Abstract
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.
Highlights
Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer and the third leading cause of death from cancer
We firstly validated the suppression of miR-214 in human HCC by quantitative RT-PCR (qRT-PCR) in paired tumor and non-tumor liver tissues from 20 HCC patients, as well as in 10 samples of histologically normal liver tissues from colorectal cancer patients with liver metastases (Fig. 1A and Figure S1)
Further analysis of HCC tissues, using qRT-PCR, of an independent cohort of 50 HCC patients of whom 29 with early recurrent disease (#2 years) and 21 with late recurrent disease demonstrated that miR-214 was significantly suppressed in samples of HCC patients with early recurrent disease, P,0.01, (Fig. 1B)
Summary
Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer and the third leading cause of death from cancer. The development of HCC is a multi-step process from chronic hepatitis, to cirrhosis, to dysplastic nodules, and to malignant tumors with various genetic and epigenetic alterations [3]. Surgery currently offers the only possibility of prolonged survival for HCC patients. Recurrence occurs in more than two-thirds of these patients despite initial curative intent and converts the situation to a dismal prognosis [1,5]. It is presently a challenge to identify patients who are at high risk of early recurrence after undergoing potentially curative treatment for HCC. Most HCC are diagnosed at the advanced stages when there is no effective treatment, so there is an urgent need to develop novel therapeutic strategies for the treatment of HCC [5]
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