miR-211 suppresses epithelial ovarian cancer proliferation and cell-cycle progression by targeting Cyclin D1 and CDK6

Epithelial ovarian cancer (EOC) is a significant cause of morbidity and mortality. MicroRNAs play important roles in cancer development and progression. The microRNA miR-211 is localized on intron 6 of the Trpm1 gene at 15q13-q14, a locus that is frequently lost in neoplasms. Its function and loss-of-function have been described in normal and cancer cells and tissues. miR-211 is known to be dysregulated in ovarian cancer: however, its function and the downstream effect of its loss-of-function in ovarian cancer have not been described before. We analyzed miR-211 expression in clinical samples of primary EOC tissues compared to normal epithelial ovarian tissues and in the EOC cell lines: OVCAR3, Caov3, OVCA429, SKOV3 and A2780 compared to human ovarian surface epithelial cells. We then investigated the effect of miR-211 on EOC cell proliferation and apoptosis by counting cell numbers, MTT, colony formation, cell cycle, and PI/Annexin V staining assays. A luciferase reporter system was developed to assess miR-211 regulation of the predicted targets. Expression level of discovered targets and correlation with miR-211 expression were analyzed in EOC tissues. Finally, OVCAR3 stably expressing miR-211 or control cells were injected subcutaneously into mice to determine in vivo effect of miR-211 on tumorigenesis. We found that the expression of miR-211 is downregulated in EOC tissues and cell lines compared to normal epithelial ovarian tissue and human ovarian surface epithelial cells, respectively. miR-211 was found to arrest cells in the G0/G1-phase, inhibit proliferation and induce apoptosis. Cyclin D1 and CDK6 were found to be direct targets of miR-211, and when overexpressed in miR-211-expressing EOC cells, could restore proliferative ability. Finally, in vitro investigation confirmed that miR-211 is a tumor suppressor that controls Cyclin D1 and CDK6 expression. Our results demonstrate that miR-211 is a tumor suppressor that controls expression of Cyclin D1 and CDK6, and that its downregulation results in overexpression of Cyclin D1 and CDK6 which increases proliferation ability of EOC cells to proliferate compared to normal cells.


Background
Ovarian cancer (OC) has a high mortality rate and low 5-year survival rate due to lack of early, safe and noninvasive detection methods. This malignancy also develops chemoresistance during recurrence after initial chemotherapy [1][2][3][4]. Therefore, new therapies, clinical biomarkers and treatment targets are in demand.
MicroRNAs (miRNAs) regulate gene expression at the post-transcriptional level [5][6][7] and miRNA dysregulation is frequently associated with cancer progression, including OC [8][9][10][11][12]. The microRNA miR-211 is localized on intron 6 of the Trpm1 gene at 15q13-q14, a locus that is frequently lost in neoplasms [13][14][15][16]. MiR-211 functions and the effect of loss-of-function have been described in normal and cancer cells and tissues. Using mouse embryonic fibroblasts, Chitnis et al. [17] found that miR-211 is a pro-survival molecule that is expressed in a PERK (aka EIF2AK3, Eukaryotic translation initiation factor 2-alpha kinase) -dependent manner and regulates the expression of chop/gadd153 by mediating temporal accumulation of the pro-apoptotic transcription factor chop. PERK is important to survival of tumor and normal cells in response to stress [18][19][20][21][22] and Chitnis et al. [17] suggested that miR-211 negatively regulates chop accumulation, allowing cells to re-establish homeostasis before having to commit to apoptosis.
In clinical melanoma samples, Mazar et al. [8] found that miR-211 targets KCNMA1, is downregulated in melanoma and that its expression is microphthalmaassociated transcription factor dependent. This transcription factor is important for melanocyte growth, maturation, apoptosis and pigmentation [23]. Bell et al. found that miR-211 contributes to melanoma adhesion by targeting the AMP-activated protein kinase-related kinase NUAKI and that inhibition of miR-211 resulted in increased NUAK1 expression and reduced adhesion [24]. In glioblastoma multiform, miR-211 was found to be downregulated with an inverse correlation of miR-211 expression and matrix metalloproteinase-9 expression [25]. The authors suggested that rescuing miR-211 expression could have therapeutic applications. Conversely, others reported that in oral carcinoma, miR-211 is upregulated, contributes to progression of oral carcinoma and correlates with poor prognosis in oral carcinoma [26].
The present study investigated the regulatory role and implications of aberrant expression of miR-211 in epithelial OC (EOC). We report that miR-211 is downregulated in EOC, inhibits proliferation and induces apoptosis in EOC cells in vitro and that overexpression of miR-211 inhibits growth of EOC xenograft tumors in vivo by repressing Cyclin D1 and CDK6 expression.

