and desmin protein expression compared with the control cells. Error bars indicate s.d. (n=3). (D) P19 cells transfected with miR-20a mimic have significantly higher apoptosis rate than the control cells. Error bars indicate s.d. (n=3). (E) P19 cells transfected with miR-20a mimics have a marked increase in Bax protein expression and an obvious decrease in Bcl-2 protein expression compared with the control cells. Error bars indicate s.d., n=3. *P<0.05, **P<0.01 and ***P<0.001 (Student’s t-test).
Fig. 3.
Fig. 3.
SMO is a direct target of miR-20a. (A) Bioinformatics-based target prediction analysis shows that SMO is a potential target of miR-20a and the binding site is on the 3′-UTR of SMO. Black lines indicate the predicted binding sites. (B) P19 cells co-transfected with plasmid containing 3′-UTR-WT regions of SMO and miR-20a mimics have significantly decreased luciferase activity compared with control, while mutation of the putative miR-20a binding sites in the 3′-UTR regions of SMO abolishes this effect. (C,D) Overexpression of miR-20a inhibits SMO mRNA and protein expression in P19 cells. (E,F) Downregulation of miR-20a enhances SMO mRNA and protein expression in P19 cells. *P<0.05, Error bars indicate s.d., n=3. **P<0.01 and ***P<0.001 (Student’s t-test).
Fig. 4.
Fig. 4.
Knockdown of SMO suppresses proliferation and differentiation and promotes apoptosis in P19 cells. (A) P19 cells transfected with si-SMO had lower SMO protein expression than control cells. Error bars indicate s.d. (n=3). (B) P19 cells transfected with si-SMO proliferate significantly slower than those transfected with si-control. Error bars indicate s.e.m. (n=3). (C) The expression level of Ki67 in P19 cells transfected with si-SMO is obviously lower than that in control. Error bars indicate s.d. (n=3). (D) P19 cells transfected with si-SMO have obvious decreases in cTnT, GATA4 and desmin protein expression compared with the control cells. Error bars indicate s.d. (n=3). (E) Knockdown of SMO results in an obviously increased apoptosis in P19 cells. Error bars indicate s.d. (n=3). (F) P19 cells transfected with si-SMO have a marked increase in Bax expression and an obvious decrease in Bcl-2 expression compared with the control cells. Error bars indicate s.d., n=3. *P<0.05, **P<0.01 and ***P<0.001 (Student’s t-test).
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