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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.


H19 targets and inhibits miR-19b expression. a The sketch map of the mmu-miR-19b-3p site in H19 3′-UTR. b Luciferase activity assay was performed in 293T cells at 48 h after transfection. miR-19b mimics were capable of significantly inhibiting luciferase expression of H19-WT reporter plasmid but not the H19-Mut reporter plasmid. c miR-19b levels were significantly up- or downregulated in P19CL6 cells transfected with shH19 or H19, respectively. **P < 0.01, ***P < 0.001
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Fig3: H19 targets and inhibits miR-19b expression. a The sketch map of the mmu-miR-19b-3p site in H19 3′-UTR. b Luciferase activity assay was performed in 293T cells at 48 h after transfection. miR-19b mimics were capable of significantly inhibiting luciferase expression of H19-WT reporter plasmid but not the H19-Mut reporter plasmid. c miR-19b levels were significantly up- or downregulated in P19CL6 cells transfected with shH19 or H19, respectively. **P < 0.01, ***P < 0.001

Mentions: Online software starBase v2.0 predicted H19 contains binding sequences complementary to miR-19b seed regions, as shown in Fig. 3a. Furtherly, the luciferase report assay indicated that miR-19b mimics inhibited luciferase activity of the wild type H19 reporter (H19-WT), but no change was observed for the luciferase activity in the mutant reporter (H19-Mut) (Fig. 3b). Moreover, the expression level of miR-19b was significantly increased in cells transfected with shH19, whereas significantly reduced in cells with pcDNA-H19. These results indicated that H19 negatively modulated miR-19b expression.Fig. 3


Downregulation of long non-coding RNA H19 promotes P19CL6 cells proliferation and inhibits apoptosis during late-stage cardiac differentiation via miR-19b-modulated Sox6
H19 targets and inhibits miR-19b expression. a The sketch map of the mmu-miR-19b-3p site in H19 3′-UTR. b Luciferase activity assay was performed in 293T cells at 48 h after transfection. miR-19b mimics were capable of significantly inhibiting luciferase expression of H19-WT reporter plasmid but not the H19-Mut reporter plasmid. c miR-19b levels were significantly up- or downregulated in P19CL6 cells transfected with shH19 or H19, respectively. **P < 0.01, ***P < 0.001
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5120414&req=5

Fig3: H19 targets and inhibits miR-19b expression. a The sketch map of the mmu-miR-19b-3p site in H19 3′-UTR. b Luciferase activity assay was performed in 293T cells at 48 h after transfection. miR-19b mimics were capable of significantly inhibiting luciferase expression of H19-WT reporter plasmid but not the H19-Mut reporter plasmid. c miR-19b levels were significantly up- or downregulated in P19CL6 cells transfected with shH19 or H19, respectively. **P < 0.01, ***P < 0.001
Mentions: Online software starBase v2.0 predicted H19 contains binding sequences complementary to miR-19b seed regions, as shown in Fig. 3a. Furtherly, the luciferase report assay indicated that miR-19b mimics inhibited luciferase activity of the wild type H19 reporter (H19-WT), but no change was observed for the luciferase activity in the mutant reporter (H19-Mut) (Fig. 3b). Moreover, the expression level of miR-19b was significantly increased in cells transfected with shH19, whereas significantly reduced in cells with pcDNA-H19. These results indicated that H19 negatively modulated miR-19b expression.Fig. 3

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.