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International Journal of Clinical and Experimental Pathology logoLink to International Journal of Clinical and Experimental Pathology
. 2017 Jul 1;10(7):7836–7846.

MicroRNA-590-5p promotes cell survival in endometrioid endometrial cancer by suppressing tumor suppressor PTEN

Miaohua Zhu 1, Xiaoming Zhou 1, Jing Zhang 1, Yingzhi Zhang 1
PMCID: PMC6965288  PMID: 31966631

Abstract

MicroRNAs (miRNAs) are known to be dysregulated in many tumors and associated with aggressive or poor prognosis phenotypes. miR-590-5p acts as an oncogene in a variety of human malignancies. However, its mechanism of action in endometrioid endometrial cancer (EEC) is poorly understood. In this study, we performed qRT-PCR to detect the miR-590-5p expression in EEC tissues, and found that miR-590-5p expression levels were significantly upregulated in EEC tissue specimens compared with the noncancerous endometrial tissues. Subsequently, we confirmed that knockdown of miR-590-5p inhibits cell proliferation, and induces cell cycle arrest and apoptosis, and activates the intrinsic apoptotic pathway including upregulating cleaved-caspase-3, Bax and cleaved-PARP. Most importantly, we identified that miR-590-5p inhibits phosphatase and tensin homolog (PTEN), a tumor suppressor gene by directly targeting its 3’-UTR. Meanwhile, our data showed that PTEN level in the cancer tissues was inversely correlated with miR-590-5p expression in 20 EEC patients. Furthermore, the tumor suppressive effects of miR-590-5p downregulation were rescued by knockdown of PTEN in EEC cells. These results demonstrated that miR-590-5p acts as an oncogene and positively regulates EEC cells by targeting PTEN, suggesting that suppression of miR-590-5p may be a novel approach for the treatment of EEC.

Keywords: miR-590-5p, endometrioid endometrial cancer, proliferation, apoptosis, cell cycle, PTEN

Introduction

Endometrial cancer (EC) is the most common gynecologic malignancy in the developed countries [1]. Statistical analysis has shown a higher incidence of EC than of cervical cancer (CC) in China, making it the leading form of female reproductive system cancer, with an increasing incidence in recent years [2]. Of these EC cases, 80-90% are endometrioid endometrial cancer (EEC) [3]. Although the integrated diagnosis and treatment provide significant insights into EEC, it still has some limitations including disease biology, morbidity and mortality. Therefore, better understanding of the molecular mechanism of EEC may be helpful to develop more effective therapies for the treatment of this disease.

Phosphatase and tensin homolog (PTEN) is a dual-specific phosphatase, dephosphorylating lipid and protein substrates [4]. It is a key molecule in the development of many diseases due to PTEN regulates cell proliferation, survival, apoptosis and metabolism through its target molecules, phosphoinositide-3 kinase (PI3K) and protein kinase B (AKT) [5,6]. PTEN is one of the most frequently altered tumor suppressor genes in a variety of human cancers [7,8]. PTEN upregulation can suppress cell proliferation and tumorigenicity [9,10], an observation attributed to the ability of PTEN to induce cell cycle arrest and apoptosis [11,12]. Previous study documented that functional inactivation of PTEN was associated with EEC initiation and progression, a 34-55% somatic mutation frequency of the PTEN gene with a 50-83% frequency of loss or decrease of the PTEN protein in EEC [13].

MicroRNAs (miRNAs) are a group of endogenous, non-coding, single-strand, small RNAs of 22-25 nucleotides, which serve as a unique regulator of gene expression at the posttranscriptional level by directly interacting with the 3’ untranslated region (UTR) of their target genes. Increasing evidence demonstrated that miRNAs are involved in a variety of biological and pathological processes, such as cellular proliferation, differentiation, apoptosis and carcinogenesis [14-16]. Recent study revealed that expression of at least 20-30% of human protein-coding genes is modulated by miRNAs [17]. Accumulating evidence demonstrated that aberrant expression of miRNAs was confirmed in a variety of human malignancies, such as human hepatocellular carcinoma (HCC) [18], lung cancer [19], human ovarian cancer [20], and is also associated with the clinical outcome of cancer patients [21,22]. They may function as tumor suppressors or oncogenes to play critical roles in carcinogenesis [23]. Previous study indicated that miR-590-5p was upregulated in renal cell carcinoma (RCC), and promoted tumorigenesis by regulating the expression of their target tumor suppressor gene, PBRM1 [24]. Jiang et al. revealed that miR-590-5p promotes proliferation and invasion in human HCC by directly targeting TGF-beta RII [25]. Recent study documented that miR-590-5p acts as an oncogene by targeting the close homologue of L1 (CHL1) gene and promotes CC proliferation [26]. However, the expression and mechanism of action of miR-590-5p were largely poor understood in EEC.

