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Keywords:

  • cyclin D1;
  • endometrioid endometrial cancer;
  • microRNA;
  • miR-503;
  • target gene

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

MicroRNAs (miRNAs) are post-transcriptional inhibitor regulators of gene expression that act by directly binding complementary mRNA and are key determinants of cancer initiation and progression. In this study, we revealed a role for the tumor-suppressor miRNA miR-503 in endometrioid endometrial cancer (EEC) cells. The miR-503 expression level gradually decreases across normal endometrial tissues, endometrial tissues with complex atypical hyperplasia, and EEC tissues. A relatively high level of miR-503 in EEC tissues indicates a longer survival time in EEC patients. The expression of a cell cycle-associated oncogene encoding cyclin D1 (CCND1) was inversely correlated with miR-503 expression in EEC tissues and cell lines. CCND1 has a binding sequence of miR-503 within its 3′ untranslated region, and was confirmed to be a direct target of miR-503 by the fluorescent reporter assays. Increasing the miR-503 level in EEC cells suppressed cell viability, colon formation activity and cell-cycle progression, and the inhibited oncogenic phenotypes induced by miR-503 were alleviated by ectopic expression of CCND1 without the untranslated region sequence. Furthermore, in vivo studies also suggested a suppressive effect of miR-503 on EEC cell-derived xenografts. miR-503 increased in cell cycle-arrested EEC cells, and was restored to a normal level in EEC cells after cell cycle re-entry, while CCND1 displayed the opposite expression pattern. Collectively, this study suggested that miR-503 plays a tumor-suppressor role by targeting CCND1. Abnormal suppression of miR-503 leads to an increase in the CCND1 level, which may promote carcinogenesis and progression of EEC.


Abbreviations
CAH

complex atypical hyperplasia

CCND1

cyclin D1

EEC

endometrioid endometrial cancer

miR-503

microRNA-503

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Endometrial carcinoma is the fourth most common cancer among women worldwide [1]. Each year, endometrial cancer develops in about 142 000 women worldwide, and an estimated 42 000 women die from this cancer, with the relative 5-year survival not having improved over many decades. Although unopposed estrogen exposure, complex atypical hyperplasia, and treatment with tamoxifen during breast cancer therapy are recognized risk factors for endometrial cancer, the etiology of endometrial cancer remains unclear. Endometrial cancer is generally considered to arise through progressive accumulation of multiple genetic abnormalities, which may activate oncogenes and inactivate tumor-suppressor genes [2]. However, these genetic alterations are not universally present in all cases of endometrial cancer, suggesting that other mutations or epigenetic alterations may also be important.

MicroRNAs (miRNAs) are a recently discovered class of non-coding small regulatory RNAs that interfere with translation of coding mRNAs in a sequence-specific manner [3]. miRNAs are frequently de-regulated in cancer, and have been suggested to play important roles in cancer initiation and development [4, 5]. miRNA profiling analysis of endometrial carcinoma unrevealed a number of dysregulated miRNAs that are commonly found in endometrial cancer [6], suggesting an important role for these small regulators in endometrial malignancy. For example, several miRNAs have been shown to repress expression of FOXO1, a crucial regulator of progesterone-dependent differentiation of the normal human endometrium [7]. miR-125b promotes proliferation and migration of endometrial carcinoma cells by targeting the TP53INP1 tumor suppressor [8]. On the other hand, some miRNAs act as tumor suppressors in endometrial carcinoma. miR-194 inhibits the epithelial to mesenchymal transition of endometrial cancer cells by targeting the oncogene BMI-1 [9]. Epigenetic silencing of miR-152 drives endometrial carcinogenesis by targeting E2F transcription factor 3 (E2F3), met proto-oncogene and Rictor [10]. Also, hyper-methylation of miR-129-2 contributes to over-expression of the SOX4 oncogene in endometrial cancer [11]. Interestingly, aberrant expression of miRNAs in endometrial carcinoma may be due to decreased expression of Drosha and Dicer, two major regulators of miRNA biogenesis [12].

