The present study was approved by the Human Studies Committee at The Second Affiliated Hospital of Zhengzhou University (Zhengzhou, China). Informed consent was obtained from each patient prior to surgery. A total of 43 pairs of EC samples (all females; age, 54±16 years) were obtained from patients; Patients that were treated with chemotherapy or radiotherapy before surgery were excluded. samples were histologically validated for type and grade. These samples were collected from May 2012 to July 2016.

Human EC cell lines HHUA and JEC were obtained from the Cell Bank of Type Culture Collection of Chinese Academy of Science (Shanghai, China) and cultured in DMEM (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) supplemented with 15% of fetal bovine serum (FBS), 100 U/ml of penicillin and 100 µg/ml of streptomycin (all from Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Cells were incubated at 37°C in a humidified atmosphere of 5% CO2. To inhibit the PI3K/AKT signaling pathway, 50 µM LY294002 (cat. no. 9901; Cell Signaling Technology, Inc., Danvers, MA, USA) was added to the cultured cells for 24 or 48 h at 37°C according to the manufacturer's protocol.

miR-494-3p mimics (5′-UGAAACAUACACGGGAAACCUC-3′), inhibitors (5′-GAGGUUUCCCGUGUAUGUUUCA-3′) and negative controls (NCs; 5′-ACAUCUGCGUAAGAUUCGAGUCUA-3′) were synthetized by Invitrogen (Thermo Fisher Scientific, Inc.). For PTEN overexpressioin, the coding sequence of PTEN was amplified by PCR and constructed into the pcDNA3 vector (Invitrogen; Thermo Fisher Scientific, Inc.) between EcoRI and XhoI. All transfections were performed using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol, at a concentration of 50 nM for miRNAs and 1 µg for pcDNA3-PTEN. After 48 h at 37°C, the transfection efficiency was validated using qRT-PCR as described below.

All animal experiments were approved by the Ethics Committee of The Second Affiliated Hospital of Zhengzhou University. Female BALB/c nude mice (4 mice per group; weight, 20.00±1.92 g), 4–6-weeks-old were obtained from Beijing Vital River Laboratories Animal Technology Co., Ltd. (Beijing, China) and were routinely housed in light-(12 h dark/12 h light) and temperature-controlled rooms (23°C). These mice were given free access to sterile food and water during the experiment process. HHUA cells (1×10⁷) were transfected with miR-494-3p inhibitors or controls resuspended in 200 µl FBS-free culture medium and subcutaneously injected into the right flanks of mice. miR-494-3p inhibitors or controls were injected into the formed tumor tissues every three days for 42 days. The tumor volume and weight were determined routinely following inoculation using direct measurement and calculated using the formula (length × width²)/2. The mice were then sacrificed; tumor tissues were obtained and tumor weights were determined.

Cell proliferation was examined using a Cell Counting Kit-8 (CCK8) assay (R&D Systems, Inc., Minneapolis, MN, USA) according to the manufacturer's protocols. Proliferation was determined through measuring absorbance at 450 nm using an ELx808 absorbance reader (BioTek Instruments, Inc., Winooski, VT, USA). And a colony formation assay was also conducted. In brief, 1,000 HHUA or JEC cells were seeded into 6-well plates and cultured for 14 days in DMEM medium at 37°C. Then the colonies were fixed with paraformaldehyde for 30 min at 25°C and stained with 0.1% crystal violet for 30 min at 25°C. Colony numbers were manually counted.

To examine cell migration, cells were plated onto a 24-well Transwell chamber (Corning Incorporated, Corning, NY, USA). A total of 2×104 cells were diluted in serum-free medium at 24 h post-transfection and subsequently inoculated onto the upper chamber. A total of 600 µl Dulbecco's modified Eagles medium (Sigma-Aldrich; Merck KGaA) with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) was added to the lower chamber at 37°C. Non-migratory cells were removed using a cotton swab following overnight incubation, while the migratory cells in the lower chamber were fixed with paraformaldehyde for 30 min at 25°C and stained with 0.1% crystal violet for 30 min at 25°C. A cell invasion assay was performed in a similar manner, but Matrigel (Collaborative Research, Bedford, MA, USA) was added to the upper chamber. Finally, the number of migrated and invasive cells were observed and counted using an optical microscope (magnification ×200, Nikon Corporation, Tokyo, Japan). Three random fields were counted per group.

