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Detection of miR-33 Expression and the Verification of Its Target Genes in the Fatty Liver of Geese.

Zheng Y, Jiang S, Zhang Y, Zhang R, Gong D - Int J Mol Sci (2015)

Bottom Line: They bind to the 3' untranslated regions of mRNA transcripts to reduce the translation of these transcripts or to cause their degradation.The expression level of miR-33 increases significantly in the liver after 19 days in comparison with the control group; (2) By using the bioinformatics software programs TargetScan, miRDB and miRCosm to predict the target genes of miR-33 according to laboratory prophase transcriptome results and references, we screen nine target genes: adenosine triphosphate binding cassette transporters A1, adenosine triphosphate binding cassette transporters G1, Neimann Pick C, carnitine O-octanoyltransferase (CROT), cyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase/enoyl-CoA hydratase, beta subunit (HADHB), AMP-activated protein kinase, alpha subunit 1 (AMPKα1), insulin receptor substrate 2, glutamic pyruvate transaminase and adipose differentiation-related protein.These genes determine the combined target site.

View Article: PubMed Central - PubMed

Affiliation: College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China. zhengyun@yzu.edu.cn.

ABSTRACT

Background: miRNAs are single-stranded, small RNA molecules with a length of 18-25 nucleotides. They bind to the 3' untranslated regions of mRNA transcripts to reduce the translation of these transcripts or to cause their degradation. The roles of these molecules differ in biological processes, such as cell differentiation, proliferation, apoptosis and tumor genesis. miRNA-33 is encoded by the gene introns of proteins that bind sterol-regulatory elements. This molecule cooperates with these proteins to control cholesterol homeostasis, fatty acid levels and the genes that are related to the expression of fat metabolism. The examination of miR-33 expression and its target genes can promote the in-depth study of the miRNA regulation mechanism in the formation process of goose fatty liver and can lay a foundation for research into human fatty liver.

Methodology/principal findings: (1) Through real-time fluorescent quantitative polymerase chain reaction (TaqMan MicroRNA Assay), we detected the expression of miR-33 during the feeding of Landes geese. The expression level of miR-33 increases significantly in the liver after 19 days in comparison with the control group; (2) By using the bioinformatics software programs TargetScan, miRDB and miRCosm to predict the target genes of miR-33 according to laboratory prophase transcriptome results and references, we screen nine target genes: adenosine triphosphate binding cassette transporters A1, adenosine triphosphate binding cassette transporters G1, Neimann Pick C, carnitine O-octanoyltransferase (CROT), cyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase/enoyl-CoA hydratase, beta subunit (HADHB), AMP-activated protein kinase, alpha subunit 1 (AMPKα1), insulin receptor substrate 2, glutamic pyruvate transaminase and adipose differentiation-related protein. The dual luciferase reporter gene system in the CHO cell line verifies that CROT, HADHB and NPC1 are the target genes of miR-33 in geese. The inhibition rate of CROT is highest and reaches 70%; (3) The seed sequence (5' 2-8 bases) is the acting site of miR-33. The two predicted target sites of CROT are the target sites of miR-33. Moreover, the predicted target site of HADHB and NPC1 is the target site of miR-33.

Conclusions/significance: (1) After 19 days of overfeeding, the expression level of miR-33 increases significantly in the livers of geese; (2) CROT, HADHB and NPC1 are the target genes of miR-33 in geese. These genes determine the combined target site.

No MeSH data available.


Related in: MedlinePlus

Detection of luciferase activity after target site mutation. (A) Luciferase activity after target site mutation in the CROT gene; (B) luciferase activity after target site mutation in the HADHB gene; (C) luciferase activity after target site mutation in the NPC1 gene. Control group: pcDNA3.1 + pmiR-report + phRL-TK; miRNA-33: pcDNA3.1-miR-33 + pmiR-report + phRL-TK; mut-miR-33: pcDNA3.1-mut-miR-33 + pmiR-report + phRL-TK; mut-Box: pcDNA3.1-miR-33 + mut-pmir-report + phRL-TK. Mut is an abbreviation of the mutant gene. Mut-miR-33 represents the mutation of miR-33, and mut-box represents the mutation of target sites. * represents p < 0.05; ** represents p < 0.01.
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ijms-16-12737-f006: Detection of luciferase activity after target site mutation. (A) Luciferase activity after target site mutation in the CROT gene; (B) luciferase activity after target site mutation in the HADHB gene; (C) luciferase activity after target site mutation in the NPC1 gene. Control group: pcDNA3.1 + pmiR-report + phRL-TK; miRNA-33: pcDNA3.1-miR-33 + pmiR-report + phRL-TK; mut-miR-33: pcDNA3.1-mut-miR-33 + pmiR-report + phRL-TK; mut-Box: pcDNA3.1-miR-33 + mut-pmir-report + phRL-TK. Mut is an abbreviation of the mutant gene. Mut-miR-33 represents the mutation of miR-33, and mut-box represents the mutation of target sites. * represents p < 0.05; ** represents p < 0.01.

Mentions: The report vectors of the 2–5 base at the 5′ end of miRNA-33 and of either the 2–3 or the 5–6 base at the 3′ end of target gene are mutated. The overexpression and internal vectors are then transfected into CHO cells to detect luciferase activity (Figure 6). The repeated experiment results show that the luciferase activity in the CROT, HADHB and NPC1 genes did not change significantly when the seed sequence points of miRNA-33 were mutated.


