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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.


miR-19b targets Sox6 gene and inhibits its expression. a The sketch map of the mmu-miR-19b-3p site in Sox6 3′-UTR. b Luciferase activity assay was performed in 293T cells at 48 h after transfection. The reporter assay indicated that miR-19b mimics were capable of significantly inhibiting luciferase expression of the Sox6-WT reporter plasmid but not the Sox6-Mut reporter plasmid. c Western blot showed that protein level of Sox6 was low from day 0 to 6, and was significantly upregulated from day 8 to 12. d The Sox6 protein levels were significantly down- and up-regulated respectively in P19CL6 cells transfected with miR-19b and anti-miR-19b at day 10. e shH19 inhibited the Sox6 expression significantly, but miR-19b enhanced the inhibitory effect of shH19 on Sox6 expression and anti-miR-19b reversed the influence of shH19 on Sox6 expression at day 10 after transfection. *P < 0.05, **P < 0.01, ***P < 0.001
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Fig4: miR-19b targets Sox6 gene and inhibits its expression. a The sketch map of the mmu-miR-19b-3p site in Sox6 3′-UTR. b Luciferase activity assay was performed in 293T cells at 48 h after transfection. The reporter assay indicated that miR-19b mimics were capable of significantly inhibiting luciferase expression of the Sox6-WT reporter plasmid but not the Sox6-Mut reporter plasmid. c Western blot showed that protein level of Sox6 was low from day 0 to 6, and was significantly upregulated from day 8 to 12. d The Sox6 protein levels were significantly down- and up-regulated respectively in P19CL6 cells transfected with miR-19b and anti-miR-19b at day 10. e shH19 inhibited the Sox6 expression significantly, but miR-19b enhanced the inhibitory effect of shH19 on Sox6 expression and anti-miR-19b reversed the influence of shH19 on Sox6 expression at day 10 after transfection. *P < 0.05, **P < 0.01, ***P < 0.001

Mentions: Bioinformatics analysis using online software TargetScan indicated that miR-19b may bind to 3′UTR of Sox6 (Fig. 4a). In addition, luciferase reporter assay indicated that miR-19b significantly suppressed luciferase expression of the wild type Sox6 reporter (Sox6-WT) but not the mutant reporter (Sox6-Mut) (Fig. 4b), confirming that Sox6 was a target of miR-19b. Western blot was used to detect the expression of Sox6 in the cell differentiation and the results indicated that the protein level of Sox6 was very low from day 0 to day 6, but increased significantly from day 8 to day 12 (Fig. 4c). Then we transfected P19CL6 cells with miR-19b mimics, miR-NC, anti-miR-19b or anti-miR-NC. The results showed that the protein level of Sox6 was significantly down-regulated in P19CL6 cells transfected with miR-19b and significantly up-regulated in P19CL6 cells transfected with anti-miR-19b (Fig. 4d). Furthermore, we transfected H19-knockdown P19CL6 cells with miR-19b mimics or anti-miR-19b. The result showed that knockdown H19 inhibited the Sox6 expression significantly, while miR-19b enhanced the inhibitory effect of knockdown H19 on Sox6 expression, and anti-miR-19b reversed the influence of knockdown H19 on Sox6 expression (Fig. 4e). All these results suggested that knockdown of H19 inhibited the Sox6 expression by modulating miR-19b.Fig. 4


Downregulation of long non-coding RNA H19 promotes P19CL6 cells proliferation and inhibits apoptosis during late-stage cardiac differentiation via miR-19b-modulated Sox6
miR-19b targets Sox6 gene and inhibits its expression. a The sketch map of the mmu-miR-19b-3p site in Sox6 3′-UTR. b Luciferase activity assay was performed in 293T cells at 48 h after transfection. The reporter assay indicated that miR-19b mimics were capable of significantly inhibiting luciferase expression of the Sox6-WT reporter plasmid but not the Sox6-Mut reporter plasmid. c Western blot showed that protein level of Sox6 was low from day 0 to 6, and was significantly upregulated from day 8 to 12. d The Sox6 protein levels were significantly down- and up-regulated respectively in P19CL6 cells transfected with miR-19b and anti-miR-19b at day 10. e shH19 inhibited the Sox6 expression significantly, but miR-19b enhanced the inhibitory effect of shH19 on Sox6 expression and anti-miR-19b reversed the influence of shH19 on Sox6 expression at day 10 after transfection. *P < 0.05, **P < 0.01, ***P < 0.001
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Fig4: miR-19b targets Sox6 gene and inhibits its expression. a The sketch map of the mmu-miR-19b-3p site in Sox6 3′-UTR. b Luciferase activity assay was performed in 293T cells at 48 h after transfection. The reporter assay indicated that miR-19b mimics were capable of significantly inhibiting luciferase expression of the Sox6-WT reporter plasmid but not the Sox6-Mut reporter plasmid. c Western blot showed that protein level of Sox6 was low from day 0 to 6, and was significantly upregulated from day 8 to 12. d The Sox6 protein levels were significantly down- and up-regulated respectively in P19CL6 cells transfected with miR-19b and anti-miR-19b at day 10. e shH19 inhibited the Sox6 expression significantly, but miR-19b enhanced the inhibitory effect of shH19 on Sox6 expression and anti-miR-19b reversed the influence of shH19 on Sox6 expression at day 10 after transfection. *P < 0.05, **P < 0.01, ***P < 0.001
Mentions: Bioinformatics analysis using online software TargetScan indicated that miR-19b may bind to 3′UTR of Sox6 (Fig. 4a). In addition, luciferase reporter assay indicated that miR-19b significantly suppressed luciferase expression of the wild type Sox6 reporter (Sox6-WT) but not the mutant reporter (Sox6-Mut) (Fig. 4b), confirming that Sox6 was a target of miR-19b. Western blot was used to detect the expression of Sox6 in the cell differentiation and the results indicated that the protein level of Sox6 was very low from day 0 to day 6, but increased significantly from day 8 to day 12 (Fig. 4c). Then we transfected P19CL6 cells with miR-19b mimics, miR-NC, anti-miR-19b or anti-miR-NC. The results showed that the protein level of Sox6 was significantly down-regulated in P19CL6 cells transfected with miR-19b and significantly up-regulated in P19CL6 cells transfected with anti-miR-19b (Fig. 4d). Furthermore, we transfected H19-knockdown P19CL6 cells with miR-19b mimics or anti-miR-19b. The result showed that knockdown H19 inhibited the Sox6 expression significantly, while miR-19b enhanced the inhibitory effect of knockdown H19 on Sox6 expression, and anti-miR-19b reversed the influence of knockdown H19 on Sox6 expression (Fig. 4e). All these results suggested that knockdown of H19 inhibited the Sox6 expression by modulating miR-19b.Fig. 4

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.