Total RNA from tissues or cells was extracted using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocols. RNA (0.5 µg) was reverse transcribed using a PrimeScript RT Kit (Takara Bio, Inc., Otsu, Japan) according to the manufacturer's protocols. Then, the transcripts were analyzed on an ABI 7300 qPCR system (Applied Biosystem; Thermo Fisher Scientific, Inc.) using specific primers synthesized by Beijing Sunbiotech Co., Ltd. (Beijing, China) using the TaqMan™ MicroRNA Assay kit (Applied Biosystems; Thermo Fisher Scientific, Inc.) for miRNAs and the Fast SYBR™-Green Master Mix (Applied Biosystems; Thermo Fisher Scientific Inc.) for mRNAs. The thermocycling conditions were as follows: Denaturation at 95°C for 10 min; followed by 40 cycles of denaturation at 95°C for 15 sec and elongation at 60°C for 1 min. Three repearts were performed. Relative expression was calculated and normalized to endogenous β-actin (ACTB) or U6. Relative expression levels were evaluated via the 2^−ΔΔCq method (17). Primer sequences were as follows: miR-494-3p, forward, 5′-AACGAGACGACGACAGAC-3′ and reverse, 5′-TGAAACATACACGGGAAACCTC-3′; U6 forward, 5′-AACGAGACGACGACAGAC-3′ and reverse, 5′-GCAAATTCGTGAAGCGTTCCATA-3′; PTEN forward, 5′-TCCCAGACATGACAGCCATC-3′ and reverse, 5′-TGCTTTGAATCCAAAAACCTTACT-3′, and ACTB forward, 5′-CGGCGCCCTATAAAACCCA-3′ and reverse, 5′-GAGGCGTACAGGGATAGCAC-3′.

Total protein was isolated from cultured HHUA and JEC cells using radioimmunoprecipitation assay buffer (Thermo Fisher Scientific, Inc), and the supernatant was collected via centrifugation at 13,282 × g for 10 min at 4°C. Protein concentration was evaluated using a Pierce BCA Protein Assay kit (Pierce; Thermo Fisher Scientific, Inc.). Subsequently, the extracted protein was mixed with loading buffer and boiled at 100°C for 5 min. Total protein (30 µg) was separated using 10% SDS-PAGE gels and transferred onto polyvinylidene fluoride membranes (EMD Milipore, Billerica, MA, USA). Subsequently, the membranes were blocked with 5% (w/v) non-fat milk for 2 h at 25°C and incubated with antibodies against PTEN (1:2,000; cat. no. 9188), AKT (1:2,000; cat. no. 4691), phosphorylated-AKT (1:2,000; cat. no. 4060), PI3K (1:,2,000; cat. no. 4249), p-PI3K (1:2,000; cat. no. 4228), BCL2 (1:2,000; cat. no. 2872), caspase-3 (1:2,000; cat. no. 9664) or GAPDH (1:2,000; cat. no. 5174) (all from Cell Signaling Technology, Inc.) for 2 h at 25°C. The membranes were then probed with a horseradish peroxidase-conjugated secondary antibody (1:5,000; cat. no. ab7090; Abcam, Cambridge, UK) at 25°C for 1 h, and signals were visualized using an enhanced chemiluminescence kit (Beyotime Institute of Biotechnology, Beijing, China) according to manufacturer's protocol.

The potential binding site for miR-494-3p in PTEN 3′-UTR region was predicted using the TargetScan7 tool (www.targetscan.org/vert_71/). Cells were seeded on a 6-well-plate at a density of 1×10⁶ cells/well and transfected with 50 nM miR-494-3p mimics or negative controls, 100 ng pGL3-PTEN 3′-UTR-Wild type or Mutant vector and 1 ng pRL-TK Renilla luciferase plasmid (Promega Corporation, Madison, WI, USA) according to the manufacturer's protocols (Lipofectamine RNAiMAX, Invitrogen; Thermo Fisher Scientific, Inc.). After 48 h, luciferase assays were performed using the dual-luciferase reporter assay system (Promega Corporation) according to the manufacturer's protocols. Luminescent signals were quantified with a luminometer (Glomax, Promega Corporation), and the value of firefly luciferase was normalized to that of Renilla luciferase.

Statistical analysis was performed using SPSS 17.0 (SPSS Inc., Chicago, IL, USA). The differences among groups, in at least three separate experiments, were analyzed using a Student's t-test or one-way analysis of variance followed by Dunnett's multiple comparison test, as appropriate. The samples were divided into miR-494-3p low and high expression groups according to the median value of miR-494-3p. Then Kaplan-Meier survival analysis and log-rank test were used for survival evaluation. Spearman's rank correlation analysis was performed to analyze correlation between miR-494-3p and PTEN expression levels. P<0.05 was considered to indicate a statistically significant difference.