In present study, we investigated the biological function and molecular mechanism of miR-590-5p in EEC. We found that miR-590-5p was dramatically upregulated in EEC clinical specimens and cell lines as compared to normal tissues and cells. Knockdown of miR-590-5p represses cell proliferation, and induces cell cycle arrest and apoptosis. Most importantly, we further confirmed that miR-590-5p inhibits PTEN expression by directly targeting its 3’-UTR. Furthermore, the suppressive effects of miR-590-5p downregulation were rescued via knockdown of PTEN in EEC cells. These data provided a better understanding of the molecular mechanism of miR-590-5p in the initiation and progression of EEC.

Materials and methods

Patient tissue specimens and cell culture

20 EECs and 20 normal endometrial specimens were collected from patients who underwent surgical resection at the Affiliated Hospital of School of Medicine of Ningbo University (Ningbo, Zhejiang, China) between June 2014 and June 2015. None of the patients had received preoperative radio therapy or chemotherapy prior to surgical resection. The tumor specimens were independently confirmed by two pathologists. Fresh specimens were snap-frozen in liquid nitrogen and stored at -80°C immediately after resection for subsequent RNA extraction. The project protocol was approved by the China Medical University Ethics Committee. All patients provided written informed consent for the use of the tumor tissues for clinical research. The human EEC cell lines KLE, RL95-2, HEC-50B and Ishikawa, HEC-1A and one normal endometrial cell (ESC) were obtained from the Tumor Cell Bank of the Chinese Academy of Medical Science (Peking, China). All cell lines were maintained in DMEM, supplemented with streptomycin (100 IU/ml), penicillin (100 IU/ml, Sigma, St. Louis, MO), 2 mM glutamine, and 10% fetal bovine serum (FBS, Gibco BRL, Grand Island, NY).

RNA extraction and quantitative real-time PCR

Total RNA was extracted from EEC tissue samples and culture cells using fTRIzol Reagent (Invitrogen) according to the manufacturer’s instructions and then both miRNA and mRNA were reverse transcribed to cDNA with the Reverse Transcriptase M-MLV kit (Takara, China). The miR-590-5p primers were purchased from Ribobio (Guangzhou, China). The U6 gene was used as a reference control for miR-590-5p. Real-time qRT-PCR was carried out on an Applied Biosystems 7500 Real-Time PCR machine with miRNA-specific primers by TaqMan Gene Expression Assay (Applied Biosystems). All reactions were performed in triplicate. The 2-ΔΔCt method was conducted to analyze the miR-590-5p relative expression.

Cell transfection

Mature miR-590-5p inhibitor and inhibitor negative control (NC) were designed and chemically synthesized by GenePharma (Shanghai, China). The small interfering RNAs (siRNA) were synthesized by BioMics. The ECC cell lines HEC-1A or Ishikawa were seeded into six-well plates and incubated overnight. When cells were grown to 60-80% confluence, the miR-590-5p inhibitor/inhibitor NC or siRNA was transfected into cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. About 48 h after transfection, the transfection efficiency was assessed using a Leica DMIRE2 microscope system (Leica Microsystems, Montreal, QC, Canada). 48 h after transfection, cells were harvested for cell proliferation, cycle, apoptosis and Western blot analysis.

Cell counting Kit-8 assay

The proliferation of cells was measured by the Cell Counting Kit-8 (CCK-8) assay according to the manufacturer’s instructions. After transfection with miR-590-5p inhibitor or inhibitor NC into HEC-1A or Ishikawa cells, cells (5 × 104 cells/well) were seeded in 96-well plate with 100 μl DMEM medium supplemented with 10% FBS. After 48 h incubation, 10 μl of CCK-8 reagent dissolved with 100 µl DMEM was added to each well and continuously cultured for 1 h in 5% CO2 (Thermo). The absorbance rate at 450 nm was measured by Microplate Reader (Bio-Rad, USA). All experiments were performed in quintuplicate on three separate occasions.

