MiR-137 and miR-34a directly target Snail and inhibit EMT, invasion and sphere-forming ability of ovarian cancer cells

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.

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.

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 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 × 10³ 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.

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).

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.

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.

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.

To investigate miRNA regulation of Snail, we first employed multiple algorithms, including TargetScan, miRSystem and DIANA-MicroT-CDS, to screen the specific miRNAs that can target the 3′-untranslated region (3′-UTR) region of the Snail mRNA. These analyses revealed six common miRNAs predicted to bind the 3′-UTR of the Snail transcript (Fig. 1a). For example, target prediction algorithms predicted one putative binding site for miR-137 in the 3′-UTR of Snail (nt 444-450) and recognized one binding site for miR-34a in Snail 3′-UTR (nt 86–93) [10].

Next, we sought to determine whether their expression is altered in human OC by measuring the expression level of these 6 miRNAs in 50 pairs of human OC samples and adjacent normal ovarian tissues. Interestingly, the levels of all 6 miRNAs were significantly decreased in OC tissues compared with adjacent normal ovarian tissues (data not shown). Among them, miR-137 and miR-34a were the most significantly down-regulated miRNAs (Fig. 1b and c), and selected to examine their effects on Snail expression. To examine whether the downregulation of miR-137 or miR-34a has any clinical significance in OC, we analyzed the association between miR-137 or miR-34a expression and patient prognosis. OC patients population were divided into two groups based on high (n = 25) or low (n = 25) expression of miR-137 or miR-34a according to the median expression levels among OC specimens, and Kaplan–Meier curves for overall survival was plotted. Patients with lower miR-137 or miR-34a expression had significantly shorter overall survival (Fig. 1d and e). Consistent with these results, both miR-137 and miR-34a were clearly reduced in two OC cell lines (SKOV-3 and ES-2) compared with normal ovarian epithelial NOEC cells (Fig. 2a).

To determine whether Snail is a direct target of miR-137 and miR-34a, we transfected a human OC ES-2 cell line (which expresses very high levels of endogenous Snail and lacks the expression of miR-137 and miR-34a) with pre-miRNA-137 or pre-miRNA-34a, together with a firefly luciferase reporter vector containing the Snail 3′-UTR. We found significant repression of luciferase activities by either miR-137 or miR-34a (Fig. 2b and c). In addition, these inhibitory effects of miR-137 or miR-34a on luciferase activities were eliminated following mutations targeting the predicted-binding sites of miR-137 or miR-34a within the Snail 3′-UTR (Fig. 2b and c), demonstrating that they bind directly to the 3′-UTR of Snail.

To directly assess if these miRNAs had a biological effect on Snail, mRNA and protein expression of Snail was examined in ES-2 and SKOV-3 cells following transfection of miRNA mimics or inhibitors, respectively. As expected, Snail mRNA and protein were down-regulated in ES-2 cells transfected with miR-137 or miR-34a mimic (Fig. 2d and f). In contrast, transfection with miR-137 or miR-34a inhibitor into SKOV-3 cells (which has relatively higher levels of miR-34a and miR-137) up-regulated Snail expression at mRNA and protein levels (Fig. 2e and f). Then we examined the expression of epithelial marker E-cadherin, mesenchymal marker N-cadherin and Vimentin in OC cells after the overexpression or knockdown of miR-137 and miR-34a, using qPCR analysis. ES-2 cells transfected with miR-137 or miR-34a mimic showed increased levels of E-cadherin and decreased expression of N-cadherin and Vimentin (Fig. 2g). However, transfection of SKOV-3 cells with miR-137 or miR-34a inhibitor reversed these effects (Fig. 2h).

