Background
The majority (75 %) of patients with ovarian cancer (OC) present with advanced disease and widely metastatic disease [
1,
2]. In OC, the acquisition of invasiveness is accompanied by a shift from epithelial to mesenchymal phenotype, also called the epithelial-to-mesenchymal transition (EMT), which endows cancer cells with increased motility and invasiveness to seed metastasis and with stem cell-like properties, such as upregulation of stem cell genes (CD44 and CD133) and self-renewal ability [
3,
4]. The EMT process can be initiated by a group of transcription factors including SNAIL (Snail), which repress the expression of epithelial markers (E-cadherin and ZO-1), and induce the levels of mesenchymal markers (Vimentin and N-cadherin) [
5]. Therefore, identification of key actors regulating Snail expression and EMT in OC cells would have tremendous clinical utility.
Growing evidence suggests that a number of epigenetic mechanisms control the expression of genes that facilitate EMT and induce metastasis [
6]. For example, microRNAs (miRNAs) have important roles in the regulation of cancer cell invasion and motility, by suppressing or promoting EMT [
7]. In addition, several miRNAs [
8‐
11] have been implicated in the regulation of Snail expression in human cancers other than OC. To date, however, little is known about controlling Snail expression in OC cells by using specific miRNAs.
In this study, we provide evidence that miR-137 and miR-34a directly bind to and down-regulate Snail levels to suppress EMT, invasion and sphere-forming ability of OC cells, and that the repression of these two miRNAs is significantly correlated with worse patient survival in OC.
Methods
Reagents and cell culture
Human OC cell lines (SKOV-3 and ES-2) were obtained from the American Type Culture Collection (Manassas, VA), and were cultured in DMEM/F12 medium (Invitrogen) supplemented with 10 % fetal bovine serum (FBS, Invitrogen). Normal ovarian epithelial cells (NOEC, Pricells, Wuhan, China) were cultured in Ham’s F-12 (Gibco) supplemented with 20 % FBS (Gibco). MiRNA mimic and miRNA inhibitor for miR-137 or miR-34a (30 nM, Ambion), Snail siRNA (5 nM, Ambion) and Snail cDNA plasmids (OriGene) were transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol.
Real-time quantitative RT-PCR (qPCR)
Total RNA was extracted using TRIzol reagents (Invitrogen) according to the manufacturer’s instructions. For mRNA and miRNA analysis, cDNAs were synthesized using the PrimeScript RT reagent kit (Takara). Then, qPCRs were performed by using the Takara SYBR Premix Ex Taq II (Takara) and the 7500 Real-Time PCR System (Applied Biosystems). The primer sequences for
Snail [
12],
E-cadherin [
12],
ZO-1 [
12],
N-cadherin [
12],
Vimentin [
12],
CD44 [
13],
CD133 [
12] and
GAPDH [
12] have been previously reported. For miRNA analysis, qPCRs were performed using the NCode miRNA qRT-PCR analysis (Invitrogen, CA, USA). Forward primer is the exact sequence of the mature miR-137 and miR-34a. The mRNA and miRNA expression data were normalized to
GAPDH and U6, respectively. Results were represented as the fold change relative to respective controls.
Cell invasion assay
OC cells were grown to 50–70 % confluence and transfected as indicated. After 24 h, cells were seeded into upper chamber of Boyden chambers coated with Matrigel as described previously [
14,
15]. After incubation for 24 h, the non-invading cells were gently removed with a cotton swab. Invasive cells located on the lower surface of chamber were stained with Giemsa and counted under a microscope. Relative cell invasion activities were expressed as the fold change over respective controls.
Single cells (1000 cells per well) were plated onto a 24-well ultra-low attachment plate (Corning) in serum-free DMEM/F12 medium supplemented with N2 plus media supplement (Invitrogen), 20 ng/ml epidermal growth factor (Invitrogen), 20 ng/ml basic fibroblast growth factor (Invitrogen) and 4 mg/ml heparin (Sigma-Aldrich). After 10 days of culture, the number of spheres larger than 50 μm was counted under an inverted microscope.
Cell viability assay and cell apoptosis assay
Cell counting kit-8 assay (Dojindo) and Caspase-Glo 3/7 assay (Promega) were used to assess cell viability and cell apoptosis as previously reported [
16]. For the cell viability assay, OC cells and NOEC cells were seeded were seeded at a density of 5 × 10
3 per well in 96-well plates for 24 h, and then transfected with 30 nM of miR-137 or miR-34a mimic or negative control mimic (Neg mimic). After 72 h, 10 μl of Cell counting kit-8 solution was added into each well and the plates were incubated for additional 4 h at 37 °C. The UV absorbance of each sample was measured in a microplate reader at 450 nm. For the apoptotic assay, caspase-3/7 activity was analyzed in accordance with the manufacturer’s protocol. Briefly, OC and NOEC cells were seeded into 96-well plates and transfected as described above. After 72 h, an equal volume of Caspase-Glo 3/7 reagent was added into each well, and the cells were incubated at room temperature in the dark. Luminescence was measured after 3 h of incubation with the caspase substrate.
