Bek Chai Heah Laboratory of Cancer Genomics, Division of Cellular and Molecular Research, Humphrey Oei Institute of Cancer Research, National Cancer Centre, Singapore, Singapore.
PLoS One. 2012;7(9):e44206. doi: 10.1371/journal.pone.0044206. Epub 2012 Sep 4.
The down-regulation of miR-214 has previously been observed in human hepatocellular carcinoma (HCC). Here, we demonstrated the down-regulation of miR-214 is associated with cell invasion, stem-like traits and early recurrence of HCC. Firstly, we validated the suppression of miR-214 in human HCC by real-time quantitative RT-PCR (qRT-PCR) in 20 paired tumor and non-tumor liver tissues of HCC patients and 10 histologically normal liver tissues from colorectal cancer patients with liver metastases. Further qRT-PCR analysis of 50 HCC tissues from an independent cohort of HCC patients of whom 29 with early recurrent disease (<2 years) and 21 with late recurrent disease demonstrated that the suppression of miR-214 was significantly more suppressed in samples from HCC patients with early recurrent disease compared those from patients with no recurrence. Re-expression of miR-214 significantly suppressed the growth of HCC cells in vitro and reduced their tumorigenicity in vivo. The enhancer of zeste homologue 2 (EZH2) and β-catenin (CTNNB1) was identified as two potential direct downstream targets of miR-214 through bioinformatics analysis and experimentally validated the miRNA-target interactions with a dual-firefly luciferase reporter assay. In corroborate with this, both EZH2 and CTNNB1 are found to be significantly overexpressed in human HCC biopsies. Since EZH2 can regulate CTNNB1, CTNNB1 can also be an indirect target of miR-214 through EZH2. Silencing EZH2 or CTNNB1 expression suppressed the growth and invasion of HCC cells and induced E-cadherin (CDH1), known to inhibit cell invasion and metastasis. Furthermore, the silencing of miR-214 or overexpression of EZH2 increased EpCAM(+) stem-like cells through the activation of CTNNB1. Interestingly, the up-regulation of EZH2, CTNNB1 and the down-regulation of CDH1 in HCC patients correlated with early recurrent disease and can be an independent predictor of poor survival. Therefore, miR-214 can directly or indirectly target CTNNB1 to modulate the β-catenin signaling pathway in HCC.
miR-214 的下调先前已在人肝癌（HCC）中观察到。在这里，我们证明 miR-214 的下调与 HCC 的细胞侵袭、干细胞样特征和早期复发有关。首先，我们通过实时定量 RT-PCR（qRT-PCR）在 20 对 HCC 患者的肿瘤和非肿瘤肝组织以及 10 例结直肠癌肝转移患者的组织学正常肝组织中验证了人 HCC 中 miR-214 的抑制。对来自另一独立 HCC 患者队列的 50 例 HCC 组织进行进一步 qRT-PCR 分析，其中 29 例为早期复发（<2 年），21 例为晚期复发，结果表明，早期复发的 HCC 患者样本中 miR-214 的抑制更为显著与无复发患者相比。miR-214 的重新表达显著抑制了 HCC 细胞的体外生长，并降低了其体内致瘤性。通过生物信息学分析鉴定出增强子的锌指同源物 2（EZH2）和β-连环蛋白（CTNNB1）是 miR-214 的两个潜在直接下游靶标，并通过双荧光素酶报告基因检测实验验证了 miRNA-靶标相互作用。与此相符的是，EZH2 和 CTNNB1 在人 HCC 活检中均显著过表达。由于 EZH2 可以调节 CTNNB1，因此 CTNNB1 也可以通过 EZH2 成为 miR-214 的间接靶标。沉默 EZH2 或 CTNNB1 的表达抑制了 HCC 细胞的生长和侵袭，并诱导了上皮钙黏蛋白（CDH1）的表达，已知其抑制细胞侵袭和转移。此外，miR-214 的沉默或 EZH2 的过表达通过激活 CTNNB1 增加了 EpCAM（+）干细胞样细胞。有趣的是，HCC 患者中 EZH2、CTNNB1 的上调和 CDH1 的下调与早期复发相关，并且是不良预后的独立预测因子。因此，miR-214 可以直接或间接靶向 CTNNB1 来调节 HCC 中的 β-连环蛋白信号通路。
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