Despite the identified roles of these miRNAs in endometrial cancer, the precise role of miRNAs in this malignant tumor is largely unknown. Therefore, we attempted to identify and functionally validate new miRNAs that are involved in the initiation and progression of endometrial cancer. The most dominant sub-type, endometrioid endometrial cancer (EEC), accounts for approximately 80% of cases [13]. In this study, cyclin D1 (CCND1), an oncogenic cell-cycle regulator, is shown to be directly and negatively regulated by miR-503 in EEC cell lines. Moreover, miR-503 suppresses the growth and cell-cycle progression of EEC cells by regulating CCND1 both in vitro and in vivo. This knowledge provides clues to understanding the role of miRNAs in regulation of endometrial cancers.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

miR-503 is down-regulated in EEC, while CCND1 is up-regulated

In a previous study, we found that miR-503 was down-regulated in EEC tissues compared to normal endometrium tissues (Y.Y. Xu and L.R. Yin, Department of Gynecology, The Second Hospital of Tianjin Medical University, unpublished data). In order to confirm the dysregulation of miR-503 in EEC, we first determined the expression levels of miR-503 in five normal endometrium samples, nine samples of endometrial tissue with complex atypical hyperplasia (CAH) and 13 samples of EEC tissues, respectively. A gradual reduction in the miR-503 level was observed across these three types of tissues, with the lowest level in EEC tissues (Fig. 1A). Further study confirmed the decrease of miR-503 in EEC tissues using paired normal endometrium and EEC tissues (10/10, Fig. 1B). In contrast, the level of CCND1, a key regulator of the cell cycle and a potential miR-503 target, was found to increase in CAH and EEC tissues compared to normal tissues by immunohistochemistry studies (Fig. 1C), which was also confirmed in paired normal and EEC tissues (8/10, Fig. 1D). Moreover, the clinical data, including miR-503 level, were obtained for 48 EEC patients. Survival analysis suggested that patients with a relatively high level of miR-503 (miR-503/U6 ≥ 0.01, = 25) exhibited a longer survival time compared to those with a lower miR-503 level (miR-503/U6 < 0.01, = 23; Fig. 1E). These results suggest a tumor-suppressor activity of miR-503 in EEC.

image

Figure 1. . The potential tumor suppressor miR-503 was down-regulated in EEC tissues, while CCND1 was up-regulated. (A) miR-503 levels in five normal endometrial tissues (Normal), nine endometrial tissues with complex atypical hyperplasia (CAH) and 13 endometrial endometrioid carcinoma (EEC) tissues measured using quantitative RT-PCR. U6 snRNA was used as the endogenous normalizer. The P value indicates the comparison of the three groups. (B) miR-503 levels in 10 pairs of normal endometrial and EEC tissues detected using quantitative RT-PCR. The P value indicates the comparison of these two groups. (C) CCND1 expression levels in the normal, CAH and EEC tissues detected using immunohistochemistry assay. (D) CCND1 protein levels in the 10 pairs of normal and EEC tissues measured using a western blot assay. GAPDH was used as the endogenous normalizer. The P value indicates the comparison of these two groups. (E) miR-503 levels in EEC tissues from 48 patients detected using quantitative RT-PCR. These cases were devided into a high miR-503 group (miR-503/U6 ≥ 0.01, = 25) and a low miR-503 group (miR-503/U6 < 0.01, = 23). Survival analysis was performed to evaluate the relationship between the miR-503 level and the patient's survival time.

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CCND1 is a direct target gene of miR-503 in EEC cells

As the CCND1 expression level exhibited inverse correlation with miR-503 level, we determined whether CCND1 was directly targeted by miR-503. Using bioinformatics analysis, we identified potential binding sites within the 3′ UTR of CCND1 mRNA (Fig. 2A). A fluorescent reporter assay was performed to validate interaction between miR-503 and the predicted binding sites. The wild-type reporter vector includes the sequences of the two potential binding sites, while the three mutant reporter vectors are point-mutated in the first, second or both binding sites (Fig. 2A). It was suggested that miR-503 mimics reduce the fluorescent intensity of the wild-type reporter vector in both HEC-1-A and Ishikawa cells. When the second site was mutated, the effect of miR-503 on the reporter gene remained. However, miR-503 no longer influenced expression of the reporter gene when the first or both two binding sites were mutated (Fig. 2B). Similarly, miR-503 blockade through mutation of the first site rather than the second aborted the enhancement of the fluorescent intensity (Fig. 2C). These data validate the role of the first predicted binding site in regulation of CCND1 expression by miR-503. The negative regulation of endogenous CCND1 by miR-503 was confirmed in HEC-1-A, Ishikawa and RL95-2 cells (Fig. 2D).