Cell cycle analysis

The HEC-1A or Ishikawa cells were seeded into 6-well plates at a concentration of 5 × 104 cells/well the day before transfection. After the transfection with miR-590-5p inhibitor or inhibitor NC, the cells were incubated for 72 h. For flow cytometric analysis (FACSCalibur, BD Biosciences), the cells were prepared by using the BD CycletestTM Plus DNA Reagent Kit (BD Biosciences) according to manufacturer’s instructions. The G0/G1 and G2/M ratios were calculated by using analysis software (Cell Quest, BD Biosciences).

Apoptosis analysis

About 48 h after transfection, 1 × 106 cells were collected and washed twice with Hepes-buffered saline. After treatment with trypsin, cells were fixed with 70% ice-cold methanol at 4°C for 30 min. Cells were then resuspended in binding buffer and stained with 5 μl of Annexin V-FITC (BD, Mountain View, CA, United States) and 1 μl of propidium iodide (PI, 50 μg/ml) (BD, Mountain View, CA, United States). Flow cytometric evaluation was performed within 5 min. Stained cells were analyzed using flow cytometry (BD, FACSCalibur, CA, United States). The measurements were performed independently for at least three times with similar results.

Western blot analysis

48 h after transfection, total protein of cultured cells was extracted using RIPA buffer with protease inhibitor Cocktail (Pierce, Rockford, IL, USA). BCA protein assay kit (Beyotime, Haimen, China) was used to detect the concentration. Total proteins (20 μg) were separated on 10% SDS-PAGE (Sigma Aldrich, St. Louis, MO) and then transferred onto polyvinylidene difluoride (PVDF) membranes (BD Pharmingen, San Diego, CA). The membranes were then blocked with a blocking buffer (5% non-fat dry milk in 1 × TBST, i.e. 20 mM Tris-HCl, pH 7.6 containing 0.8% NaCl and 0.1% Tween-20) at room temperature for 1 h. Subsequently, the PVDF membranes were incubated with primary antibodies against cleaved-caspase-3 (1:1000, Cell Signaling, Danvers, MA, USA), Bax (1:1000, Cell Signaling), cleaved-PARP (1:1000, Cell Signaling Technology, Beverley, MA, USA), PTEN (1:1000, Cell Signaling) at 4°C overnight, β-actin (1:1000, Sigma, St. Louis, MO) was used as an internal control for protein loading. The goat anti-mouse IgG horseradish peroxidase antibody was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Thereafter, the protein bands were visualized on the X-ray film using the enhanced chemiluminescence detection system (PerkinElmer Life and Analytical Sciences, Boston, MA). The gels shown in figures are representatives of results from three separate experiments.

Luciferase reporter assay

The possible binding site between PTEN and miR-590-5p was searched in TargetScan (http://www.targetscan.org). The miR-590-5p mimics and NC mimics were designed and synthesized GenePharma (Shanghai, China). The fragment of the 3’-UTR of PTEN (wild-type or mutant, respectively) was amplified and cloned into the pMIR-REPORT luciferase vector (Ambion, USA). All PCR products were verified by DNA sequencing. For the luciferase assay, HEC-1A cells at a density of 2 × 105 per well were seeded into 24-well plates and co-transfected with 0.8 μg of pMIR-PTEN-3’-UTR or pMIR-PTEN-mut-3’-UTR, 50 nM miR-590-5p mimic or corresponding mimic NC using Lipofectamine 2000 reagent (Invitrogen). The relative firefly luciferase activity normalized with Renilla luciferase was measured 48 h after transfection by using the Dual-Light luminescent reporter gene assay (Applied Biosystems). All experiments were repeated three times in triplicate.

Statistical analysis

All statistical analysis was performed using SPSS 14.0 software (Chicago, IL). Numerical data presented as the mean ± standard deviation. The difference between means was analyzed with Student’s t test. Probability value of < 0.05 was considered significant and < 0.01 was considered very significant.

Results

miR-590-5p is upregulated in EEC tissues and cell lines

To clarify the biological role of miR-590-5p in EEC, we detected the expression of miR-590-5p using qRT-PCR in 20 pairs of human EEC tissue specimens and their normal endometrial specimens. We observed that the miR-590-5p expression levels were significantly upregulated in EEC tissue specimens compared with the noncancerous endometrial tissues (P < 0.01; Figure 1A). To further verify this differential expression of miR-590-5p, we also measured the miR-590-5p level in five kinds of EEC cell lines (KLE, RL95-2, HEC-50B and Ishikawa and HEC-1A) and one normal endometrial cell (ESC). Consistent with the results in EEC tissues, miR-590-5p expression levels were higher in all EEC cell lines than that ESC cell (P < 0.01; Figure 1B). These results indicated that miR-590-5p is increased in EEC tissues and may be serve as oncogene involved in endometrial carcinogenesis.

