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Mir-34a is upregulated during liver regeneration in rats and is associated with the suppression of hepatocyte proliferation.

Chen H, Sun Y, Dong R, Yang S, Pan C, Xiang D, Miao M, Jiao B - PLoS ONE (2011)

Bottom Line: In BRL-3A cells, INHBB was identified as a direct target of miR-34a by luciferase reporter assay.A decrease of INHBB and Met was detected in regenerating liver.MiR-34a expression was upregulated during the late phase of liver regeneration.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Second Military Medical University, Shanghai, China.

ABSTRACT

Background: MicroRNAs are a class of small regulatory RNAs that modulate a variety of biological processes, including cellular differentiation, apoptosis, metabolism and proliferation. This study aims to explore the effect of miR-34a in hepatocyte proliferation and its potential role in liver regeneration termination.

Methodology/principal finding: MiR-34a was highly induced after partial hepatectomy. Overexpression of miR-34a in BRL-3A cells could significantly inhibit cell proliferation and down-regulate the expression of inhibin βB (INHBB) and Met. In BRL-3A cells, INHBB was identified as a direct target of miR-34a by luciferase reporter assay. More importantly, INHBB siRNA significantly repressed cell proliferation. A decrease of INHBB and Met was detected in regenerating liver.

Conclusion/significance: MiR-34a expression was upregulated during the late phase of liver regeneration. MiR-34a-mediated regulation of INHBB and Met may collectively contribute to the suppression of hepatocyte proliferation.

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Related in: MedlinePlus

Analyses of candidate target genes of miR-34a.(A) miR-34a decreased mRNA expression of inhibin beta B (INHBB) and Met by qRT-PCR. (B) miR-34a decreased protein expression of INHBB and Met by westernblot analysis. Actin was used as sample control. (C) miR-34a-binding site in the 3′-UTR (top) and mutated sites in 3′-UTR (bottom) of INHBB constructed in a luciferase system. (D) The 3′-UTR of INHBB mediates INHBB repression. BRL-3A cells were co-transfected with a luciferase reporter vector containing the 3′-UTR or mutated sequence of INHBB and miR-34a mimics (miR-34a) or negative control (NC). pGL3-control vector was used as control. **P<0.01 vs NC.
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pone-0020238-g003: Analyses of candidate target genes of miR-34a.(A) miR-34a decreased mRNA expression of inhibin beta B (INHBB) and Met by qRT-PCR. (B) miR-34a decreased protein expression of INHBB and Met by westernblot analysis. Actin was used as sample control. (C) miR-34a-binding site in the 3′-UTR (top) and mutated sites in 3′-UTR (bottom) of INHBB constructed in a luciferase system. (D) The 3′-UTR of INHBB mediates INHBB repression. BRL-3A cells were co-transfected with a luciferase reporter vector containing the 3′-UTR or mutated sequence of INHBB and miR-34a mimics (miR-34a) or negative control (NC). pGL3-control vector was used as control. **P<0.01 vs NC.

Mentions: INHBB, which encodes a subunit of activin AB and activin B, was identified by bioinformatic analysis. Although Met has been proved to be a target of miR-34a in HeLa cells and HepG2 cells [17], [18], it is still involved in validation in BRL-3A cells. qRT-PCR and westernblot analysis revealed that miR-34a drastically inhibited the expression of INHBB and Met on both mRNA and protein levels (Figure 3A,B). To detect whether the regulation of INHBB was direct, we fused the 3′-UTR region of INHBB to a luciferase system. As shown in Figure 3C,D, miR-34a remarkably repressed the expression of luciferase containing an original miR-34a binding site (INHBB-UTR) but not a mutant binding site (INHBB-Mu-UTR). And mutations in seed complementary sites of the 3′-UTR region of INHBB could restore the luciferase expression.


Mir-34a is upregulated during liver regeneration in rats and is associated with the suppression of hepatocyte proliferation.

Chen H, Sun Y, Dong R, Yang S, Pan C, Xiang D, Miao M, Jiao B - PLoS ONE (2011)

Analyses of candidate target genes of miR-34a.(A) miR-34a decreased mRNA expression of inhibin beta B (INHBB) and Met by qRT-PCR. (B) miR-34a decreased protein expression of INHBB and Met by westernblot analysis. Actin was used as sample control. (C) miR-34a-binding site in the 3′-UTR (top) and mutated sites in 3′-UTR (bottom) of INHBB constructed in a luciferase system. (D) The 3′-UTR of INHBB mediates INHBB repression. BRL-3A cells were co-transfected with a luciferase reporter vector containing the 3′-UTR or mutated sequence of INHBB and miR-34a mimics (miR-34a) or negative control (NC). pGL3-control vector was used as control. **P<0.01 vs NC.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3105003&req=5

pone-0020238-g003: Analyses of candidate target genes of miR-34a.(A) miR-34a decreased mRNA expression of inhibin beta B (INHBB) and Met by qRT-PCR. (B) miR-34a decreased protein expression of INHBB and Met by westernblot analysis. Actin was used as sample control. (C) miR-34a-binding site in the 3′-UTR (top) and mutated sites in 3′-UTR (bottom) of INHBB constructed in a luciferase system. (D) The 3′-UTR of INHBB mediates INHBB repression. BRL-3A cells were co-transfected with a luciferase reporter vector containing the 3′-UTR or mutated sequence of INHBB and miR-34a mimics (miR-34a) or negative control (NC). pGL3-control vector was used as control. **P<0.01 vs NC.
Mentions: INHBB, which encodes a subunit of activin AB and activin B, was identified by bioinformatic analysis. Although Met has been proved to be a target of miR-34a in HeLa cells and HepG2 cells [17], [18], it is still involved in validation in BRL-3A cells. qRT-PCR and westernblot analysis revealed that miR-34a drastically inhibited the expression of INHBB and Met on both mRNA and protein levels (Figure 3A,B). To detect whether the regulation of INHBB was direct, we fused the 3′-UTR region of INHBB to a luciferase system. As shown in Figure 3C,D, miR-34a remarkably repressed the expression of luciferase containing an original miR-34a binding site (INHBB-UTR) but not a mutant binding site (INHBB-Mu-UTR). And mutations in seed complementary sites of the 3′-UTR region of INHBB could restore the luciferase expression.

Bottom Line: In BRL-3A cells, INHBB was identified as a direct target of miR-34a by luciferase reporter assay.A decrease of INHBB and Met was detected in regenerating liver.MiR-34a expression was upregulated during the late phase of liver regeneration.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Second Military Medical University, Shanghai, China.

ABSTRACT

Background: MicroRNAs are a class of small regulatory RNAs that modulate a variety of biological processes, including cellular differentiation, apoptosis, metabolism and proliferation. This study aims to explore the effect of miR-34a in hepatocyte proliferation and its potential role in liver regeneration termination.

Methodology/principal finding: MiR-34a was highly induced after partial hepatectomy. Overexpression of miR-34a in BRL-3A cells could significantly inhibit cell proliferation and down-regulate the expression of inhibin βB (INHBB) and Met. In BRL-3A cells, INHBB was identified as a direct target of miR-34a by luciferase reporter assay. More importantly, INHBB siRNA significantly repressed cell proliferation. A decrease of INHBB and Met was detected in regenerating liver.

Conclusion/significance: MiR-34a expression was upregulated during the late phase of liver regeneration. MiR-34a-mediated regulation of INHBB and Met may collectively contribute to the suppression of hepatocyte proliferation.

Show MeSH
Related in: MedlinePlus