image

Figure 2. miR-503 directly and negatively regulates CCND1. (A) As predicted using the TargetScanHuman database, the wild-type CCND1 3′ UTR (wt UTR) contains two potential binding sites for miR-503. The mutated CCND1 3′ UTR sequences, with mutations (underlined nucleotides) within the first (mut1), second (mut2) or both predicted binding sites, are also shown. (B, C) The four fluorescent reporter vectors including either wild-type (wt) or mutated (mut1, mut2 or mut1&2) binding sites were transfected together with miR-503 mimics (B) or miR-503 antisense oligonucleotides (ASO) (C) into HEC-1-A and Ishikawa cells, and the luciferase intensity within the cells was measured. The histograms show the normalized luciferase intensity (luciferase/Renilla). (D) HEC-1-A, Ishikawa and RL95-2 cells were transfected with miR-503 mimics or antisense oligonucleotides, and the endogenous CCND1 protein levels were detected by western blot assay.

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miR-503 reduces the proliferative activities of EEC cells by negatively regulating CCND1

In order to assess the tumor-suppressor role of miR-503 in EEC cells, we increased the miR-503 level in HEC-1-A or Ishikawa cells, and detected proliferation-associated cellular phenotypes. Simultaneously, a CCND1 ectopic expression vector pCMV6/CCND1 (without any UTR sequence to avoid potential regulation by miRNAs) was constructed to rescue the decrease in CCND1 caused by miR-503 (Fig. 3A) without influencing the miR-503 level (Fig. 3B). Using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cellular activity detection, we found that miR-503 decreased the viability of both HEC-1-A and Ishikawa cells. Moreover, when CCND1 was expressed subsequently, the decreased cellular activity was rescued (Fig. 3C). Vigorous colony formation activity is a key characteristic of cancer cells. Our experiment also revealed inhibition of colony formation activity by miR-503, and alleviation of this effect by subsequent transfection of the CCND1 expression vector (Fig. 3D). miR-503 increases the number of cells in G1 phase, and decreases the number of cells in S phase in the HEC-1-A and Ishikawa lines, implying blockage of the G1/S transition. CCND1 ectopic expression relieved the G1/S blockage caused by miR-503 (Fig. 3E). These data demonstrate a tumor-suppressor role for miR-503 in EEC cells. The influence of miR-503 on EEC phenotypes was achieved through negative regulation of CCND1.

image

Figure 3. miR-503 suppresses EEC cell growth and cell-cycle progression by targeting CCND1. (A, B) HEC-1-A and Ishikawa cells were transfected with miR-503 mimics alone or together with CCND1 expression vector (pCMV6/CCND1), and CCND1 protein (A) and miR-503 (B) levels were detected using western blot or quantitative RT-PCR assays, respectively. (C) The viabilities of the cells shown in (A) were measured by the MTT assay. Values are the relative cellular viability 1–5 days after transfection. (D) The colony formation activities of the cells as treated in (A) were detected using a colony formation assay. The crystal violet-stained cell colonies are also shown. (E) The cell-cycle distribution of cells treated as in (A) was measured using propidium iodide staining and flow cytometry analyses. The proportion of the cells in each cell-cycle phase is shown.

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miR-503 and CCND1 exhibited inverse dynamic expression patterns during serum starvation and cell cycle re-entry in EEC cells

It has been suggested that when serum-starved NIH-3T3 cells are induced to re-enter the cell cycle by serum addition, the level of miR-503 significantly decreases [14]. In view of the observation of regulation of the cell cycle and CCND1, a key factor of cell-cycle progression, by miR-503 in EEC cells, we next measured the expression levels of both miR-503 and its target CCND1 in serum-starved EEC cells that were subsequently re-supplied with serum. Serum starvation led to an increase in miR-503 and a decrease in CCND1 at 6 and 12 h of serum deprivation in both HEC-1-A and Ishikawa cells (Fig. 4A). Moreover, when the cells were re-supplied with serum, miR-503 expression decreased to a level comparable to that of normally cultured cells, and the CCND1 level increased (Fig. 4B). The opposite expression patterns of miR-503 and CCND1 suggest a possible regulation of the cell cycle by miR-503 by suppression of CCND1.

image

Figure 4. miR-503 and CCND1 exhibit inverse dynamic expression patterns during serum starvation and cell cycle re-entry in EEC cells. (A, B) HEC-1-A and Ishikawa cells were cultured in serum-free medium for 12 h, and then in serum-filled complete culture medium for another 12 h. The miR-503 (A) and CCND1 protein (B) levels were detected at various time points using Northern and western blot assays, respectively.