Figure 1.

Figure 1

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miR-590-5p expression is upregulated in endometrioid endometrial cancer (EEC) tissues and cell lines. A. miR-590-5p expression was quantified by qRT-PCR analysis in 20 pairs of human EEC tissue specimens and their normal endometrial specimens. B. qRT-PCR analysis of miR-590-5p expression in EEC cell lines (KLE, RL95-2, HEC-50B and Ishikawa and HEC-1A) and one normal endometrial cell (ESC). Results were normalized against the expression level of U6 messenger RNA (mRNA) in each sample. **P < 0.01 vs control.

Knockdown of miR-590-5p represses EEC cells growth

Among the five EEC cell lines analyzed, the HEC-1A and Ishikawa cells showed relatively higher levels of miR-590-5p expression. Subsequently, these two cell lines were selected for further study. To explore the role of miR-590-5p in proliferation and cycle of EEC cells in vitro, the HEC-1A and Ishikawa cells were transfected with miR-590-5p inhibitor or inhibitor NC and performed CCK-8 assay and flow cytometric analysis to measure cell proliferation and cycle, respectively. Cells did not receive transfection as blank control. The inhibitory effect of miR-590-5p inhibitor was assessed using qRT-PCR. We found that the miR-590-5p levels were dramatically decreased in HEC-1A and Ishikawa cells following transfection with miR-590-5p inhibitor when compared with NC (P < 0.01; Figure 2A and 2B). The CCK-8 assay showed that cells transfected with miR-590-5p inhibitor markedly reduced cell viability compared with NC (P < 0.01; Figure 2C and 2D). In consistent with this result, we confirmed the effect of miR-590-5p inhibitor on cell cycle. As shown in Figure 2E and 2F, downregulation of miR-590-5p resulted in a marked delay in the ability of arrested cells to progress beyond the G2/M phase block. The data indicated that knockdown of miR-590-5p suppresses cell growth through inhibiting cell proliferation and inducing G2/M phase cell-cycle arrest.

Figure 2.

Figure 2

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Knockdown of miR-590-5p represses EEC cells growth. A and B. The EEC cell lines HEC-1A and Ishikawa cells were transfected with miR-590-5p inhibitor or inhibitor NC and performed qRT-PCR to determine miR-590-5p expression. C and D. The EEC cell lines HEC-1A and Ishikawa cells were transfected with miR-590-5p inhibitor or inhibitor NC and performed CCK-8 assay to measure cell proliferation, respectively. E and F. The cell cycle was measured using flow cytometric analysis in HEC-1A and Ishikawa cells following transfection with miR-590-5p inhibitor or inhibitor NC, respectively. **P < 0.01 vs control.

Knockdown of miR-590-5p induces cell apoptosis

To further identify the potential mechanisms underlying the inhibitory effects of miR-590-5p inhibitor on the apoptosis of EEC cell lines, we analyzed its impact on apoptosis using flow cytometric analysis in HEC-1A and Ishikawa cells. Compared with the cell transfected with inhibitor NC, downregulation of miR-590-5p led to a significant increase in cell apoptosis (P < 0.01; Figure 3A). Subsequently, we performed Western blot to measure cleaved-caspase-3, Bax and cleaved-PARP protein expression levels after transfection with miR-590-5p inhibitor or inhibitor NC in HEC-1A and Ishikawa cells. The protein expression of cleaved-caspase-3, Bax and cleaved-PARP were significantly increased by miR-590-5p inhibitor compared with NC (P < 0.01; Figure 3B and 3C). Taken together, these results suggested that knockdown of miR-590-5p induces cell apoptosis by enhancing the expression levels of cleaved-caspase-3, Bax and cleaved-PARP.

Figure 3.