miR-211 is downregulated in EOC tissues and cell lines
Searching the literature, we found that miR-211 is downregulated in OC tissues [9]. We further used a public data base to investigate miR-211 expression in EOC tissues and found that the of miR-211 expression was significantly lower in clear-cell OC (CCOC, n = 9) and high-grade serous ovarian carcinomas (HGSC, n = 12) than in ovarian surface epithelial cells (OSES, n = 9) ( Figure 1A, GSE47841, p < 0.001) [27]. Next, we explored this finding in clinical samples by comparing miR-211 expression in normal epithelial ovarian tissues with primary EOC tissues. Consistent with the mentioned literature, miR-211 expression was significantly lower in EOC tissues than in normal epithelial ovarian tissues ( Figure 1B, p < 0.01). We extended our investigations to six EOC cell lines (OVCAR3, Caov3, OVCA429, SKOV3, A2780 and COV644) and found that their miR-211 expression levels were significantly lower than those of human ovarian surface epithelial (HOSE) cells ( Figure 1C). These findings suggest that downregulation of miR-221 may affect EOC development.

miR-211 inhibits proliferation and induces apoptosis of EOC cells
To investigate the function of miR-211 in EOC tumorigenesis, we transfected miR-211 or miR-Ctrl in OVCAR3 and SKOV3 cell lines and determined their miR-211 levels 48 hours after transfection. Results showed increased miR-211 levels in both miR-211 transfected cell lines compared to miR-Ctrl transfected cells ( Figure 2A). We then investigated the effect of miR-211 on OVCAR3 and SKOV3 cell proliferation. As shown in Figure 2B, miR-211 significantly inhibited EOC proliferation. MTT assay further confirmed that miR-211 had a negative effect on EOC cell proliferation ( Figure 2C). To examine the effect of miR-211 on long-term EOC cell proliferation, we performed colony formation assays. We first constructed a miR-211 lentiviral vector (LV-miR-211), then infected OVCAR3 and SKOV3 cells with LV-miR-211  to establish stably expressing miR-211 cells ( Figure 2D). LV-miR-211 and LV-miR-Ctrl cells were subjected to colony formation assay for two weeks. As expected, miR-211 significantly reduced colony numbers to 20% in both EOC cell lines ( Figure 2E).
Next, we performed cell cycle analysis and noted that miR-211 transfection significantly arrested significantly more cells in the G0/G1 phase ( Figure 3A) than miR-211-Ctrl transfection. To investigate whether miR-211 affects EOC cell apoptosis, OVCAR3 and SKOV3 cells were transfected with miR-211 and apoptosis assessed 48 hours later. As shown in Figure 3B, miR-211 transfected cells had a higher incidence of apoptosis than miR-211-Ctrl transfected cells. These results suggest that miR-211 inhibits EOC cell proliferation and induces apoptosis.
Cyclin D1 and CDK6 are direct target of miR-211 We used microRNA.org, Targetscan and miRWalk databases to predict potential miR-211 targets. Hundreds of potential targets were found, but we selected transcription factor 4 (TCF4), Cyclin D1, Cyclin D2, Cyclin D3 and Cyclin dependent kinase 6 (CDK6) for further analysis since these genes have previously been reported to affect EOC cell proliferation [28][29][30][31] (Figure 4A). We inserted TCF4, CDK6, Cyclin D1, Cyclin D2 and Cyclin D3 3′UTR into luciferase reporter vectors and cotransfected with miR-211 expression plasmid into OVCAR3 and SKOV3 cells ( Figure 4A). Forty-eight hours after transfection, only cells transfected with CDK6 and Cyclin D1 3′UTR plasmids had lower luciferase activity than their controls. Luciferase activity in both of these cell lines was 70% less than that of the miR-Ctrl groups ( Figure 4B). Next, we compared the 3′ UTR sequence of Cyclin D1 and CDK6 with miR-211. Both genes have two potential complementary miR-211 binding sites ( Figure 4C, D). We next constructed Cyclin D1 and CDK6 3′UTR mutant plasmid (Additional file 1: Figure S1A-B), cotransfected with miR-211 and then performed luciferase assay. The data revealed that miR-211 targets two sites in Cyclin D1 and CDK6 (Additional file 1: Figure S1C-D). To confirm that Cyclin D1 and CDK6 are specifically targeted by miR-211, OVCAR3 and SKOV3 cells transfected with miR-211 or infected with LV-miR-211 were subjected to western blot analysis. We found that the protein levels of Cyclin D1 and CDK6 were lower in miR-211 and LV-miR-211 transfected cells compared to control ( Figure 4E, F). The  mRNA level of Cyclin D1 and CDK6 and the levels of miR-211 were measured in 60 EOC tissues using qRT-PCR to analyze their correlation. Spearman correlation analysis revealed a reverse correlation between miR-211 expression and Cyclin D1 or miR-211 and CDK6 expression ( Figure 4G, H). The high expression of Cyclin D1 and CDK6 in OEC tissues compared to normal tissue was consistent with results from the public database ( Figure 4I, J) [32]. Taken together, these results indicate that miR-211 can repress the expression of Cyclin D1 and CDK6 in EOC by directly targeting the 3′UTR of Cyclin D1 and CDK6 mRNA.