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miR-503 suppresses the growth of xenografts derived from EEC cells in vivo

As miR-503 suppresses proliferation of EEC cells in vitro, we next explored the role of miR-503 in growth of EEC cell-derived allogenetic tumors in nude mice. HEC-1-A cells stably expressing miR-503 were induced and injected into the axillary fossae of nude mice. The tumors derived from miR-503-expressing HEC-1-A cells were smaller than those from the control cell line (Fig. 5A). The negative effect of miR-503 on the EEC-derived xenografts was probably due to suppression of CCND1, as the tumors of the miR-503 over-expression group exhibited a higher level of miR-503 and a lower level of CCND1 (Fig. 5B). These data confirm the negative regulation role of miR-503 in EEC tumor growth in vivo.

image

Figure 5. miR-503 suppresses the growth of xenografts derived from EEC cells in vivo. (A) Ishikawa cells stably expressing miR-503 were inoculated at the axillary fossae of female athymic nude mice aged 6–8 weeks. On the 16th day after injection, the mice were killed and the tumors were isolated. (B) The RNA and protein of the tumor tissues were extracted, and the levels of miR-503 and CCND1 were detected using quantitative RT-PCR and western blot assays, respectively.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

In this study, to elucidate the regulation pathways involving miRNAs in EEC cells, we focused on the regulation of CCND1 by miR-503. We chose miR-503 for the following reasons. First, miRNA microarray analysis revealed that miR-503 is down-regulated in EEC tissues compared to the normal endometrial tissues (Y.Y. Xu and L.R. Yin, Department of Gynecology, The Second Hospital of Tianjin Medical University, unpublished data). Second, the level of miR-503 exhibited a dramatic decrease across normal endometrial, CAH and EEC tissues, suggesting potential association of this miRNA with tumor malignancy. Third, according to clinical data, a relatively high level of miR-503 in tumor tissues implied a longer survival time for EEC patients, highlighting a tumor-suppressor role of miR-503 in EEC progression.

miR-503 is an intragenic miRNA clustered with miR-424 at chromosomal location Xq26.3 [15]. miR-503 belongs to the ‘extended’ miR-16 family, because the seed region of miR-503, which indicates the 5′ end of the miRNA, differs only at nucleotide 8 from that of the canonical miR-16 family (miR-15a/b, miR-16, miR-195, miR-424 and miR-497) [16]. This results in a CG dinucleotide at positions 7 and 8 of miR-503; CG dinucleotides are strongly depleted in mammalian miRNAs. Thus, miR-503 has few predicted targets containing 7-mer sites, and hence few predicted targets not also recognized by the canonical miR-16 family [14]. Several studies have identified miR-503 as involved in malignant tumors. miR-503 expression was up-regulated in human parathyroid carcinomas [17] and adenocortical carcinomas [18]. Moreover, miR-503 induced G1 arrest by targeting an overlapping set of cell-cycle regulators during monocyte differentiation into macrophages [19]. miR-503 was also induced during myogenesis and promotes cell-cycle arrest through cdc25A degradation [20]. Given the down-regulation of miR-503 in EEC tissues, and the better prognosis for EEC patients with higher miR-503, we determined whether miR-503 is involved in cell-cycle regulation, and which genes are functional targets of miR-503 in EEC cells.

A systematic validation study revealed that CCND1, a key cell-cycle regulator, is targeted and suppressed by miR-503 in human head and neck carcinoma cells [21]. We determined whether regulation of CCND1 by miR-503 also occurs in EEC cells. Evidence for their direct interaction comes from the following data. First, the miR-503 level was inversely correlated with the CCND1 level in EEC and normal endometrial tissues. miR-503 showed a lower level and CCND1 showed a higher level in EEC tissues compared to paired normal endometrial tissues. Second, the predicted miR-503 binding sequence within the CCND1 3′ UTR was bound efficiently by miR-503, as confirmed by a fluorescent reporter assay. Third, alteration of miR-503 levels in EEC cells resulted in a reciprocal alteration in the level of endogenous CCND1, indicating negative regulation. Interestingly, we noted that, in the paired tissue samples, despite the inverse relationship between miR-503 and CCND1, the most significant down-regulation of miR-503 (sample number 7 in Fig. 1B) did not correspond to the greatest increase in CCND1 (Fig. 1D). Thus we assume that CCND1 may be co-regulated by other miRNAs and even other regulating factors [21].

