From Jan, 2016 to Jun, 2016, a total of 16 pairs of tissue samples from 16 patients with endometrial cancer were used in the present study, each of which consisted of human endometrial cancer tissue and matched adjacent normal tissue from the same patient. The matched normal tissue samples were obtained from the distal end of the surgical excisions, distant from the tumor. The samples were obtained from the Department of Gynecology, Maternal and Child Healthcare Hospital (Huaian, China). All the patients were females, the average age was 58.82±8.21-years-old. No patients had suffered from previous tumors. The average cancer tissue size was 4.23±1.08 mm³, and the average weight was 1.37±0.24 g. In addition, the average adjacent normal tissue size was 4.44±1.02 mm³, and the average weight was 1.45±0.28 g. The present study was approved by the ethics committee of the Maternal and Child Healthcare Hospital.

The Ishikawa, HEC-1A and HEC-1B human endometrial cancer cell lines were purchased from America Type Culture Collection (Manassas, VA, USA). The cells were maintained in Dulbecco's modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) in a humidified incubator at 37°C with 5% CO₂. The transfection was performed using the Lipofectamine 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol.

RNA was extracted from patient tissue specimens or cells 48 h following transfection using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Small RNA (5 µg) was reverse transcribed into cDNA (2 µg) using M-MLV reverse transcriptase (Promega Corporation, Madison, WI, USA) with the specific primers (0.5 µg). The cDNA was used as template to amplify either mature miR-409 or an endogenous control U6 snRNA using real time PCR kit (Takara Bio, Inc., Otsu, Japan). The reaction mixture was prepared: SYBR Premix Ex Taq II 10 µl, forward primer 0.8 µl, reverse primer 0.8 µl, cDNA template 2 µl, and dH₂O 6.4 µl. The primer sequences used were as follows: miR-424-forward: 5′-CAGCAGCAATTCATGT-3′, miR-424-reverse: 5′-TGGTGTCGTGGAGTCG-3′; Smad2-forward: 5′-CAGGACGGTTAGATGAGCTTGAGA-3′, Smad2-reverse: 5′-CCCACTGATCTACCGTATTTGCTG-3′; β-actin-forward: 5′-TCTGGCAACGGTGAAGGTGACA-3′, β-actin-reverse: 5′-CACCTCCCCTGTGTGGACTT-3′; U6-forward: 5′-CTCGCTTCGGCAGCACA-3′, U6-reverse: 5′-AACGCTTCACGAATTTGCGT-3′; miR-409-forward: 5′-TATATCCAGCTGGGTGCTAATTTGCCG-3′, universal primer 5′-TGGTGTCGTGGATAC-3′. The qPCR was performed as follows: 94°C for 3 min, followed by 40 cycles of 94°C for 30 sec, 50°C for 30 sec and 72°C for 30 sec. The RT-qPCR analysis was performed using SYBR Premix Ex Taq (Takara Bio, Inc.) on the iQ5 Real-Time PCR detection system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The relative expression levels of miR-409 and Smad2 were defined as follows: Quantity of miR-409/quantity of U6 within the same sample; quantity of Smad2/quantity of β-actin within the same sample. The 2^−ΔΔCq method was used to analyses the relative gene expression (16).

To determine the viability and proliferative capacity of the cells, the cells were examined using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and colony formation assays, as described previously (17). Following transfection, Ishikawa and HEC-1B cells were seeded in 96-well plates at a density of 8,000 cells per well. At different time points following transient transfection, the cells were incubated with 10 µl MTT at a final concentration of 0.5 mg/ml at 37°C for another 4 h. The medium was then removed, and the precipitated formazan was dissolved in 100 µl DMSO. Following shaking for 20 min, the absorbance at 570 nm (A570) was detected using a uQuant Universal microplate spectrophotometer (Bio-Tek Instruments, Inc., Winooski, VT, USA). For the colony formation assay, the numbers of viable cell colonies were determined 15 days following the inoculation of 150 cells/well in triplicate into 12-well plates. The cells were stained with 0.1% crystal violet at room temperature for 20 min. The plates were observed under a FastScan atomic force microscope (Bruker AXS, Bruker Corporation Santa-Barbara, CA, USA). The rate of colony formation was calculated using the following equation: Colony formation rate=(number of colonies/number of seeded cells) ×100%.

