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Silencing of microRNA-122 is an early event during hepatocarcinogenesis from non-alcoholic steatohepatitis.

Takaki Y, Saito Y, Takasugi A, Toshimitsu K, Yamada S, Muramatsu T, Kimura M, Sugiyama K, Suzuki H, Arai E, Ojima H, Kanai Y, Saito H - Cancer Sci. (2014)

Bottom Line: Expression of miR-122 in non-tumor LC at the age of 18 weeks was significantly lower than that in LC at the age of 12 weeks.Expression of miR-122 was further decreased in HCCs relative to non-tumor LC at the age of 18 weeks.DNA methylation analysis revealed that silencing of miR-122 was not mediated by DNA hypermethylation of the promoter region.

View Article: PubMed Central - PubMed

Affiliation: Division of Pharmacotherapeutics, Keio University Faculty of Pharmacy, Tokyo, Japan.

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Promoter assay of microRNA-122 (miR-122) expression. (a) The promoter region of miR-122, which contains a TATA-box, a CCAAT-box, and DR-1 and DR-2 elements. DNA methylation status was determined by bisulfite pyrosequencing at the CpG sites indicated by asterisks. Arrow indicates the transcription start site (TSS), as described previously.21 (b) Promoter assay of miR-122 expression using a Dual Luciferase Reporter Assay System. Fragments of the human miR-122 promoter with or without the DR-1 and DR-2 elements were inserted between the SacI and HindIII sites within pGL4.10. Plasmids with or without Sss I CpG methylase treatment were cotransfected with Renilla luciferase expression vector into HepG2 cells. Forty-eight hours after transfection, luciferase activities were measured. *P < 0.01; **P < 0.005.
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fig05: Promoter assay of microRNA-122 (miR-122) expression. (a) The promoter region of miR-122, which contains a TATA-box, a CCAAT-box, and DR-1 and DR-2 elements. DNA methylation status was determined by bisulfite pyrosequencing at the CpG sites indicated by asterisks. Arrow indicates the transcription start site (TSS), as described previously.21 (b) Promoter assay of miR-122 expression using a Dual Luciferase Reporter Assay System. Fragments of the human miR-122 promoter with or without the DR-1 and DR-2 elements were inserted between the SacI and HindIII sites within pGL4.10. Plasmids with or without Sss I CpG methylase treatment were cotransfected with Renilla luciferase expression vector into HepG2 cells. Forty-eight hours after transfection, luciferase activities were measured. *P < 0.01; **P < 0.005.

Mentions: To reveal the molecular mechanism underlying regulation of miR-122, we analyzed the DNA methylation status of the miR-122 promoter region, which contains a TATA-box, a CCAAT-box, and DR-1 and DR-2 elements21 (Fig.5a). We carried out the promoter assay using fragments of the human miR-122 promoter with or without the DR-1 and DR-2 elements (Fig.5b). Plasmids with or without Sss I CpG methylase treatment were used to cotransfect HepG2 cells with the Renilla luciferase expression vector. Forty-eight hours after transfection, luciferase activities were measured. The relative luciferase activity of the construct containing the DR-1 and DR-2 elements was significantly higher than that of the construct lacking these elements (*P < 0.01, Fig.5b). After treatment with CpG methylase, the relative luciferase activities were significantly decreased in the constructs both with and without the DR-1 and DR-2 elements (**P < 0.005, Fig.5b). These results indicate that the DR-1 and DR-2 elements in the miR-122 promoter are essential for regulation of miR-122 expression and that DNA methylation around the DR-1 and DR-2 elements suppress miR-122 expression.


Silencing of microRNA-122 is an early event during hepatocarcinogenesis from non-alcoholic steatohepatitis.

Takaki Y, Saito Y, Takasugi A, Toshimitsu K, Yamada S, Muramatsu T, Kimura M, Sugiyama K, Suzuki H, Arai E, Ojima H, Kanai Y, Saito H - Cancer Sci. (2014)

Promoter assay of microRNA-122 (miR-122) expression. (a) The promoter region of miR-122, which contains a TATA-box, a CCAAT-box, and DR-1 and DR-2 elements. DNA methylation status was determined by bisulfite pyrosequencing at the CpG sites indicated by asterisks. Arrow indicates the transcription start site (TSS), as described previously.21 (b) Promoter assay of miR-122 expression using a Dual Luciferase Reporter Assay System. Fragments of the human miR-122 promoter with or without the DR-1 and DR-2 elements were inserted between the SacI and HindIII sites within pGL4.10. Plasmids with or without Sss I CpG methylase treatment were cotransfected with Renilla luciferase expression vector into HepG2 cells. Forty-eight hours after transfection, luciferase activities were measured. *P < 0.01; **P < 0.005.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4462359&req=5

fig05: Promoter assay of microRNA-122 (miR-122) expression. (a) The promoter region of miR-122, which contains a TATA-box, a CCAAT-box, and DR-1 and DR-2 elements. DNA methylation status was determined by bisulfite pyrosequencing at the CpG sites indicated by asterisks. Arrow indicates the transcription start site (TSS), as described previously.21 (b) Promoter assay of miR-122 expression using a Dual Luciferase Reporter Assay System. Fragments of the human miR-122 promoter with or without the DR-1 and DR-2 elements were inserted between the SacI and HindIII sites within pGL4.10. Plasmids with or without Sss I CpG methylase treatment were cotransfected with Renilla luciferase expression vector into HepG2 cells. Forty-eight hours after transfection, luciferase activities were measured. *P < 0.01; **P < 0.005.
Mentions: To reveal the molecular mechanism underlying regulation of miR-122, we analyzed the DNA methylation status of the miR-122 promoter region, which contains a TATA-box, a CCAAT-box, and DR-1 and DR-2 elements21 (Fig.5a). We carried out the promoter assay using fragments of the human miR-122 promoter with or without the DR-1 and DR-2 elements (Fig.5b). Plasmids with or without Sss I CpG methylase treatment were used to cotransfect HepG2 cells with the Renilla luciferase expression vector. Forty-eight hours after transfection, luciferase activities were measured. The relative luciferase activity of the construct containing the DR-1 and DR-2 elements was significantly higher than that of the construct lacking these elements (*P < 0.01, Fig.5b). After treatment with CpG methylase, the relative luciferase activities were significantly decreased in the constructs both with and without the DR-1 and DR-2 elements (**P < 0.005, Fig.5b). These results indicate that the DR-1 and DR-2 elements in the miR-122 promoter are essential for regulation of miR-122 expression and that DNA methylation around the DR-1 and DR-2 elements suppress miR-122 expression.

Bottom Line: Expression of miR-122 in non-tumor LC at the age of 18 weeks was significantly lower than that in LC at the age of 12 weeks.Expression of miR-122 was further decreased in HCCs relative to non-tumor LC at the age of 18 weeks.DNA methylation analysis revealed that silencing of miR-122 was not mediated by DNA hypermethylation of the promoter region.

View Article: PubMed Central - PubMed

Affiliation: Division of Pharmacotherapeutics, Keio University Faculty of Pharmacy, Tokyo, Japan.

Show MeSH
Related in: MedlinePlus