CCND1 is a cell-cycle regulator that is essential for progression through G1 phase and is a candidate proto-oncogene. Abnormal expression of this protein has been implicated in the pathogenesis of several human tumor types, such as breast cancer [22, 23], lymphoma [24] and ovarian cancer [25]. Moreover, targeting CCND1 appears to be feasible in cancer prognostic and therapy [26, 27]. The oncogenic activity of CCND1 is due to its acceleration of cell cycle, promoting cell proliferation. In this study, the decreased miR-503 level in EEC cells resulted in an increase in its target CCND1, which contributed to malignant cellular phenotypes both in vitro and in vivo. The blockage of the G1/S transition caused by miR-503 mimics may, at least in part, explain the suppressed proliferation and colony formation activities of EEC cell lines induced by miR-503. Importantly, all the alterations of cellular phenotypes caused by miR-503 mimics were restored by ectopic expression of CCND1 lacking its 3′ UTR to avoid regulation by any miRNA. The restoration of CCND1 expression by the ectopic expression vectors reversed the decreased CCND1 level induced by miR-503, thus rescuing the decreased proliferation activities of the EEC cells. Thus, we conclude that the tumor-suppressor role of miR-503 is due to its negative regulation of CCND1. In other words, suppression of miR-503 alleviated repression of its target gene CCND1, allowing proliferation of EEC cells, which may be a mechanism for miR-503-involved tumor initiation and progression.

miR-503 expression increases in response to serum starvation in mesenchymal stem cells [28], NIH-3T3 cells [14] and endothelial cells cultured at high glucose concentrations [29]. Moreover, in NIH-3T3 cells G1 arrested by serum starvation, the increased miR-503 level returns to normal upon cell cycle re-entry, due to the constitutive instability and rapid degradation of miR-503 [14]. Similar results were observed in EEC cells. A rapid increase of the miR-503 level was observed during serum starvation in HEC-1-A and Ishikawa cells. When the cells were re-supplied with serum, i.e. re-entered the cell cycle, the miR-503 level decreased to a level comparable with normally cultured cells. The CCND1 level was also examined during serum deprivation and re-supply. As expected, CCND1 expression showed reciprocal alteration compared with miR-503. We presume that, in EEC cells, cell cycle re-entry is triggered by degradation of miR-503 after the supply of serum, which in turn alleviated suppression of CCND1 and enhanced cellular proliferation.

Collectively, our research reveals the miR-503/CCND1 cell-cycle regulation pathway in EEC cells. The decreased miR-503 level in EEC cells relieved suppression of its target CCND1, and in turn accelerated cell-cycle progression and cellular growth activity. This knowledge may shed light on the mechanism of regulation by miRNA in EEC cells, and may also provide suggestions for new therapeutic strategies for EEC. Although we mainly focused on CCND1 in this study, we presume that other potential genes act as functional targets of miR-503 in EEC, some of which may also be associated with the cell cycle and affect cell proliferation.

Experimental procedures

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Tissue samples, cell lines and transfection

Normal endometrial tissues (= 5, patient age 59.2 ± 9.26 years), endometrial tissues with CAH (= 9, patient age 62.9 ± 8.64 years) and EEC tissues (= 13, patient age 64.7 ± 7.93 years), as well as 10 pairs of normal endometrial and EEC tissues (patient age 62.5 ± 7.74 years), were obtained from patients in the Department of Pathology, Second Hospital of Tianjin Medical University, China, with the patients' informed consent. The normal endometrial tissues used in this research were the distal end of the operative excision far from the localized tumors. An additional 48 EEC samples from patients with complete follow-up data (high miR-503, = 25, patient age 60.7 ± 8.23 years; low miR-503, = 23, patient age 63.0 ± 9.72 years) were collected from the same source. The pathological type and the International Federation of Gynecology and Obstetrics (FIGO) stage of all clinical samples was confirmed by pathological analysis (Table S1). All the samples were snap-frozen in liquid nitrogen immediately after surgical resection and then stored at −80 °C until use. This study was approved by the Ethics Committee of Tianjin Medical University.

Three human EEC cell lines, HEC-1-A, Ishikawa and RL95-2, were maintained in McCoy's 5a, RPMI-1640 and DMEM/F12 media (all from Gibco, Carlsbad, CA, USA), respectively, supplemented with 10% fetal bovine serum (serum-free medium was used when necessary), 100 IU·mL−1 penicillin and 100 mg·mL−1 streptomycin. The medium for RL95-2 cells was supplemented with 5 μg·mL−1 insulin. All cells were cultured at 37 °C in a humidified chamber supplemented with 5% CO2.

