Limits...
Downregulation of long non-coding RNA H19 promotes P19CL6 cells proliferation and inhibits apoptosis during late-stage cardiac differentiation via miR-19b-modulated Sox6

View Article: PubMed Central - PubMed

ABSTRACT

Background: Regulating 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.

Methods: To 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.

Results: The 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.

Conclusion: Downregulation 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.

No MeSH data available.


The expression levels of GATA4, Nkx-2.5, α-MHC, MLC-2v and H19 are significantly upregulated and miR-19b was downregulated at indicated time points. a mRNA levels of early cardiac-specific markers (GATA4 and Nkx-2.5α-MHC) were increased significantly in P19CL6 cells at day 4 and 6. b Protein levels of GATA4 and Nkx-2.5α-MHC were augmented significantly in P19CL6 cells at day 4 and 6. c mRNA levels of cardiac contractile protein genes (α-MHC and MLC-2v) were increased significantly in P19CL6 cells at day 10 and 12. d Protein levels of α-MHC and MLC-2v were elevated significantly in P19CL6 cells at day 10 and 12. e Level of H19 was augmented significantly from day 8 to day 12. f Level of miR-19b was dropped significantly from day 8 to day 12. **P < 0.01, ***P < 0.001
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC5120414&req=5

Fig1: The expression levels of GATA4, Nkx-2.5, α-MHC, MLC-2v and H19 are significantly upregulated and miR-19b was downregulated at indicated time points. a mRNA levels of early cardiac-specific markers (GATA4 and Nkx-2.5α-MHC) were increased significantly in P19CL6 cells at day 4 and 6. b Protein levels of GATA4 and Nkx-2.5α-MHC were augmented significantly in P19CL6 cells at day 4 and 6. c mRNA levels of cardiac contractile protein genes (α-MHC and MLC-2v) were increased significantly in P19CL6 cells at day 10 and 12. d Protein levels of α-MHC and MLC-2v were elevated significantly in P19CL6 cells at day 10 and 12. e Level of H19 was augmented significantly from day 8 to day 12. f Level of miR-19b was dropped significantly from day 8 to day 12. **P < 0.01, ***P < 0.001

Mentions: 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 (an late stage of differentiation) than those in P19CL6 cells at day 8 (Fig. 1c and d). In order to define the temporal expression profile of H19 during cardiomyocyte differentiation, we carried out qRT-PCR for H19. The expression of H19 was low at day 0, 4 and 6, but elevated significantly from day 8 to day 12 (Fig. 1e). Moreover, the level of miR-19b was significantly reduced from day 8 to day 12, suggesting that H19 and miR-19b might play some biological roles during the late stage of cardiac differentiation of P19CL6 cells (Fig. 1f).Fig. 1


Downregulation of long non-coding RNA H19 promotes P19CL6 cells proliferation and inhibits apoptosis during late-stage cardiac differentiation via miR-19b-modulated Sox6
The expression levels of GATA4, Nkx-2.5, α-MHC, MLC-2v and H19 are significantly upregulated and miR-19b was downregulated at indicated time points. a mRNA levels of early cardiac-specific markers (GATA4 and Nkx-2.5α-MHC) were increased significantly in P19CL6 cells at day 4 and 6. b Protein levels of GATA4 and Nkx-2.5α-MHC were augmented significantly in P19CL6 cells at day 4 and 6. c mRNA levels of cardiac contractile protein genes (α-MHC and MLC-2v) were increased significantly in P19CL6 cells at day 10 and 12. d Protein levels of α-MHC and MLC-2v were elevated significantly in P19CL6 cells at day 10 and 12. e Level of H19 was augmented significantly from day 8 to day 12. f Level of miR-19b was dropped significantly from day 8 to day 12. **P < 0.01, ***P < 0.001
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC5120414&req=5

Fig1: The expression levels of GATA4, Nkx-2.5, α-MHC, MLC-2v and H19 are significantly upregulated and miR-19b was downregulated at indicated time points. a mRNA levels of early cardiac-specific markers (GATA4 and Nkx-2.5α-MHC) were increased significantly in P19CL6 cells at day 4 and 6. b Protein levels of GATA4 and Nkx-2.5α-MHC were augmented significantly in P19CL6 cells at day 4 and 6. c mRNA levels of cardiac contractile protein genes (α-MHC and MLC-2v) were increased significantly in P19CL6 cells at day 10 and 12. d Protein levels of α-MHC and MLC-2v were elevated significantly in P19CL6 cells at day 10 and 12. e Level of H19 was augmented significantly from day 8 to day 12. f Level of miR-19b was dropped significantly from day 8 to day 12. **P < 0.01, ***P < 0.001
Mentions: 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 (an late stage of differentiation) than those in P19CL6 cells at day 8 (Fig. 1c and d). In order to define the temporal expression profile of H19 during cardiomyocyte differentiation, we carried out qRT-PCR for H19. The expression of H19 was low at day 0, 4 and 6, but elevated significantly from day 8 to day 12 (Fig. 1e). Moreover, the level of miR-19b was significantly reduced from day 8 to day 12, suggesting that H19 and miR-19b might play some biological roles during the late stage of cardiac differentiation of P19CL6 cells (Fig. 1f).Fig. 1

View Article: PubMed Central - PubMed

ABSTRACT

Background: Regulating 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.

Methods: To 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 &alpha;-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.

Results: The expression levels of &alpha;-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.

Conclusion: Downregulation 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.

No MeSH data available.