Liver Carcinogenesis Section, Laboratory of Human Carcinogenesis, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland, USA.
Cell Biosci. 2011 Jan 18;1(1):4. doi: 10.1186/2045-3701-1-4.
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.
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.
Taken together, our results demonstrate that microRNA-181s are transcriptionally activated by the Wnt/beta-catenin signaling pathway in HCC.
肝细胞癌（HCC）是一种恶性肿瘤，具有明显的异质性，microRNAs 与 HCC 的发生密切相关。我们最近发现，从甲胎蛋白（AFP）阳性 HCC 样本中分离出的 EpCAM（CD326）阳性细胞是肝癌干细胞（HepCSCs）。EpCAM+AFP+HepCSCs 具有激活的 Wnt/β-catenin 信号通路，同时平行增加了四个 microRNA-181 家族成员的表达。我们假设 Wnt/β-catenin 信号通路转录激活 HCC 中的 microRNA-181。
通过 Western blot 和定量逆转录-PCR 分析，我们发现 HCC 细胞系中所有四个 microRNA-181 家族成员的表达均与 β-catenin 表达呈正相关。Wnt/β-catenin 信号的激活可直接诱导 microRNA-181 的表达，包括 Wnt10B 过表达、LiCl 抑制 GSK3β 信号或强制表达 β-catenin/Tcf4。此外，Wnt/β-catenin 信号的失活可通过 APC 表达的诱导或通过 RNA 干扰沉默 β-catenin 来抑制 microRNA-181 的表达。此外，在 microRNA-181a-2 和 microRNA-181b-2 转录物的启动子区域鉴定出七个推定的 β-catenin/Tcf4 结合位点。一致地，我们使用染色质免疫沉淀测定发现 Tcf4 在体内与这些区域相互作用。
总之，我们的结果表明，microRNA-181s 是由 HCC 中的 Wnt/β-catenin 信号通路转录激活的。
