Limits...
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

Wnt/β-catenin signaling inhibition reduces the expression of miR-181. (A) HT29-APC and HT29-GAL cells were incubated with 100 μM of ZnCL2 for different time points (0 hours, 24 hours and 48 hours). Then qRT-PCR analysis of all four mature miR-181s was performed in these cells. (B, C) β-catenin siRNA and control siRNA was transfected in HuH7 and HuH1 cells using lipofectamine 2000. Western blotting analysis of β-catenin and β-actin (left panel) and qRT-PCR analysis of CTNNB1 (right panel) were performed (B). qRT-PCR analysis of all four mature miR-181s (C). All these experiments were done in triplicates and the level of miRNAs and genes were shown as mean ± standard deviation. * refers to p < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3116242&req=5

Figure 3: Wnt/β-catenin signaling inhibition reduces the expression of miR-181. (A) HT29-APC and HT29-GAL cells were incubated with 100 μM of ZnCL2 for different time points (0 hours, 24 hours and 48 hours). Then qRT-PCR analysis of all four mature miR-181s was performed in these cells. (B, C) β-catenin siRNA and control siRNA was transfected in HuH7 and HuH1 cells using lipofectamine 2000. Western blotting analysis of β-catenin and β-actin (left panel) and qRT-PCR analysis of CTNNB1 (right panel) were performed (B). qRT-PCR analysis of all four mature miR-181s (C). All these experiments were done in triplicates and the level of miRNAs and genes were shown as mean ± standard deviation. * refers to p < 0.05.

Mentions: Several known proteins such as adenomatosis polyposis coli (APC) can promote β-catenin degradation [14]. To investigate the effect of Wnt/β-catenin inactivation on the expression of miR-181s, we used HT29-APC cells where the APC gene is under the control of a zinc-activated metallothionein promoter [14]. The levels of all four miR-181s were significantly decreased in HT29-APC cells following addition of zinc chloride (ZnCl2), but not in control HT29-GAL cells (figure 3A). Furthermore, β-catenin siRNA was employed to suppress Wnt/β-catenin activity in HuH1 and HuH7 cells. Consistently, declined β-catenin expression by siRNA (Figure 3B and 3C) significantly reduced the expression of all four miR-181 transcripts (figure 3D). These data demonstrate that an inactivation of Wnt/β-catenin signaling results in an inhibition of miR-181 expression.


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

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

Wnt/β-catenin signaling inhibition reduces the expression of miR-181. (A) HT29-APC and HT29-GAL cells were incubated with 100 μM of ZnCL2 for different time points (0 hours, 24 hours and 48 hours). Then qRT-PCR analysis of all four mature miR-181s was performed in these cells. (B, C) β-catenin siRNA and control siRNA was transfected in HuH7 and HuH1 cells using lipofectamine 2000. Western blotting analysis of β-catenin and β-actin (left panel) and qRT-PCR analysis of CTNNB1 (right panel) were performed (B). qRT-PCR analysis of all four mature miR-181s (C). All these experiments were done in triplicates and the level of miRNAs and genes were shown as mean ± standard deviation. * refers to p < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Wnt/β-catenin signaling inhibition reduces the expression of miR-181. (A) HT29-APC and HT29-GAL cells were incubated with 100 μM of ZnCL2 for different time points (0 hours, 24 hours and 48 hours). Then qRT-PCR analysis of all four mature miR-181s was performed in these cells. (B, C) β-catenin siRNA and control siRNA was transfected in HuH7 and HuH1 cells using lipofectamine 2000. Western blotting analysis of β-catenin and β-actin (left panel) and qRT-PCR analysis of CTNNB1 (right panel) were performed (B). qRT-PCR analysis of all four mature miR-181s (C). All these experiments were done in triplicates and the level of miRNAs and genes were shown as mean ± standard deviation. * refers to p < 0.05.
Mentions: Several known proteins such as adenomatosis polyposis coli (APC) can promote β-catenin degradation [14]. To investigate the effect of Wnt/β-catenin inactivation on the expression of miR-181s, we used HT29-APC cells where the APC gene is under the control of a zinc-activated metallothionein promoter [14]. The levels of all four miR-181s were significantly decreased in HT29-APC cells following addition of zinc chloride (ZnCl2), but not in control HT29-GAL cells (figure 3A). Furthermore, β-catenin siRNA was employed to suppress Wnt/β-catenin activity in HuH1 and HuH7 cells. Consistently, declined β-catenin expression by siRNA (Figure 3B and 3C) significantly reduced the expression of all four miR-181 transcripts (figure 3D). These data demonstrate that an inactivation of Wnt/β-catenin signaling results in an inhibition of miR-181 expression.

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