MiR-199a regulates cell proliferation and survival by targeting FZD7 - PubMed (original) (raw)

. 2014 Oct 14;9(10):e110074.

doi: 10.1371/journal.pone.0110074. eCollection 2014.

Liucun Gao 2, Guang Yang 3, Shanhong Tang 4, Huahong Xie 5, Yongji Wang 6, Jingbo Wang 5, Yanping Zhang 7, Jiang Jin 5, Yawen Gou 5, Zhiping Yang 5, Zheng Chen 5, Kaichun Wu 5, Jie Liu 8, Daiming Fan 5

Affiliations

MiR-199a regulates cell proliferation and survival by targeting FZD7

Jiugang Song et al. PLoS One. 2014.

Abstract

A growing amount of evidence indicates that miRNAs are important regulators of multiple cellular processes and, when expressed aberrantly in different types of cancer such as hepatocellular carcinoma (HCC), play significant roles in tumorigenesis and progression. Aberrant expression of miR-199a-5p (also called miR-199a) was found to contribute to carcinogenesis in different types of cancer, including HCC. However, the precise molecular mechanism is not yet fully understood. The present study showed that miR-199a is frequently down-regulated in HCC tissues and cells. Importantly, lower expression of miR-199a was significantly correlated with the malignant potential and poor prognosis of HCC, and restoration of miR-199a in HCC cells led to inhibition of the cell proliferation and cell cycle in vitro and in vivo. Furthermore, Frizzled type 7 receptor (FZD7), the most important Wnt receptor involved in cancer development and progression, was identified as a functional target of miR-199a. In addition, these findings were further strengthened by results showing that expression of FZD7 was inversely correlated with miR-199a in both HCC tissues and cells and that over-expression of miR-199a could significantly down-regulate the expression of genes downstream of FZD7, including β-catenin, Jun, Cyclin D1 and Myc. In conclusion, these findings not only help us to better elucidate the molecular mechanisms of hepatocarcinogenesis from a fresh perspective but also provide a new theoretical basis to further investigate miR-199a as a potential biomarker and a promising approach for HCC treatment.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. Down-regulation of miR-199a in HCC tissues and cell lines was detected by qRT-PCR.

(A) The expression level of miR-199a was lower in 40 HCC tissues than in their pair-matched adjacent normal liver tissues (P<0.01). Each sample was analyzed in triplicate and normalized to the U6 snRNA. (B) The relative miR-199a expression in HCC cell lines was much lower compared to the normal Chang liver cell line. The relative expression of miR-199a was normalized to the endogenous control U6 snRNA. Each sample was analyzed in triplicate.

Figure 2

Figure 2. Lower miR-199a expression in HCC tissues is correlated with poorer survival of HCC patients.

Five-year cumulative survivals of the HCC patients were analyzed by Kaplan-Meier survival analysis and log-rank tests. The 40 HCC patients were split into high and low miR-199a expression groups based on the median (2.8) for miR-199a. The relative expression of miR-199a in each patient was shown in the supporting information (Table S1).

Figure 3

Figure 3. Over-expression of miR-199a represses growth and tumorigenicity in vitro and in vivo.

(A) qRT-PCR analysis confirmed that miR-199a expression was significantly up-regulated in HepG2-199a cells compared with matched controls. U6 snRNA was used as the internal control. (B) The growth curves are plotted based on the MTT assay results. The value shown is the mean of three experiments. *Statistical significance. (C) Cell cycle distribution of HepG2, HepG2-CN, and HepG2-199a cells by flow cytometry. (D) The proliferation index (PI) of HepG2, HepG2-CN, and HepG2-199a cells, as determined by flow cytometry. The PI values that are shown are the mean of three repetitions and are expressed as the mean ± SD. (E) Colony numbers of HepG2-CN and HepG2-199a cells in soft agar. Each soft agar assay was performed in triplicate, and the results are expressed as the mean number of colonies ± SD. (F) Tumor size of HepG2-CN and HepG2-199a cells in nude mice. (G) MiR-199a expression in whole tumor tissue extracts by qRT-PCR analysis. (H) FZD7 expression in whole tumor tissue extracts by Western blot analysis.

Figure 4

Figure 4. MiR-199a inhibits cell proliferation by directly targeting FZD7.

(A) Dual luciferase assays were performed in HepG2-199a and HepG2-NC cells transfected with the firefly luciferase reporter and the control vectors containing Renilla luciferase. The results showed that miR-199a could significantly suppress the luciferase activity of the reporter containing the 3′UTR of FZD7 but had no significant effect on the reporter containing the mutated binding site of FZD7. The data shown are the means ± SD of three independent experiments. (B) The sequences of the miR-199a binding sites within the 3′UTR of FZD7 and the mutated binding site are presented. (C) qRT-PCR analysis showing that the mRNA of FZD7 was significantly decreased in HepG2-199a cells compared with HepG2-NC cells. (D) Western blot analysis confirming that the expression of FZD7 and of its downstream genes was significantly inhibited by miR-199a. β-actin was used as the internal control. (E) The expression of the FZD7 protein in HepG2 and SMMC-7721 cells was higher compared with that in normal Chang liver cells.

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