Abstract
BackgroundRegulating cardiac differentiation to maintain normal heart development and function is very important. At present, biological functions of H19 in cardiac differentiation is not completely clear.MethodsTo explore the functional effect of H19 during cardiac differentiation. Expression levels of early cardiac-specific markers Nkx-2.5 and GATA4, cardiac contractile protein genes α-MHC and MLC-2v were determined by qRT-PCR and western lot. The levels of lncRNA H19 and miR-19b were detected by qRT-PCR. We further predicted the binding sequence of H19 and miR-19b by online softwares starBase v2.0 and TargetScan. The biological functions of H19 and Sox6 were evaluated by CCK-8 kit, cell cycle and apoptosis assay and caspase-3 activity.ResultsThe expression levels of α-MHC, MLC-2v and H19 were upregulated, and miR-19b was downregulated significantly in mouse P19CL6 cells at the late stage of cardiac differentiation. Biological function analysis showed that knockdown of H19 promoted cell proliferation and inhibits cell apoptosis. H19 suppressed miR-19b expression and miR-19b targeted Sox6, which inhibited cell proliferation and promoted apoptosis in P19CL6 cells during late-stage cardiac differentiation. Importantly, Sox6 overexpression could reverse the positive effects of H19 knockdown on P19CL6 cells.ConclusionDownregulation of H19 promoted cell proliferation and inhibited cell apoptosis during late-stage cardiac differentiation by regulating the negative role of miR-19b in Sox6 expression, which suggested that the manipulation of H19 expression could serve as a potential strategy for heart disease.
Highlights
Regulating cardiac differentiation to maintain normal heart development and function is very important
H19 was highly expressed in the late stage of cardiac differentiation in P19CL6 cells We treated P19CL6 cells with 1% dimethyl sulfoxide (DMSO) to induce differentiation. qRT-PCR was carried out to detect the expression levels of early cardiac-specific markers (GATA4 and Nkx-2.5), cardiac contractile protein genes (α-MHC and MLC-2v) in P19CL6 cells at indicated time points
The results showed that mRNA and protein levels of GATA4 and Nkx2.5 were very low at day 0, but increased significantly at day 4 and day 6, an early stage of differentiation (Fig. 1a and b). mRNA and protein levels of α-MHC and MLC-2v in P19CL6 cells were significantly higher at day 10 and 12 than those in P19CL6 cells at day 8 (Fig. 1c and d)
Summary
Regulating cardiac differentiation to maintain normal heart development and function is very important. Biological functions of H19 in cardiac differentiation is not completely clear. Growing research has indicated that lncRNAs are often abnormally expressed in many cancers and play a variety of biological functions in cell proliferation, apoptosis, or invasion [7, 8]. Gabory et al demonstrated that H19−/−mice presented an overgrowth phenotype, while overexpressing H19 in transgenic mice caused postnatal growth reduction [11]. It is important for understanding the cardiac development to illuminate the mechanisms of gene regulation. The precise function of H19 in cardiac differentiation is yet unascertained
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