Abstract
Objective: microRNAs have been shown to play important roles in cellular behavior and lineage specification including cardiogenic differentiation. However, full understanding of their roles in cardiomyocyte differentiation has been impeded due to lack of proper cellular model. Here, we used an embryonic stem cell (ESC) that is lacking the important microprocessor Dgcr8 (or Pasha), which allows the introduction of individual miRNAs to study their role in cardiac differentiation and for more precise target selection. Methods: Dgcr8 KO ESC was cultured in LIF-supplemented ESC medium with mouse embryonic fibroblast feeders and cardiac differentiation was induced using an embryonic body-based differentiation protocol. Differentiation was monitored by measuring mRNA and protein levels of cardiogenic markers and heterochromatin changes using immunofluorescent staining and semi-quantitative PCR. Results and conclusion: We showed that Dgcr8 KO ESCs indeed are lacking a large population of small RNAs, including but not limited to mature microRNAs. The KO cells had a lower proliferation rate and were unable to differentiate into the cardiac lineage. To our surprise, in addition to a defect in microRNA processing, Dgcr8 KO embryonic stem cells are unable to form proper heterochromatin and to inactivate genotoxic centromeric repetitive elements. Our results argue that, in addition to controlling microRNA processing, Dgcr8 may serve a previously unrecognized role in heterochromatin silencing.
Highlights
MicroRNAs have been shown to play important roles in cardiac differentiation [1,2,3,4,5,6], pathological development and progression of cardiac diseases [7,8,9,10,11,12]
Our results show that DiGeorge syndrome critical region 8 (Dgcr8) KO embryonic stem cells (ESC) proliferate slower and are lacking a large population of small RNA species ranging from 4-40 bp, including mature miRNAs (~20 bp)
Dgcr8 KO ESCs are unable to fully inactivate their heterochromatin regions like WT ESCs, which is likely leading to the defect in cardiac differentiation
Summary
MicroRNAs (miRNAs) have been shown to play important roles in cardiac differentiation [1,2,3,4,5,6], pathological development and progression of cardiac diseases [7,8,9,10,11,12] Full understanding of their role in these conditions requires identification of their precise target genes and studying their function in the relevant cell types. Knocking out Dgcr in embryonic stem cells (ESC) has become the first choice to create a versatile miRNA-free cell model In this cell model, the miRNA population will be eliminated, the activity for precursor processing will be kept intact and the cells may hold their potential to differentiate into any cell type of interest, e.g. cardiomyocytes. Inactivation of Dgcr was used to explore the role of the total miRNA population in a particular cell type or tissue in vivo [22,23,24,25], the consequence of the loss of Dgcr on cardiac lineage specification of ESCs, and whether or not we can create the "simple cardiomyocyte" from Dgcr KO ESCs, is unknown
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