miR-211 affects ovarian cell proliferation and apoptosis through suppression of Cyclin D1 and CDK6
Our previous data suggests that miR-211 inhibits EOC cell proliferation and that Cyclin D1 and CDK6 are direct targets of miR-211. To demonstrate that miR-211 regulates cell proliferation through Cyclin D1 and CDK6, we tested whether Cyclin D1 and CDK6 could rescue the impaired proliferative phenotype in miR-211 overexpressing OVCAR3 and SKOV3 cells. Western blot was performed to examine Cyclin D1 and CDK6 levels in cells transfected with LV-miR-Ctrl, LV-miR-211, LV-miR-211 + Cyclin D1 and LV-miR-211 + CDK6 ( Figure 5A). Cell counting and MTT assay were performed to analyze cell proliferation. The results indicate that miR-211 inhibited proliferation, while Cyclin D1 or CDK6 partly, and Cyclin D1 + CDK6 almost completely restored cell proliferation ( Figure 5B-F). Furthermore, we performed cell cycle assays and found that Cyclin D1 or CDK6 partly rescued the cell proliferation that had been inhibited by miR-211 ( Figure 6A). MiR-211 overexpression led to more cell apoptosis, while Cyclin D1 and CDK6 each significantly reduced apoptosis ( Figure 6B). When we overexpressed Cyclin D1 and CDK6 in miR-211-overexpressing cells, miR-211-induced cell cycle arrest and apoptosis were completely abrogated ( Figure 6C and D). These results together demonstrate that miR-211 affects EOC cell proliferation, at least in part, through suppression of Cyclin D1 and CDK6.

miR-211 reduces EOC tumorigenesis in vivo
We performed in vivo experiments to confirm our in vitro results that suggested that miR-211 inhibited EOC cell proliferation by targeting Cyclin D1 and CDK6. Sixteen mice were randomly divided into two groups. OVCAR3 cells stably expressing miR-211 or control cells were injected subcutaneously into mice in each group. We found that tumor growth was slower in the LV-miR-211 group compared to the LV-miR-Ctrl group ( Figure 7A). The tumor weights and sizes were smaller in LV-miR-211 group compared to LV-miR-Ctrl group ( Figure 7B, C). Finally, these tumor tissues were assessed with immunohistochemistry. We observed that Cyclin D1 and CDK6 staining in LV-miR-211 group was weaker than in the control group ( Figure 7D). These in vivo results further indicated that miR-211 inhibits EOC growth and reduces Cyclin D1 and CDK6 expression.