The apoptotic ratios of the cells were determined using the Annexin V-7-ADD apoptosis detection kit (Roche Diagnostics, Basel, Switzerland). Briefly, 48 h following transfection, the cells were collected and washed twice with cold PBS buffer, resuspended in 200 µl of binding buffer, and incubated with 20 µl of Annexin-V-R-PE for in an ice bath for 20 min in the dark. This was followed by the addition of 10 µl 7-AAD prior to analyzing using flow cytometry. Cells treated with DMSO were used as a negative control. Following transfection for 48 h, the cells were collected and fixed with 70% ethanol, stained with propidium iodide, and analyzed by flow cytometry. The data were analyzed using CellQuest Pro software 5.1 (BD Biosciences, Franklin Lakes, NJ, USA). The experiments were repeated at least three times.

Total cellular extracts were extracted using radioimmunoprecipitation assay buffer (2 µl). Adjust the density of relative cells to 1×10⁵/ml and inoculated with a six-well plate. Following cell adherence to the wells, cells were collected after 48 h. Cells were lysed with RIPA buffer; proteins were then separated. A Bicinchoninic Acid assay was used to determine the relative protein concentrations. Aliquotes of proteins (50 µg in 25 µl) were separated by 10% SDS-PAGE electrophoresis for 1.5 h under 110 V at 4°C. Polyvinylidene difluoride membranes were blocked with 5% non-fat dry milk in Tris-buffered saline with 0.1% Tween-20 (TBST) for 10 min at room temperature prior to the transfer of proteins. The membrane was incubated with rabbit anti-human phospho-Smad2 antibody (ab53100, 1:1,000, Abcam, Cambridge, UK) or rabbit anti-human GAPDH antibody (ab9485, 1:1,000, Abcam) overnight at 4°C, rinsed three times with TBST for 5 min, and incubated with horseradish peroxidase-labeled goat anti-rabbit IgG (ab205718, 1:5,000, Abcam) at 37°C for 2 h. The membrane was soaked in an enhanced chemiluminescence solution (Sigma-Aldrich; Merck KGaA) in the darkroom for color development. The grey value was analyzed using Image J 1.48 (National Institutes of Health, Bethesda, MD, USA).

Based on bioinformatics predictions using the DNA Intelligent Analysis 2.0 (DIANA) (http://diana.imis.athena-innovation.gr/DianaTools/index.php), miR Database (miRDB) 5.0 (http://www.mirdb.org/mirdb/policy.html), TargetScan 7.1, (http://www.targetscan.org/vert_71/) and miRwalk databases 6.0 (http://129.206.7.150/), Smad2 was selected as a candidate target of miR-409. The 3′untranslated region (UTR) segments of Smad2 containing putative binding sites for miR-409 were obtained by PCR and inserted into the pmirGLO vector (20 µg, Promega Corporation). The wild-type reporter construct pmirGLO/Smad2-3′UTR and the mutant reporter construct pmirGLO/Smad2-3′UTR mut, in which the site of perfect complementarity to miR-409 was mutated (CAA CAU U to CAA GGA A) using site-directed mutagenesis PCR as follows: PCR-SD reactant was mixed in a sterile centrifuge tube on ice, 2.5 U Taq DNA polymerase and 2.5 U Taq Extender PCR Additive were added. Following PCR amplification (denaturation at 94°C for 2 min, followed by 12 cycles of 94°C, 20 sec; 51°C 30 sec; and 72°C, 3 min, followed by 72°C for 10 min.), digestion and purification of the PCR-SDM products, the reactant was placed on the ice for 2 min, and the 25 µl of amplification products were mixed with 1 µl Dpn I restriction endonuclease (10 U/µl) and 1 µl of Pfu DNA polymerase (2.5 U/µl) was centrifuged at 15,000 × g, 4°C for 1 min. Then the reactant was incubated at 37°C for 30 min; 100 µl of H₂O, 10 µl of SDM buffer and 5 µl of ATP (10 mmol/l) were then added into the reactant. The reactants were centrifuged at 15,000 × g, 4°C for 1 min. T4 DNA ligase (4 U/µl) was added and then the reactants was placed at 37°C for 1 h. The heat-shocked cells were thawed lightly on the ice and then 40 µl of cells were removed into a pre-cooled FALCOL 2059 polypropylene tube. Ligase-treated DNA (1 µl) was then added to the cells then incubated on ice for 30 min. Subsequently, the reactants were placed at 42°C for 30 sec and at ice for 2 min. The competent cells were immediately placed on the LB agar plate, which was then incubated overnight at 37°C. The plasmid DNA was extracted via alkaline lysis, and DNA was quantified with a UV spectrophotometer and identified via digestion by the restriction enzymes DpnI and HindIII. Sequence identification was performed by Genscript Biotech (Nanjing, China). For the luciferase reporter experiments, HEC-1B cells (2×10⁵/ml) were co-transfected with miR-409 mimics or miR-409 control in a 48-well plate followed by the pmirGLO/Smad2-3′UTR reporter vector or the pmirGLO/Smad2-3′UTR mut. Firefly luciferase and Renilla luciferase levels were measured 48 h following transfection. Each experiment was repeated at least three times.

