| Background:During embryonic development of vertebrates,the heart is the first formed and functional organ.Heart development is a complex course involving series of complicated morphological events,including: cell fate commitment,formation of original heart tube and the following looping,chamber septation and valves formation,which results a four-chamberd mature heart.Correspondingly,the molecular mechanism underlying is also intricate related with the expression of numerous genes and proteins that form various of signaling pathways,such as BMP,Notch and Wnt signaling pathway.All these molecular events are precisely regulated,both temporally and spatial.For the past years,research on mechanism of heart development have made great progress,the knowledge about the whole regulatory network is still limited,however.Discovery of the regulatory function of miRNAs has just paved a new road for researchers studying heart development regulation.Micro RNAs are a class of small RNAs consisting approximately 18-22 non-coding nucleotides that critically modulate gene expression at a post-transcriptional level,inducing degradation of target m RNA or depressing its translation,thereby reducing protein production.It has been reported that miRNAs are involved in many biological processes,such as: cell proliferation,apoptosis,metablism,immunity and signal transduction.More and more research have proved the same important regulatory function of miRNAs in heart development but leave the mechanism obscured which makes the mechanism of miRNAs regulating heart development a new hotspot.Our previous work revealed that miR-29c is significantly upregulated in pregnant women with fetal CHD,indicating that miR-29c is likely to be associated with heart anamorphosis.We checked the literature but found no records concerning miR-29c regulating heart development.In this study,we continued our previous work to explore the role of miR-29c in heart development.Objective:To explore the effect of miR-29c inhibition on P19 cell proliferation,apoptosis and differentiation and the regulatory mechanism underlying.Methods:(a)construct miR-29c inhibition spongy and transfect P19 cell,filter cells that miR-29c is steadily repressed with puromycin and check the repression efficiency with q RT-PCR and fluorescent microscopy.(b)examine proliferation with CCK-8 kit and cell cycle using flow cytometry.(c)induce P19 cell to apoptosis and check the apoptosis rate with hoechst staining and flow cytometry,test the expression level of BCL-2/BAX using q RT-PCR and Westem blot(d)induce P19 cell to differentiate with DMSO and check the expression level of GATA4,c Tn T,MEF2 C with methods of q RT-PCR and Westem blot.(e)find target gene of miR-29c using bioinformatic analysis and luciferase assays,examine expression level of the target gene during differentiation of P19 cell using qRT-PCR.Results: Compared with the control group,cells in miR-29c inhibition group showed a faster proliferation rate and higher S cycle percentage;Apoptosis rate of miR-29c inhibition group was lower than that of the control group.Expression of BCL-2 of miR-29c inhibition group was significantly higher than that of control group with no difference seen in BAX expression;GATA4,c Tn T,MEF2 C expression level of miR-29c inhibition group was significantly lower than those of control group at day 6 and day 10;Wnt4 was proved to be a target of miR-29c and Wnt4/?-catenin expression level during differentiation of miR-29c inhibition group(steady high expression level)was higher than the control group(gradually increased expression level).Conclusions: Inhibition of miR-29c promotes proliferation and inhibits apoptosis and differentiation in P19 embryonal carcinoma cells.Wnt4 is the target gene of miR-29c and miR-29c inhibition stimulates WNT4/?-catenin signaling pathway. |
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