Discussion
MiRNAs are undoubtedly pivotal to tumorigenesis and understanding their functions may help provide new cancer therapies [33][34][35][36]. In the present study, we performed a database search for miR-211 expression in human ovarian cancer tissues compared to healthy control tissue, and found that miR-211 was significantly downregulated in clear-cell and high-grade serous carcinomas. This was further confirmed in clinical primary EOC samples and in EOC cell lines.
We further investigated the significance of miR-211 expression in EOC in vitro and found that miR-211 significantly modulated EOC cell proliferation and colony formation. Cell cycle analysis showed that miR-211 arrested cells in the G0/G1 phase, resulting in apoptosis. Using bioinformatics, we identified several miR-211 targets and confirmed with luciferase assay that miR-211 directly binds to sequences in Cyclin D1 and CDK6 mRNA, repressing their translation into protein. Further in vitro investigations showed that miR-211 affected EOC cell proliferation and apoptosis through suppressing the expression of Cyclin D1 and CDK6.
We confirmed our in vitro observations in vivo with a mouse tumor model. As expected, we found that Cyclin D1 and CDK6 were downregulated in vivo by miR-211 and that EOC tumor growth was reduced significantly by miR-211 overexpression.
We did not investigate the effect of dysregulated CDK6 and Cyclin D1 on downstream gene expression; however, both have been ascribed several functions. Cyclin D1 controls CDK6 activity and is known to affect angiogenesis, respond to growth factor stimulation and stimulates G1 progression. Overexpression of Cyclin D1 (and other Cyclins) was found to shorten the G1-phase of the cell cycle in various cell types [43][44][45] and inhibiting Cyclin D1 in human fibroblasts was found to inhibit progression through G1 [45,46], which is consistent with our observations in EOC. Also, D-type Cyclin overexpression can surmount the G1 growth arrest caused by retinoblastoma tumor suppressor protein (Rb) in Saos-2 osteosarcoma cells [47,48]. Although Cyclin D1 interacts with CDK6 to exert many of its functions, it also performs CDK6-independent functions such as: transcriptional regulation leading to cell growth, tissuespecific differentiation and cell cycle progression, as well as chromatin modifications and interaction with nuclear hormone receptors which both lead to differentiation and androgen-receptor-dependent cell cycle progression (reviewed by [37]).
CDK6 is a kinase catalytic subunit of a protein kinase complex that is involved in G1 progression and G1/S transition. CDK6 activity first occurs in mid G1-phase, is controlled by D-type Cyclins (i.e. Cyclin D1) and INK4 family members, and regulates Rb activity by phosphorylation [49]. Phosphorylation of Rb leads to the release of E2fs, which then activate transcription of genes required for S-phase entry [50]. Very recently, Handschick et al. [51] reported that CDK6 is a co-factor of NF-κB that interacts physically with the NF-κB subunit p65 and is found at promoters of NF-κB target genes. Thus, dysregulated CDK6 and Cyclin D1 expression is significant as it is likely to affect expression of S-phase entry proteins, and the cytokine and chemokine expression profile of EOC, contributing to oncogenesis and tumorigenesis.
CDK6 overexpression increases cell proliferation and reduces DNA repair activity by accelerating G1/S -phase progression. In glioma, CDK6 knockdown was found to increase sensitivity to chemotherapy [52]. On this basis, it is possible that miR-211-mediated inhibition of CDK6 expression in EOC could be a useful epigenetic therapeutic approach, although further experiments would be required to determine this.
In summary, we found that miR-211 negatively regulates CDK6 and Cyclin D1 activity and that miR-211 is downregulated in EOC, leading to aberrant expression of CDK6 and Cyclin D1, which results in loss of cell cycle control. Cyclin D1 and CDK6 appear to be key players in EOC tumorigenesis, and our discovery of correlated expression of miR-211 and CDK6/Cyclin D1 provides new insight that presents tentative methods for diagnosis, prognosis and therapy for EOC, and a rational for further investigation into the potential use of miR-211 for diagnosis and therapy.