Next, we examined the correlation between the expression of miR-137, miR-34a and Snail mRNA expression. We found that Snail mRNA level was significantly increased in OC samples compared with their non-tumor counterparts (Fig. 2i). Next, we chose to compare our data with existing published gene expression database on OC. Importantly, our analysis of the TCGA OC samples revealed significantly higher Snail transcript levels in OC samples, relative to normal ovaries (Fig. 2j), supporting a negative correlation between miR-137 or miR-34a and Snail expression in human OCs. Collectively, these results suggest that both miR-137 and miR-34a suppress Snail by targeting specific sites within the 3′-UTR of Snail.

We also examined the potential effects of miR-137 and miR-34a on EMT features, invasive and sphere-forming abilities of OC cells. For this, we transiently reduced or overexpressed miR-137 and miR-34a in SKOV-3 and ES-2 cells, which express relatively higher or lower expression of miR-137 and miR-34a, respectively (Fig. 2a). The down-regulation of miR-137 or miR-34a in SKOV-3 cells induced a spindle-like mesenchymal morphology (Fig. 3a), and the overexpression of miR-137 or miR-34a in ES-2 cells resulted in an epithelial morphology change (Fig. 3b). We further investigated the effects of knockdown and overexpression of these two miRNAs on EMT, OC cell invasion and sphere formation ability. Matrigel invasion assay and sphere formation assay demonstrated that knockdown of miR-137 or miR-34a using antisense oligonucleotides in SKOV-3 cells significantly promoted cell invasion and sphere formation, whereas overexpression of miR-137 or miR-34a with miRNA mimics in ES-2 cells inhibited these malignant features (Fig. 3c and d). Then we examined the expression of epithelial marker ZO-1 and cancer stemness markers (CD44 and CD133) in OC cells after the knockdown and overexpression of miR-137 and miR-34a, using qPCR assays. SKOV-3 cells transfected with miR-137 or miR-34a inhibitor showed decreased levels of ZO-1 and increased expression of CD44 and CD133 (Fig. 3e). However, transfection of ES-2 cells with miR-137 or miR-34a mimic increased the mRNA expression of ZO-1 and suppressed that of CD44 and CD133 (Fig. 3f). These data suggest that miR-137 and miR-34a inhibit mesenchymal characteristics and reduces self-renewal properties of OC cells.

To further corroborate the above observations, we investigated whether silencing of Snail with specific siRNA could repress the miR-137 or miR-34a inhibitor-induced OC cell invasion and sphere formation, or whether transient over-expression of a Snail open reading frame (ORF) could reverse the inhibitory effects of miR-137 or miR-34a mimic on OC cell invasion and sphere formation. The miR-137 or miR-34a inhibitor-induced SKOV-3 cell invasion and sphere formation were significantly reduced by Snail siRNA (Fig. 4a and b). The overexpression of Snail ORF in ES-2 cells partially rescued miR-137 or miR-34a mimic-suppressed invasion and sphere formation (Fig. 4a and b). Consistent with these data, inhibiting Snail expression using siRNA in SKOV-3 cells transfected with miR-137 or miR-34a inhibitor elevated the expression of E-cadherin but reduced the expression of N-cadherin and Vimentin (Fig. 4c). Moreover, rescuing Snail expression with Snail ORF in the presence of miR-137 or miR-34a mimic resulted in down-regulation of E-cadherin and up-regulation of N-cadherin and Vimentin (Fig. 4c). These data suggest that miR-137 and miR-34a inhibits invasive and stem cell-like properties of OC cells by suppressing Snail expression via its 3′-UTR.

Next, we investigated if overexpressing miR-137 and miR-34a could affect the viability and apoptosis of OC cells and normal NOEC cells using cell viability and cell apoptosis assays. The data demonstrated that the restoration of miR-137 and miR-34a expression in ES-2 and SKOV-3 cells led to a significant reduction in cell viability and the induction of cell apoptosis, but had no significant toxicity to NOEC cells (Fig. 5), indicating the feasibility of inhibiting the growth of OC cells via the therapeutic delivery of miR-137 or miR-34a mimics, without causing significant toxicity to normal ovarian tissues.