Figure 3

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Knockdown of miR-590-5p induces cell apoptosis. A. The EEC cell lines HEC-1A and Ishikawa cells were transfected with miR-590-5p inhibitor or inhibitor NC and performed flow cytometric analysis to measure apoptotic cells. B and C. Western blot analysis was used to detect cleaved-caspase-3, Bax and cleaved-PARP protein expression levels after transfection with miR-590-5p inhibitor or inhibitor NC in HEC-1A and Ishikawa cells, respectively. β-actin was used as an internal control for protein loading. **P < 0.01 vs control.

miR-590-5p suppresses PTEN expression by directly targeting its 3’-UTR

We further predicted the target genes of miR-590-5p using TargetScan, and identified PTEN as a potential target of miR-590-5p. To verify this bioinformatic predication, the wild type or mutant type of PTEN-3’-UTR was constructed and inserted into the firefly luciferase expressing vector pMIR-REPORT. The reporters were co-transfected with either miR-590-5p mimic or mimic NC to HEC-1A cells, and measured the luciferase activity. We found that miR-590-5p mimics dramatically reduced the luciferase activity compared with the mimic NC in the presence of the wild-type 3’-UTR (P < 0.01; Figure 4B), whereas miR-590-5p did not reduce the luciferase activity of the reporter vector containing 3’-UTR of PTEN with mutations in the miR-590-5p-binding site (Figure 4B). To further verify that the PTEN expression is regulated by miR-590-5p, we transfected HEC-1A and Ishikawa cells with either miR-590-5p mimic or mimic NC and performed qRT-PCR and Western blot analysis to determine the mRNA and protein level for PTEN, respectively. Our results showed that upregulation of miR-590-5p significantly suppresses the mRNA and protein level of PTEN in human EEC cell lines tested compared with the mimic NC (P < 0.01; Figure 4C and 4D). To explore the association between miR-590-5p and PTEN in EEC patients, their expressions in EEC patient tissues were quantified using qRT-PCR. We observed that PTEN level in the cancer tissues was inversely correlated with miR-590-5p expression in 20 EEC patients (R2 = -0.8242, P < 0.01; Figure 4E). These data collectively suggested that miR-590-5p exerts an inhibitory effect on PTEN expression by directly targeting its 3’-UTR in EEC cells.

Figure 4.

Figure 4

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miR-590-5p represses PTEN expression by directly targeting its 3’-UTR. A. The PTEN 3’-UTR region containing the wild type or mutant binding site for miR-590-5p. B. The relative luciferase activity of PTEN wild type or mutant 3’-UTR in HEC-1A cells after transfection with the miR-590-5p mimic or corresponding mimic NC. C. The PTEN mRNA level was measured using qRT-PCR in HEC-1A cells transfected with the miR-590-5p mimic or corresponding mimics NC. D. Western blot was conducted to detect the protein level of PTEN in HEC-1A and Ishikawa cells transfected with miR-590-5p mimic or corresponding mimic NC, β-actin was used as an internal control. E. miR-590-5p expression and PTEN level in EEC tissues showed an inverse correlated trend from 20 EEC patients (R2 = -0.8242). **P < 0.01 vs control.

Knockdown of PTEN rescues the suppressive effects of miR-590-5p downregulation on EEC cells

To investigate whether PTEN knockdown can rescue the suppressive effect of miR-590-5p downregulation, HEC-1A and Ishikawa cells were transfected with miR-590-5p inhibitor (miR-590-5p inhibitor group) or were transfected with miR-590-5p inhibitor and si-PTEN (miR-590-5p inhibitor + si-PTEN group). The cell which did not receive transfection was serviced as control group. Subsequently, we performed CCK-8 assay to determine the cell proliferation in each group. We found that miR-590-5p downregulation significantly inhibited cell proliferation in HEC-1A and Ishikawa cells, but knockdown of PTEN led to a marked increase in cell proliferation after co-transfection with miR-590-5p inhibitor and si-PTEN (Figure 5A and 5B). To further verify this result, the flow cytometric analysis was conducted to measure cell apoptosis. As expected, miR-590-5p downregulation also markedly increased apoptotic ratio compared with control group, but the promotion effects of miR-590-5p downregulation on EEC cells apoptosis were rescued after co-transfection with miR-590-5p inhibitor and si-PTEN (Figure 5C and 5D). Taken together, these data indicated that miR-590-5p functioned as oncogene in EEC cells through suppressing PTEN.

Figure 5.

Figure 5

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The suppressive effects of miR-590-5p downregulation on EEC cells were abrogated by knockdown of PTEN. HEC-1A and Ishikawa cells were transfected with miR-590-5p inhibitor (miR-590-5p inhibitor group) or were transfected with miR-590-5p inhibitor and si-PTEN (miR-590-5p inhibitor + si-PTEN group). The cell which did not receive transfection was serviced as control (control group). A and B: The CCK-8 assay was used to determine the proliferation in HEC-1A and Ishikawa cells, respectively. C and D: The flow cytometric analysis was conducted to measure apoptotic cells in HEC-1A and Ishikawa cells, respectively. **P < 0.01 vs control. ##P < 0.01 vs miR-590-5p inhibitor group.

