Effect of Raldh2 silence on the differentiation of P19 cells into cardiomyocyte-like cells / 复旦学报（医学版）

ABSTRACT

Objective To study the effect of retinaldehyde dehydrogenase 2 (Raldh2) on the differentiation of P19 cell to cardiomyocyte-like cells and explore the potential mechanism.Methods A miRNA expression plasmid specific to Raldh2 was packaged and constructed by RNA interference (RNAi) method.The P19 stable cell line carried Raldh2 miRNA expression was selected by adding blasticidin and induced to differentiate towards cardiomyocyte-like cells.The mRNA levels of myocardium development-related markers were determined by qPCR at different stages during the differentiation process.Results The miRNA expression plasmid specific to Raldh2 could effectively suppress Raldh2 expression,and the MiRaldh2 group,a P19 stable cell line was established successfully in which the knockdown efficiency on Raldh2 was 91％ (t =25.52,P＜0.000 1,95％ CI0.81-1.01).When compared with P19 group,the mRNA levels of cardiac transcription factors were generally decreased in the MiRaldh2 group during the whole differentiation process.In detail,on the 7th day,the relatively low expression rates of these cardiac markers including Gata4,Tef-1,N-myc,α-mhc and Ctnt was0.16±0.01 (t=17.29,P＜0.000 1),0.51 ±0.02 (t=3.564,P=0.023 5),0.23 ±0.01 (t=13.17,P =0.000 2),0.20 ± 0.02 (t=17.76,P＜0.000 1) and 0.59 ± 0.06 (t =3.642,P =0.021 9) in MiRaldh2 group when compared with the P19 group.Conversely,the mRNA levels of Nkx2.5 and Hand2 were dramatically increased in MiRaldh2 group on day 2 to 7 and the expression rates on the 7th day was 2.25 ± 0.35 (t =3.526,P =0.024 3) compared with the P19 group while Hand2 was 3.58 ± 0.20 (t =9.214,P =0.011 6).Conclusions Knockdown of Raldh2 inhibits the P19 cells differentiated into cardiomyocyte-like cells,which suggests that Raldh2 may play a potential role in early development of heart.The low expression of Raldh2 might be an explanation of the cardiac malformations associated with retinoic acid deficiency.