Transfection of plasmids and oligonucleotides was performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Briefly, cells were trypsinized, counted and seeded in plates the day before transfection to ensure a suitable cell confluence on the day of transfection. Plasmids or oligonucleotides were used at a final concentration of 5 ng·μL−1 or 200 nm, respectively, in antibiotic-free Opti-MEM medium (Invitrogen). Transfection efficiency was monitored by use of fluorescent protein-expressing vectors or Cy5-labeled oligonucleotides.

Extractions of RNA and protein

The large and small RNA fractions of the tissue samples or cell lines were isolated using a mirVana™ miRNA isolation kit (Ambion, Austin, TX, USA) according to the manufacturer's instructions. Large RNA (larger than 200 nt) and small RNA (smaller than 200 nt) were separated and purified. The integrity of the large RNA was confirmed by resolution on a 1% agarose gel by gel electrophoresis, and the purity of RNA was evaluated by ratio of the absorbance at 260 nm (A260) to that at 280 nm (A280).

Tissue protein was extracted using TRIzol reagent (Invitrogen) from the residual organic phase after RNA extraction of tissue samples. To extract the cellular protein, the cells were lysed using radio-immunoprecipitation assay lysis buffer (150 mm NaCl, 50 mm Tris/HCl pH 7.2, 1% Triton X-100 and 0.1% SDS) supplemented with Complete protease inhibitor cocktail (Roche, Basel, Switzerland). After centrifugation at 12 000 g for 10 min at 4 °C, the undissolved cell components were removed and the cellular proteins were obtained.

Northern blot assay

Small RNAs (40 μg) were separated in a 18% polyacrylamide gel containing 8 m urea, electrophoretically transferred to a BrightStar-Plus nylon membrane (Ambion), cross-linked using 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride, pre-hybridized, and then hybridized to the labeled probes. A fragment generated using probes against the mature miRNA was γ-32P-labeled using T4 polynucleotide kinase (Fermentas, Pittsburgh, PA, USA). The membrane was then washed 4 times in the washing solution (6 × saline sodium citrate, 0.5% SDS) for 5 min at room temperature and exposed to X-ray film (Fujifilm, Tokyo, Japan). U6 snRNA was simultaneously measured as a loading control.

Western blot assay

The proteins were resolved by SDS/PAGE and then transferred onto nitrocellulose membrane. Antibodies against CCND1 or GAPDH were incubated with the membranes overnight at 4 °C. The membranes were then washed 4 times in the washing solution (Tris-buffered saline with Tween-20) for 5 min at room temperature and incubated with horseradish peroxidase-conjugated secondary antibodies. Protein expression was assessed by enhanced chemiluminescence and exposure to chemiluminescent film. LabWorks™ image acquisition and analysis software (UVP, Upland, CA, USA) was used to quantify the band intensities. The antibodies were purchased from Abcam (Cambridge, MA, USA).

Immunohistochemistry assay

For immunohistochemistry, formalin-fixed, paraffin-embedded tissue sections were de-paraffined in xylol and rehydrated in an ethanol gradient (100%, 95%, 85%, 70% and then 50%). Antigen retrieval was performed by heating the tissue sections for 20 min in 1 mm EDTA buffer (pH 8.0). Endogenous peroxidase activity was eliminated by incubation in 0.3% hydrogen peroxide in methanol for 30 min. The slides were first incubated in non-immune serum for 30 min followed by incubation with antibody to CCND1 (Abcam) overnight at 4 °C. After washing in Tris-buffered saline with Tween-20, the sections were incubated with horseradish peroxidase-conjugated secondary antibody for 20 min. The tissue sections were then visualized by staining with 3,3′-diaminobenzidine, and counterstained with hematoxylin, and photographs were taken.