Detection of miR-33 Expression and the Verification of Its Target Genes in the Fatty Liver of Geese.

Zheng Y, Jiang S, Zhang Y, Zhang R, Gong D - Int J Mol Sci (2015)

Detection of luciferase activity after target site mutation. (A) Luciferase activity after target site mutation in the CROT gene; (B) luciferase activity after target site mutation in the HADHB gene; (C) luciferase activity after target site mutation in the NPC1 gene. Control group: pcDNA3.1 + pmiR-report + phRL-TK; miRNA-33: pcDNA3.1-miR-33 + pmiR-report + phRL-TK; mut-miR-33: pcDNA3.1-mut-miR-33 + pmiR-report + phRL-TK; mut-Box: pcDNA3.1-miR-33 + mut-pmir-report + phRL-TK. Mut is an abbreviation of the mutant gene. Mut-miR-33 represents the mutation of miR-33, and mut-box represents the mutation of target sites. * represents p < 0.05; ** represents p < 0.01.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4490470&req=5

ijms-16-12737-f006: Detection of luciferase activity after target site mutation. (A) Luciferase activity after target site mutation in the CROT gene; (B) luciferase activity after target site mutation in the HADHB gene; (C) luciferase activity after target site mutation in the NPC1 gene. Control group: pcDNA3.1 + pmiR-report + phRL-TK; miRNA-33: pcDNA3.1-miR-33 + pmiR-report + phRL-TK; mut-miR-33: pcDNA3.1-mut-miR-33 + pmiR-report + phRL-TK; mut-Box: pcDNA3.1-miR-33 + mut-pmir-report + phRL-TK. Mut is an abbreviation of the mutant gene. Mut-miR-33 represents the mutation of miR-33, and mut-box represents the mutation of target sites. * represents p < 0.05; ** represents p < 0.01.
Mentions: The report vectors of the 2–5 base at the 5′ end of miRNA-33 and of either the 2–3 or the 5–6 base at the 3′ end of target gene are mutated. The overexpression and internal vectors are then transfected into CHO cells to detect luciferase activity (Figure 6). The repeated experiment results show that the luciferase activity in the CROT, HADHB and NPC1 genes did not change significantly when the seed sequence points of miRNA-33 were mutated.

Bottom Line: They bind to the 3' untranslated regions of mRNA transcripts to reduce the translation of these transcripts or to cause their degradation.The expression level of miR-33 increases significantly in the liver after 19 days in comparison with the control group; (2) By using the bioinformatics software programs TargetScan, miRDB and miRCosm to predict the target genes of miR-33 according to laboratory prophase transcriptome results and references, we screen nine target genes: adenosine triphosphate binding cassette transporters A1, adenosine triphosphate binding cassette transporters G1, Neimann Pick C, carnitine O-octanoyltransferase (CROT), cyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase/enoyl-CoA hydratase, beta subunit (HADHB), AMP-activated protein kinase, alpha subunit 1 (AMPKα1), insulin receptor substrate 2, glutamic pyruvate transaminase and adipose differentiation-related protein.These genes determine the combined target site.

View Article: PubMed Central - PubMed

Affiliation: College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China. zhengyun@yzu.edu.cn.

ABSTRACT

Background: miRNAs are single-stranded, small RNA molecules with a length of 18-25 nucleotides. They bind to the 3' untranslated regions of mRNA transcripts to reduce the translation of these transcripts or to cause their degradation. The roles of these molecules differ in biological processes, such as cell differentiation, proliferation, apoptosis and tumor genesis. miRNA-33 is encoded by the gene introns of proteins that bind sterol-regulatory elements. This molecule cooperates with these proteins to control cholesterol homeostasis, fatty acid levels and the genes that are related to the expression of fat metabolism. The examination of miR-33 expression and its target genes can promote the in-depth study of the miRNA regulation mechanism in the formation process of goose fatty liver and can lay a foundation for research into human fatty liver.

Methodology/principal findings: (1) Through real-time fluorescent quantitative polymerase chain reaction (TaqMan MicroRNA Assay), we detected the expression of miR-33 during the feeding of Landes geese. The expression level of miR-33 increases significantly in the liver after 19 days in comparison with the control group; (2) By using the bioinformatics software programs TargetScan, miRDB and miRCosm to predict the target genes of miR-33 according to laboratory prophase transcriptome results and references, we screen nine target genes: adenosine triphosphate binding cassette transporters A1, adenosine triphosphate binding cassette transporters G1, Neimann Pick C, carnitine O-octanoyltransferase (CROT), cyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase/enoyl-CoA hydratase, beta subunit (HADHB), AMP-activated protein kinase, alpha subunit 1 (AMPKα1), insulin receptor substrate 2, glutamic pyruvate transaminase and adipose differentiation-related protein. The dual luciferase reporter gene system in the CHO cell line verifies that CROT, HADHB and NPC1 are the target genes of miR-33 in geese. The inhibition rate of CROT is highest and reaches 70%; (3) The seed sequence (5' 2-8 bases) is the acting site of miR-33. The two predicted target sites of CROT are the target sites of miR-33. Moreover, the predicted target site of HADHB and NPC1 is the target site of miR-33.

Conclusions/significance: (1) After 19 days of overfeeding, the expression level of miR-33 increases significantly in the livers of geese; (2) CROT, HADHB and NPC1 are the target genes of miR-33 in geese. These genes determine the combined target site.

No MeSH data available.


Related in: MedlinePlus