MiR-214 Targets β-Catenin Pathway to Suppress Invasion, Stem-Like Traits and Recurrence of Human Hepatocellular Carcinoma
Stem-Like Traits and Recurrence of Human Hepatocellular
Carcinoma. PLoS ONE 7(9): e44206. doi:10.1371/journal.pone.0044206
MiR-214 Targets b-Catenin Pathway to Suppress Invasion, Stem-Like Traits and Recurrence of Human Hepatocellular Carcinoma
Hongping Xia
London Lucien P. J. Ooi
Kam M. Hui
Cara Gottardi, Northwestern University Feinberg School of Medicine, United States of America
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 b-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 bcatenin signaling pathway in HCC.
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Funding: This work was supported by by grants from the National Medical Research Council, Biomedical Research Council of Singapore and The Singapore
Millennium Foundation, http://www.nmrc.gov.sg/content/nmrc_internet/home.html. The funders had no role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Hepatocellular carcinoma (HCC) is the most common type of
primary liver cancer and the third leading cause of death from
cancer. A variety of etiological and risk factors including hepatitis
virus (HBV or HCV) infection, alcohol abuse and aflatoxin
ingestion have been associated with hepatocarcinogenesis
[1,2,3,4]. 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]. Although numerous studies have been devoted to delineate the
molecular pathogenesis of HCC, the incidence and mortality of
HCC has not been reduced over the past few decades. Surgery
currently offers the only possibility of prolonged survival for HCC
patients. Unfortunately, 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. Various
surrogate clinicopathologic features such as lymphovascular
invasion, capsular invasion, satellite lesions, and tumour numbers
are often used but with varying reliability reported [4].
Additionally, 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].
MicroRNAs (miRNAs) are a class of highly conserved, small
non-coding RNAs that play essential roles in the
post-transcriptional regulation of gene expression through base pairing with the
39-untranslated region (39-UTR) of target mRNAs. Because
miRNAs have been discovered to target a large proportion of
mammalian genes, many studies have indicated that miRNAs play
critical roles in the regulation of many biological functions and
consequently, miRNAs play crucial roles in the development of
many human diseases, including cancer [6,7]. The dsyregulation
of miRNAs in HCC have been reported using miRNA expression
profiling studies with several mi (...truncated)