Human samples
This study was approved by the Medical Ethics Committee of Harbin Medical University Cancer Hospital and all patients provided informed consent. Tissues were collected from patients who underwent surgery at the Department of Obstetrics and Gynecology of Harbin Medical University Cancer Hospital between 2012 and 2013, including 60 epithelial EOC tissues and 20 normal epithelial ovarian tissue sections. Patients with previous radiation therapy, chemotherapy, or immunotherapy, were excluded from the study. The histopathological diagnostics was performed according to the World Health Organization criteria. All fresh specimens were stored at −80°C for further use. Patients' characteristics including presenting age, clinical stage, pathological stage and tumor size are available in Table 1.

Cancer cell lines and primary normal epithelial cells
The human EOC cell lines (OVCAR3, Caov3, OVCA429, SKOV3 and A2780) and normal Human Ovarian Surface Epithelial (HOSE) cells were acquired from the China Center for Type Culture Collection (CCTCC). The COV644 cell line was purchased from Sigma (St. Louis, MO). EOC cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco-BRL, Gaithersburg, MD) supplemented with 10% fetal bovine serum and antibiotics (Gibco). HOSE cells were cultured in medium containing 1:1 mixture of MCDB 105 and M199 medium (Sigma). All cells were incubated at 37°C in a humidified atmosphere containing 5% CO 2 .

Colony formation assay
Forty-eight hours after infection with LV-miR-Ctrl or LV-miR-211, the EOC cells were seeded in 6-well plates (500 cells per well) and incubated for 2 weeks for the colony formation assay. The cells were then washed with PBS, fixed with 10% formalin, and stained with 0.5% crystal violet (Sigma). The assay was repeated in triplicates.

Cell cycle assay
Forty-eight hours after transfection with miRNA mimics, EOC cells were seeded in 6-well plates. Two days later, the cells were collected and fixed in 70% ethanol, washed in PBS, re-suspended in 200 μl of PBS containing 0.5 mg/ml RNase, 0.05% Triton X-100 and 10 μg/ml propidium iodide (Sigma), incubated for 1 hour at 37°C in the dark, and analyzed immediately using a Flow Cytometer (BD Biosciences, San Jose, CA). The experiment was done in triplicates.

Luciferase reporter assay
The cells were seeded in triplicate in 24-well plates one day before transfection for the luciferase assays. Plasmids inserted into the Renilla lucifearse vector (Promega) with Cyclin D1 or CDK6 3′UTR inserts were co-transfected with miR-Ctrl or miR-211 plasmids. Forty eight hours after transfection, the cells were harvested and lysed, and the luciferase activity assayed using the dualluciferase assay kit (Promega). Normalized luciferase activity was reported as luciferase activity/Renilla luciferase activity. Three independent experiments were performed.

Animal studies
Animal studies were approved by the Institutional Animal Care and Use Committee of Harbin Medical University. Nude mice (5 weeks old) were randomly divided into two groups (n = 8 per group). A suspension of OVCAR3 cells (1 × 10 7 ) stably expressing miR-211 or cells infected with miR-Ctrl were injected subcutaneously into the left flank of each group. Tumor volumes were measured every 5 days using a caliper. Thirty days after implantation, the mice were sacrificed and the subcutaneous tumors excised and weighed.

Immunohistochemistry
Tumor samples were fixed in 4% formaldehyde, embedded in paraffin wax, and then cut into 5 μm sections. Samples were deparaffinized in clearite and rehydrated. After blocking endogenous peroxidase and performing antigen retrieval, tissue slides were blocked in goat serum for 30 min and incubated with antibodies against Cyclin D1 or CDK6 (1:100 dilution) overnight at 4°C, followed by biotinylated secondary antibody (Santa Cruz) for 30 min. Staining was performed in parallel using a Vectastain ABC kit (Vector Laboratories).

Statistical analysis
The statistical analyses were performed using SPSS Windows version 19. Data is expressed as mean ± SEM of triplicate experiments. One-Way ANOVA was performed to determine significant differences between groups. Differences were considered significant when p < 0.05 (*) and highly significant when p < 0.01 (**).