All the data in the present study was analyzed by SPSS software 22.0 (IBM Corp., Armonk, NY, USA). Data are expressed as the mean ± standard deviation. P≤0.05 was considered to indicate a statistically significant difference using the Students-Newman-Keuls test.

To examine the role of miR-409 in the development of endometrial cancer, the present study measured the expression of miR-409 in 16 paired endometrial cancer samples using RT-qPCR analysis (Fig. 1A), which showed significantly reduced expression levels in the tumor tissues, compared with the adjacent normal tissues. The expression of miR-409 was significantly downregulated in the endometrial cancer cell lines (Ishikawa, HEC-1A and HEC-1B), compared with that in the adjacent normal tissues, also determined using RT-qPCR analysis (Fig. 1B).

To determine the role of miR-409 in tumor cell proliferation, the miR-409 mimics were used to induce the ectopic expression of miR-409 in Ishikawa and HEC-1B cells (Fig. 2A). Using an MTT assay, the overexpression of miR-409 was shown to suppress cell viability in the Ishikawa and HEC-1B cells (Fig. 2B and C). The colony formation rates of the Ishikawa and HEC-1B cells transfected with miR-409 mimics, were significantly lower, compared with those in the control group (Fig. 2D and E). These results indicated that miR-409 suppressed the ability of Ishikawa and HEC-1B cells to proliferate.

Flow cytometry revealed that the percentages of Ishikawa and HEC-1B cells in the of S phase of the cell cycle were markedly higher in the miR-409 mimic-transfected groups, compared with those in the control, suggesting that miR-409 initiated S phase arrest (Fig. 3A). The fluorescence-activated cell sorting analysis revealed that the forced expression of miR-409 led to endometrial cancer cell apoptosis. In the Ishikawa and HEC-1B cells, the percentage of apoptotic cells was significantly increased in response to the overexpression of miR-409, compared with that in the mimic control (Fig. 3B). These data demonstrated that miR-409 may inhibit proliferation by inducing S phase arrest and promoting apoptosis in endometrial cancer.

Based on the miR-409-induced suppression of proliferation in the endometrial cancer cells, the present study hypothesized that miR-409 inhibited the malignancy of endometrial cancer cells by regulating oncogenes and/or genes involved in cell proliferation or apoptosis. Therefore, five bioinformatics algorithms (DIANA, miRDB, TargetScan, RNA22 and miRwalk) were used to identify potential target genes of miR-409 (Fig. 4A). As a result, Smad2, a signal transducer and transcriptional modulator mediating multiple signaling pathways, was predicated to have a putative miR-409 binding site within its 3′UTR (Fig. 4A) and was selected for further evaluation. The binding sites between miR-409 and Smad2 were also conserved among species (Fig. 4B). To confirm that miR-409 directly targeted Smad2, luciferase reporter assays were performed to examine whether miR-409 interacts directly with its target Smad2. A series of 3′UTR fragments were constructed, including the wild-type Smad2 3′UTR and a binding site mutant. These fragments were then inserted into the pmirGLO luciferase reporter plasmid. In the Ishikawa cells, cotransfection with miR-409 and the wild-type Smad2 3′UTR caused a significant decrease in luciferase activity, compared with that in the control. However, the cotransfection with mutant Smad2 3′UTR and miR-409 mimics did not alter the luciferase intensity (Fig. 4C). The overexpression of miR-409 reduced the mRNA and protein expression levels of Smad2 in the Ishikawa and HEC-1B cells (Fig. 4D and E). Taken together, these results suggested that miR-409 binds directly to the 3′UTR of Smad2, repressing gene expression.

To determine the expression of Smad2 in endometrial cancer and adjacent normal tissues, RT-qPCR analysis of Smad2 was performed on the 16 paired endometrial cancer tissues, each consisting of an endometrial cancer and adjacent normal tissue specimen. In general, the expression levels of Smad2 were significantly higher in the endometrial cancer tissues, compared with those in the matched normal tissues (Fig. 5A). miR-409 was negatively correlated with Smad2 in the endometrial cancer tissues (Fig. 5B).