Quantitative RT-PCR for miRNAs

Quantification of miRNAs was performed using stem-loop RT-PCR [30]. Briefly, 2 μg small RNA was reverse-transcribed into cDNA using M-MLV reverse transcriptase (Promega, Madison, WI, USA) using the following primers: miR-503-RT (5′-GTCGTATCCAGTGCAGGGTCCGAGGTGCACTGGATACGACCTGCAG-3′) and U6-RT (5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAAAATATGGAAC-3′), which folds into a stem-loop structure. The cDNA was used for amplification of mature miR-503 and the endogenous control, U6 snRNA, by PCR. The corresponding PCR primers were as follows: gene-specific forward primers miR-503-Fwd (5′-TGCGGTAGCAGCGGGAACAGTTC-3′) and U6-Fwd (5′-TGCGGGTGCTCGCTTCGGCAGC-3′), and a universal downstream primer (reverse: 5′-CCAGTGCAGGGTCCGAGGT-3′). The PCR conditions comprised initial denaturation at 95 °C for 3 min, followed by 40 cycles of 95 °C for 30 s, 56 °C for 30 s, and 72 °C for 30 s.

For quantification of CCND1 mRNA, 5 μg large RNA was reverse-transcribed into cDNA using an oligo(dT) primer. The cDNA was used for amplification of CCND1 mRNA and the endogenous control, β-actin, by PCR. The corresponding PCR primers were CCND1-qPCR-sense (5′-CTGTGTTATTCTTTGCGTG-3′), CCND1-qPCR-antisense (5′-GCTTCATTGAGATTTGGAG-3′), β-actin-sense (5′-CGTGACATTAAGGAGAAGCTG-3′) and β-actin-antisense (5′-CTAGAAGCATTTGCGGTGGAC-3′). The PCR conditions comprised initial denaturation at 95 °C for 3 min, followed by 40 cycles of 95 °C for 30 s, 58 °C for 30 s, and 72 °C for 30 s.

Quantitative PCR was performed using SYBR Premix Ex Taq (TaKaRa, Otsu, Japan) on an iQ5 real-time PCR detection system (Bio-Rad, Hercules, CA, USA). All of the primers were purchased from AuGCT Inc. (Beijing, China). The fold change in miR-503 or CCND1 expression was calculated using the inline image method [31].

Prediction of miRNA targets

A bioinformatics method was used to predict the target genes of miRNA. The database used was TargetScanHuman, release 6.2 (http://www.targetscan.org).

Vector construction

To construct the fluorescent reporter vectors, four 57 bp double-stranded fragments were obtained via annealing reactions. The oligonucleotides used were wt-Top (5′-CGCGTCCATTTTCTTATTGCGCTGCTAGGATCCTCTTTCACATTGTTTGCTGCTATA-3′), wt-Bottom (5′-AGCTTATAGCAGCAAACAATGTGAAAGAGGATCCTAGCAGCGCAATAAGAAAATGGA-3′), mut1-Top (5′-CGCGTCCATTTTCTTATTGCCCAGGTTGGATCCTCTTTCACATTGTTTGCTGCTATA-3′), mut1-Bottom (5′-AGCTTATAGCAGCAAACAATGTGAAAGAGGATCCAACCTGGGCAATAAGAAAATGGA-3′), mut2-Top (5′-CGCGTCCATTTTCTTATTGCGCTGCTAGGATCCTCTTTCACATTGTTTCCAGGTTTA-3′), mut2-Bottom (5′-AGCTTAAACCTGGAAACAATGTGAAAGAGGATCCTAGCAGCGCAATAAGAAAATGGA-3′), mut1&2-Top (5′-CGCGTCCATTTTCTTATTGCCCAGGTTGGATCCTCTTTCACATTGTTTCCAGGTTTA-3′) and mut1&2-Bottom (5′-AGCTTAAACCTGGAAACAATGTGAAAGAGGATCCAACCTGGGCAATAAGAAAATGGA-3′). These frag-ments were then cloned into pMIR-REPORT vector (Ambion) at the MluI and HindIII sites, respectively. The constructed vectors were named pMIR/CCND1-wt, -mut1, -mut2 and -mut1&2.

To construct the CCND1 ectopic expression vector, the complete coding sequence of CCND1 was amplified by PCR using a cDNA library from HEC-1-A cells as the template. The PCR primers used were CCND1-sense (5′-CAGAGAAGCTTCCATGGAACACCAGCTCCTGTG-3′) and CCND1-antisense (5′-TGGTGCTCGAGGATGTCCACGTCCCGCAC-3′). The amplified fragment was cloned into the pCMV6-Entry vector (AMS Biotechnology, Abingdon, UK) at the HindIII and XhoI sites. The constructed vector was named pCMV6/CCND1.