To investigate the function of miR-494-3p in EC, 43 EC samples were employed and the expression of miR-494-3p was determined by RT-qPCR in the present study. The results indicated that miR-494-3p was significantly upregulated in EC tissues compared with adjacent normal tissues (Fig. 1A). Then, these tissues were divided into low- or high-expression subgroups according to miR-494-3p expression, followed by Kaplan-Meier survival analysis. As presented in Fig. 1B, increased expression of miR-494-3p in patients with EC indicated poorer prognosis.

To further determine the effects of miR-494-3p on EC cells, miR-494-3p was overexpressed in EC cell lines (HHUA and JEC; Fig. 2A). Then, CCK8 and colony formation assays were performed using HHUA and JEC cells transfected with miR-494-3p mimics or controls. The results revealed that overexpression of miR-494-3p significantly promoted cellular proliferation and increased the number of the colonies compared with the control group (Fig. 2B and C). In addition, the effects of miR-494-3p on cellular apoptosis were evaluated by analysing the protein expression levels of caspase-3 and B-cell lymphoma-2 (Bcl-2). The results revealed that overexpression of miR-494-3p did not affect caspase-3 and Bcl-2 expression (data not shown), indicating that apoptosis was not affected by miR-494-3p. Tumor metastasis is one of the main causes of tumor malignancy (18). The effects of miR-494-3p on tumor cell metastasis were investigated using a Transwell assay. As presented, overexpression of miR-494-3p significantly promoted the migration and invasion of HHUA and JEC cells compared with the control group (Fig. 2D and E). Furthermore, the effects of miR-494-3p inhibition were evaluated by CCK8 and Transwell assays. miR-494-3p expression levels were significantly inhibited following transfection with miR-494-3p inhibitors (Fig. 2F). In addition, the results demonstrated that miR-494-3p inhibitor significantly suppressed the proliferation and invasion of HHUA and JEC cells compared with the control (Fig. 2G and H).

To further investigate the physiological function of miR-494-3p in vivo, a xenograft experiment was performed using HHUA cells. miR-494-3p-silenced or control HHUA cells were injected into nude mice. At indicative time points following injection, the tumor volumes were measured; miR-494-3p knockdown significantly delayed tumor growth in vivo compared with the control (Fig. 3A). In addition, the tumor weights were determined at the endpoints of experiments. As presented in Fig. 3B, miR-494-3p knockdown resulted in significantly reduced tumor weight compared with the control.

The miR-494-3p-regulated molecular mechanisms associated with EC were investigated. According to bioinformatic prediction, PTEN was identified to be a potential target gene of miR-494-3p. There were two conserved potential binding sites in the 3′-UTR of PTEN mRNA (Fig. 4A); then, luciferase reporter assays were conducted. As presented in Fig. 4B, overexpression of miR-494-3p significantly inhibited luciferase activity in HHUA and JEC cells compared with the control. Furthermore, the present study reported that overexpression of miR-494-3p significantly downregulated the mRNA expression levels of PTEN in HHUA and JEC cells compared with the control (Fig. 4C). Then, the expression levels of miR-494-3p and PTEN in EC tissues were evaluated. The results of RT-qPCR indicated that there was an inverse correlation between the expression levels of miR-494-3p and PTEN in EC tissues (Fig. 4D). A recent study revealed that the downstream signaling of PTEN comprised the PI3K/AKT pathway (8), which was widely involved in various human cancers, such as EC (19,20). Furthermore, the effects of miR-494-3p on PTEN protein expression and PI3K/AKT activation were evaluated using western blot analysis. Overexpression of miR-494-3p notably downregulated the protein expression levels of PTEN, and upregulated the phosphorylation of PI3K and AKT (Fig. 4E). In summary, the aforementioned data indicated that miR-494-3p activated the PI3K/AKT pathway by inhibiting PTEN in EC cells.

To determine whether miR-494-3p affects EC cell proliferation, migration and invasion via the PTEN/PI3K/AKT pathway, the protein expression of PTEN was restored or the PI3K/AKT pathway was inhibited using a specific inhibitor (LY294002) in miR-494-3p-overexpressed HHUA and JEC cells (Fig. 5A). Then, CCK8 and colony formation assays were performed to evaluate cellular proliferation. As presented in Fig. 5B and C, overexpression of miR-494-3p signficantly promoted cell proliferation compared with the control; however, restoration of PTEN or inhibition of the PI3K/AKT pathway abrogated the effects of miR-494-3p overexpression on EC cells. Similarly, overexpression of miR-494-3p significantly promoted cellular migration and invasion compared with the control; however restoration of PTEN or inhibition of the PI3K/AKT pathway in miR-494-3p-overexpressed HHUA and JEC cells reversed these effects (Fig. 5D and E). In summary, the results in the present study demonstrated that miR-494-3p promoted the progression of EC via the PTEN/PI3K/AKT signaling pathway.