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Wnt/beta-catenin signaling activates microRNA-181 expression in hepatocellular carcinoma.

Ji J, Yamashita T, Wang XW - Cell Biosci (2011)

Bottom Line: Using both western blot and quantitative reverse transcriptase-PCR analyses, we found that the expression of all four microRNA-181 family members was positively correlated with β-catenin expression in HCC cell lines.Consistently, we found that Tcf4 interacted with these regions in vivo using chromatin immunoprecipitation assay.Taken together, our results demonstrate that microRNA-181s are transcriptionally activated by the Wnt/beta-catenin signaling pathway in HCC.

View Article: PubMed Central - HTML - PubMed

Affiliation: Liver Carcinogenesis Section, Laboratory of Human Carcinogenesis, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland, USA. xw3u@nih.gov.

ABSTRACT

Background: Hepatocellular carcinoma (HCC) is a malignant cancer with an observable heterogeneity and microRNAs are functionally associated with the tumorigenesis of HCC. We recently identified that EpCAM (CD326)-positive cells isolated from alpha-fetoprotein (AFP)-positive HCC samples are hepatic cancer stem cells (HepCSCs). EpCAM+AFP+ HepCSCs have an activated Wnt/β-catenin signaling with a parallel increased expression of all four microRNA-181 family members. We hypothesized that Wnt/β-catenin signaling transcriptionally activates microRNA-181s in HCC.

Results: Using both western blot and quantitative reverse transcriptase-PCR analyses, we found that the expression of all four microRNA-181 family members was positively correlated with β-catenin expression in HCC cell lines. MicroRNA-181 expression could be directly induced upon an activation of Wnt/β-catenin signaling, which includes Wnt10B overexpression, inhibition of GSK3β signaling by LiCl, or forced expression of β-catenin/Tcf4. Moreover, microRNA-181 expression was inhibited upon an inactivation of Wnt/β-catenin signaling by an induction of adenomatosis polyposis coli (APC) expression or silencing β-catenin via RNA interference. In addition, seven putative β-catenin/Tcf4 binding sites were identified in the promoter region of the microRNA-181a-2 and microRNA-181b-2 transcripts. Consistently, we found that Tcf4 interacted with these regions in vivo using chromatin immunoprecipitation assay.

Conclusions: Taken together, our results demonstrate that microRNA-181s are transcriptionally activated by the Wnt/beta-catenin signaling pathway in HCC.

No MeSH data available.


Related in: MedlinePlus

Tcf4 associates with the promoter of miR-181s. (A) Predicted Tcf4 binding sites in the promoter region of the miR-181a-2/miR-181b-2 gene. (B) PCR amplicons for ChIP assay were designed within 200-bp windows in the ~700-bp upstream of the transcription start site (amplicon U), and in the ~500-bp downstream of transcription start site (amplicon D). (C) PCR analysis of Tcf4 chromatin immunoprecipitate. Actin chromatin immunoprecipitate was used as negative control. PCR products were run on a 3.0% agarose gel.
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Figure 4: Tcf4 associates with the promoter of miR-181s. (A) Predicted Tcf4 binding sites in the promoter region of the miR-181a-2/miR-181b-2 gene. (B) PCR amplicons for ChIP assay were designed within 200-bp windows in the ~700-bp upstream of the transcription start site (amplicon U), and in the ~500-bp downstream of transcription start site (amplicon D). (C) PCR analysis of Tcf4 chromatin immunoprecipitate. Actin chromatin immunoprecipitate was used as negative control. PCR products were run on a 3.0% agarose gel.

Mentions: Studies have shown that the β-catenin/Tcf complex transcriptionally regulates their target gene expression through binding to a consensus core TCF/LEF-binding site (5'-A/T A/T CAAAG-3') within the promoter regions [15,16]. GenBank database analyses indicate that miR-181a-1 and miR-181b-1 are located in the intron 2 of a novel host gene (RP11-31E23.1-001) on 1q31 (data not shown), while miR-181a-2 and miR-181b-2 are located in the intron 2 of a novel transcript, RP11-348K2.1-001, on 9q33 (figure 4A). We searched the core TCF/LEF binding site within 4kb genomic sequences of the predicted promoter regions of these two genes. Seven TCF/LEF binding elements were identified in the promoter region of RP11-348K2.1-001 (figure 4A), and three elements were in the promoter region of RP11-31E23.1-001 (data not shown). MiR-181c and miR-181d are present in the intergenic region of chromosome 19 and consequently no promoter region is available for analysis. Furthermore, RP11-348K2.1-001 was selected for chromatin immuoprecipitation (ChIP) assay using Tcf4 antibody in Hep3B cells where both β-catenin and miR-181s are highly elevated. We designed two amplicons within a 200-bp window, i.e., amplicon U (in the upstream of transcription site) and amplicon D (in the downstream of transcription site) (figure 4B). The results showed that Tcf4 specifically bound to both amplicons in the miR-181a-2/miR-181b-2 promoter (figure 4C), indicating that the miR-181a-2/miR-181b-2 transcript is a direct transcriptional target of Wnt/β-catenin canonical signaling pathway in HCC.