To construct the miR-503 ectopic expression vector, 76 bp double-stranded fragments were obtained via annealing reactions. The oligonucleotides used were pre-miR-503-Top (5′-AGCTTGCCCTAGCAGCGGGAACAGTTCTGCAGTGAGCGATCGGTGCTCTGGGGTATTGTTTCCGCTGCCAGGGTAC-3′) and pre-miR-503-Bottom (5′-TCGAGTACCCTGGCAGCGGAAACAATACCCCAGAGCACCGATCGCTCACTGCAGAACTGTTCCCGCTGCTAGGGCA-3′). The fragment was then cloned into the pCMV6-Entry vector at the HindIII and XhoI sites. The constructed vector was named pCMV6/miR-503.

Fluorescent reporter assay

The cells were transfected with one of the fluorescent reporter vectors (pMIR/CCND1-wt, -mut1, -mut2 or -mut1&2) together with miR-503 antisense oligonucleotides (2′-O-methyl-modified, 5′-cugcagaacuguucccgcugcua-3′, purchased from GenePharma, Shanghai, China) or miR-503 mimics (double-stranded RNA, sense: 5′-uagcagcgggaacaguucugcag-3′, antisense: 5′-cugcagaacuguucccgcugcua-3′, purchased from GenePharma). An identical amount of Renilla expression vector pRL-TK (Promega) was co-transfected in all cases for normalization. At 48 h after transfection, protein was extracted from the cells and luciferase activity was measured using the Dual-Glo luciferase assay system (Promega).

MTT assay

For the MTT assay, cells were seeded and transfected in 96-well plates. At 1–5 days after transfection, MTT [(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] was added to the cells, and the attenuance at 490 nm was measured using a μQuant Universal microplate spectrophotometer (BioTek, Winooski, VT, USA).

Colony formation assay

To measure the colony formation activity of the ovarian cells, transfected cells were counted and seeded in 12-well plates at 100 cells per well. The culture medium was replaced every 3 days. At the 12th day after seeding, colonies were counted only if they contained more than 50 cells. Finally, the cells were stained using crystal violet, and images were obtained.

Flow cytometry analyses for cell-cycle staging

For determination of cell cycle stages, transfected cells were detached from the plates by trypsin incubation, rinsed with NaCl/Pi and fixed in 70% ethanol. Before detection, the cells were rehydrated in NaCl/Pi and incubated with RNase (100 μg·mL−1) and propidium iodide (60 μg·mL−1; Sigma-Aldrich, St Louis, MO, USA). Cells in different phases were analyzed using a Beckman Coulter flow cytometer (Brea, CA, USA).

In vivo tumor xenograft studies

To establish a HEC-1-A cell line that stably over-expresses miR-503, HEC-1-A cells were transfected with pCMV6/miR-503 or the pCMV6 vector, followed by selection for 20–30 days in McCoy's 5a medium supplemented with 10% fetal bovine serum and 800 μg·mL−1 G418 (Invitrogen). Single colonies were picked and amplified, and the expression level of miR-503 was detected by real-time RT-PCR. HEC-1-A cells stably over-expressing miR-503 or control cells were inoculated with 4 × 106 cells per site bilaterally on the axillary fossae of female athymic nude mice aged 6–8 weeks, with six animals in each group. Tumor size was monitored every 2 days from the 8th day after injection by measuring the length and width using calipers, and volumes were calculated using the formula (× W2) × 0.5, where L is the length and W is the width of each tumor. On the 16th day after injection, the mice were killed and the tumors were isolated. RNA and protein were extracted from the tumor tissues, and the levels of miR-503 and CCND1 were detected. The mice used in this experiment were maintained under specific pathogen-free conditions and handled in accordance with National Institutes of Health Animal Care and Use Committee Regulations.

Statistical analysis

All experiments were performed in triplicate. Values are means ± SD. Student's t test was used for hypothesis testing for significance between two groups, and one-way ANOVA was used for hypothesis testing for significance between three or more groups, followed by the Student–Newman–Keuls q test for comparison of pairs of groups. The Kaplan–Meier estimator with log rank test was used in the survival analysis. All statistical analyses were performed using graphpad prismversion 5.0 software (GraphPad Software, La Jolla, CA, USA), and statistical significance was set as  0.05.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

This work was supported by the National Natural Science Foundation of China (grant number 30901742).

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  4. Results
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  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information
FilenameFormatSizeDescription
febs12365-sup-0001-TableS1.zipZip archive215KTable S1. Detailed information for all patients included in this study, including age and FIGO stage.

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