Western blot analysis
Cells were harvested 24 h after transfections. Equal amounts of protein lysates (30 μg) were separated by 10 % SDS-PAGE for immunoblots with antibodies to Snail (Abcam), E-cadherin (GenScript), N-cadherin (BD Biosciences), Vimentin (GenScript) and GAPDH (Santa Cruz). Primary antibodies were used at a dilution of 1:1000. A horseradish peroxidase-conjugated anti-rabbit or anti-mouse immunoglobulin-G antibody was used as the secondary antibody (1:5000; Santa Cruz). Signals were detected using enhanced chemiluminescence reagents (Amersham Biosciences).
Dual luciferase reporter assay
The
Snail 3′-UTR luciferase vector was purchased from OriGene. Mutations in the miR-137 or miR-34a-binding sequence were generated by using the QuickChange Mutagenesis Kit (Stratagene). For luciferase assay, OC cells were seeded onto 24-well plates and transfected after 24 h with 100 ng of firefly luciferase reporter plasmid, 10 ng of Renilla report plasmid as normalization control, together with miR-137 or miR-34a mimic or Neg mimic. After 24 h, a Dual Luciferase Reporter Assay (Promega) was performed as previously reported [
17]. The firefly luciferase activity was normalized to the Renilla luciferase activity.
Clinical samples
Matched serous OC and corresponding adjacent normal ovarian tissues were obtained from 50 patients undergoing resection at the Department of Gynecology, State Key Laboratory of Oncology in South China, Sun Yat-sen University Cancer Center (Guangzhou, China). Tumor and non-cancerous tissues were confirmed histologically by Hematoxylin and Eosin staining. All samples were collected from consenting individuals according to the protocols approved by the Ethics Review Board at Sun Yat-sen University Cancer Center. All tissue samples were immediately snap-frozen in liquid nitrogen. They were kept in a -80 °C freezer and total RNA was isolated using TRIzol reagents.
Statistical analysis
Results are expressed as mean ± s.e.m. from at least three independent experiments performed in triplicate. 2-tailed Student’s t-test was used for statistical analysis. The log-rank test was used for survival analysis. The value of P < 0.05 were considered as significant.
Discussion
Despite advances in our understanding of the mechanisms underlying Snail up-regulation, little is known about the endogenous miRNA suppressors of Snail in OC cells. We report in this study that in OC cells, both miR-137 and miR-34a act as novel tumor repressors that directly target Snail, which plays a pivotal role in controlling the various cellular functions during cancer metastasis, such as EMT, cell invasion, sphere-forming ability and chemoresistance [
18‐
23].
We show that miR-137 and miR-34a are downregulated in OC samples, and we found a significant association between decreased miR-137 or miR-34a expression and worse patient prognosis. Furthermore, our in vitro data have confirmed that reduced expression of miR-137 and miR-34a is critical for enhanced OC cell invasiveness and self-renewal, suggesting that these two miRNAs can be potential therapeutic targets. Therefore, attenuating the oncogenic functions of Snail by the use of miR-137 and miR-34a could provide an exciting opportunity for the development of therapy against OC.
Since a single mRNA might be targeted by multiple miRNAs, we sought to newly identify crucial miRNAs that reduce the expression of Snail in OC cells. We demonstrate for the first time that miR-137 can directly repress Snail expression through its binding to the specific binding site in the 3′-UTR of the human
Snail gene, thereby negatively regulating EMT, invasion and self-renewal of OC cells. It is interesting to note that a tumor-suppressive role for miR-137 has also been shown in a variety of human tumors [
24‐
26]. Thus, these data and our current results reveal that, the anti-invasive effects of miR-137 described here for OC—possibly mediated by Snail suppression—might be relevant in other tumor types. Therefore, we postulate that replacing tumor suppressive miR-137 targeting Snail might be a promising approach for treating metastatic and recurrent OC.
As an important tumor suppressor, miR-34a controls the expression of a host of target proteins involved in cell proliferation, apoptosis, cancer stemness, metastasis and chemoresistance [
27], and it is often down-regulated in numerous tumor types [
28‐
32]. A previous study has shown that miR-34a acts as a suppressor of Snail in colon cancer [
10], but to our knowledge, there are also opposite findings showing its tumor-promoting roles in other cancer type [
33]. Whether miR-34a affects Snail expression in OC cells have remained elusive. Here, we have demonstrated that miR-34a is a direct inhibitor of Snail in OC cells. Thus, the down-regulation of miR-34a may be essential for Snail to induce EMT and OC metastasis.
Our observation that upregulating tumor suppressive miR-137 and miR-34a via miRNA mimics restored tumor suppressor activity, with successful inhibition of OC cell viability and invasiveness, provided a rationale to investigate “miRNA replacement therapy for miR-137 and miR-34a”. However, there are concerns regarding potential toxicity and off-target effects in normal tissues. We showed here that the viability and apoptosis of normal NOEC cells was not altered after transfection with miR-137 and miR-34a mimics, which display a high selectivity for killing OC cells. These findings may be explained by several mechanisms [
34]. Especially, large differences in miR-137 and miR-34a levels between normal NOEC cells and OC cells might account for the tolerance of NOEC cells to these two miRNAs. The in vivo anti-tumor efficacy and toxicity of miR-137 and miR-34a warrants further investigation.
Conclusions
We identified a new mechanism whereby decreased expression of both miR-137 and miR-34a contributes to enhanced Snail levels, which in turn promotes EMT, invasion and sphere-forming of OC cells. Our results suggest that these two miRNAs might become candidate targets for the treatment of Snail-overexpressing OC.
Acknowledgements
We thank Dr. Zhujie Xu for technical assistance.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (
http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (
http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.