Wnt/beta-catenin signaling activates microRNA-181 expression in hepatocellular carcinoma.

Ji J, Yamashita T, Wang XW - Cell Biosci (2011)

Tcf4 associates with the promoter of miR-181s. (A) Predicted Tcf4 binding sites in the promoter region of the miR-181a-2/miR-181b-2 gene. (B) PCR amplicons for ChIP assay were designed within 200-bp windows in the ~700-bp upstream of the transcription start site (amplicon U), and in the ~500-bp downstream of transcription start site (amplicon D). (C) PCR analysis of Tcf4 chromatin immunoprecipitate. Actin chromatin immunoprecipitate was used as negative control. PCR products were run on a 3.0% agarose gel.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3116242&req=5

Figure 4: Tcf4 associates with the promoter of miR-181s. (A) Predicted Tcf4 binding sites in the promoter region of the miR-181a-2/miR-181b-2 gene. (B) PCR amplicons for ChIP assay were designed within 200-bp windows in the ~700-bp upstream of the transcription start site (amplicon U), and in the ~500-bp downstream of transcription start site (amplicon D). (C) PCR analysis of Tcf4 chromatin immunoprecipitate. Actin chromatin immunoprecipitate was used as negative control. PCR products were run on a 3.0% agarose gel.
Mentions: Studies have shown that the β-catenin/Tcf complex transcriptionally regulates their target gene expression through binding to a consensus core TCF/LEF-binding site (5'-A/T A/T CAAAG-3') within the promoter regions [15,16]. GenBank database analyses indicate that miR-181a-1 and miR-181b-1 are located in the intron 2 of a novel host gene (RP11-31E23.1-001) on 1q31 (data not shown), while miR-181a-2 and miR-181b-2 are located in the intron 2 of a novel transcript, RP11-348K2.1-001, on 9q33 (figure 4A). We searched the core TCF/LEF binding site within 4kb genomic sequences of the predicted promoter regions of these two genes. Seven TCF/LEF binding elements were identified in the promoter region of RP11-348K2.1-001 (figure 4A), and three elements were in the promoter region of RP11-31E23.1-001 (data not shown). MiR-181c and miR-181d are present in the intergenic region of chromosome 19 and consequently no promoter region is available for analysis. Furthermore, RP11-348K2.1-001 was selected for chromatin immuoprecipitation (ChIP) assay using Tcf4 antibody in Hep3B cells where both β-catenin and miR-181s are highly elevated. We designed two amplicons within a 200-bp window, i.e., amplicon U (in the upstream of transcription site) and amplicon D (in the downstream of transcription site) (figure 4B). The results showed that Tcf4 specifically bound to both amplicons in the miR-181a-2/miR-181b-2 promoter (figure 4C), indicating that the miR-181a-2/miR-181b-2 transcript is a direct transcriptional target of Wnt/β-catenin canonical signaling pathway in HCC.

Bottom Line: Using both western blot and quantitative reverse transcriptase-PCR analyses, we found that the expression of all four microRNA-181 family members was positively correlated with β-catenin expression in HCC cell lines.Consistently, we found that Tcf4 interacted with these regions in vivo using chromatin immunoprecipitation assay.Taken together, our results demonstrate that microRNA-181s are transcriptionally activated by the Wnt/beta-catenin signaling pathway in HCC.

View Article: PubMed Central - HTML - PubMed

Affiliation: Liver Carcinogenesis Section, Laboratory of Human Carcinogenesis, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland, USA. xw3u@nih.gov.

ABSTRACT

Background: Hepatocellular carcinoma (HCC) is a malignant cancer with an observable heterogeneity and microRNAs are functionally associated with the tumorigenesis of HCC. We recently identified that EpCAM (CD326)-positive cells isolated from alpha-fetoprotein (AFP)-positive HCC samples are hepatic cancer stem cells (HepCSCs). EpCAM+AFP+ HepCSCs have an activated Wnt/β-catenin signaling with a parallel increased expression of all four microRNA-181 family members. We hypothesized that Wnt/β-catenin signaling transcriptionally activates microRNA-181s in HCC.

Results: Using both western blot and quantitative reverse transcriptase-PCR analyses, we found that the expression of all four microRNA-181 family members was positively correlated with β-catenin expression in HCC cell lines. MicroRNA-181 expression could be directly induced upon an activation of Wnt/β-catenin signaling, which includes Wnt10B overexpression, inhibition of GSK3β signaling by LiCl, or forced expression of β-catenin/Tcf4. Moreover, microRNA-181 expression was inhibited upon an inactivation of Wnt/β-catenin signaling by an induction of adenomatosis polyposis coli (APC) expression or silencing β-catenin via RNA interference. In addition, seven putative β-catenin/Tcf4 binding sites were identified in the promoter region of the microRNA-181a-2 and microRNA-181b-2 transcripts. Consistently, we found that Tcf4 interacted with these regions in vivo using chromatin immunoprecipitation assay.

Conclusions: Taken together, our results demonstrate that microRNA-181s are transcriptionally activated by the Wnt/beta-catenin signaling pathway in HCC.

No MeSH data available.


Related in: MedlinePlus