Discussion

It has been well reported that miRNAs can function as tumor suppressors or oncogenes to play important roles in the initiation, promotion and development of various cancers [27] and aberrant expression of miRNAs might be of potential use as a diagnostic and prognostic biomarker for human cancer including EEC [28]. Previous study confirmed that a set of EEC-associated miRNAs in tissue and plasma of EEC patients were identified using next-generation sequencing, such as miR-499, miR-135b, miR-205, miR-10b, miR-195, miR-30a-5p, miR-30a-3p and miR-21, which were associated with pathological characteristics, and could distinguish EEC from normal endometrium samples with high accuracy [28]. Increasing evidence demonstrated that miR-590-5p acts as an oncogene in a variety of human malignancies, such as RCC [24], HCC [25] and cervical cancer (CC) [26]. However, the role of miR-590-5p in EEC has yet to be elucidated. In the present study, we confirmed that miR-590-5p was obviously upregulated in EEC clinical specimens and cell lines as compared to normal tissues and cells. Moreover, downregulation of miR-590-5p significantly suppressed cell growth in EEC cells through inhibiting cell proliferation and inducing cell cycle arrest and apoptosis. Additionally, our results demonstrated that miR-590-5p acted as oncogene in EEC cells via inhibiting a tumor suppressor gene, PTEN. These findings provided new insights to understand the carcinogenic functions of miR-590-5p in EEC.

Previous studies revealed that miR-590-5p was upregulated in human ovarian cancer [29], and acted as an oncogene through targeting CHL1 gene in CC [26]. Thus, we speculate that miR-590-5p may act as an oncogene in EEC. In this study, we identified miR-590-5p has significant oncogenic activity. We performed qRT-PCR to detect the expression of miR-590-5p in 20 pairs of human EEC tissue specimens and their normal endometrial specimens. Our data showed that the miR-590-5p expression levels were significantly upregulated in EEC tissue specimens compared with the noncancerous endometrial tissues. Moreover, miR-590-5p upregulation was further confirmed in EEC cells, which suggested miR-590-5p may play the role of oncogene in EEC. To elucidate its functional role in EEC cells, we performed CCK-8 assay and flow cytometric analysis to detect miR-590-5p inhibitor on cell proliferation and cycle. We observed that knockdown of miR-590-5p represses cell proliferation and induced cell cycle arrest. Meanwhile, our further study confirmed that knockdown of miR-590-5p promotes cell apoptosis and activates the intrinsic apoptotic pathway via upregulating cleaved-caspase-3, Bax and cleaved-PARP. These results indicated that miR-590-5p acts as an oncogene in EEC and miR-590-5p downregulation inhibits EEC cell growth and induces cell apoptosis. Our report is consistent with the previous studies showing the oncogene role of miR-590-5p in other types of tumors [24-26]. However, the possible molecular mechanism need further research to be understood deeply.

PTEN, a tumor suppressor gene, plays an important role in the regulation of the cell cycle, apoptosis and formation of many types of solid tumors [30], and its tumor suppressor activity is dependent on its lipid phosphatase activity, which negatively regulates the PI3K/AKT/mTOR pathway [31]. Recent study revealed that PTEN expression level was significantly downregulated in the EEC tumor tissues compared with the non-tumor tissues [3], and the functional inactivation of PTEN was associated with EEC initiation and progression [13]. miRNAs have been demonstrated to regulate the expression of PTEN in tumorigenesis or metabolic disorders [31]. Qin et al. demonstrated that overexpression miR-21 regulates EEC cell proliferation through suppressing PTEN expression [3]. In present study, we performed bioinformatic analysis to predicate the putative targets of miR-590-5p, and found that PTEN might be a potential target of miR-590-5p. Our data showed that miR-590-5p represses the protein and mRNA levels for PTEN by targeting its 3’-UTR in EEC cell. Moreover, our results demonstrated that PTEN level in the cancer tissues was inversely correlated with miR-590-5p expression in 20 EEC patients. To further investigate whether the suppressive effect of miR-590-5p downregulation on EEC cell via modulating PTEN expression level, we performed CCK-8 assay and flow cytometric analysis to determine cell proliferation and apoptosis after downregulation of miR-590-5p and PTEN in EEC cell. We found that knockdown of miR-590-5p significantly inhibited cell proliferation and induced cell apoptosis, but the suppressive effects of miR-590-5p downregulation on EEC cell was rescued by knockdown of PTEN after co-transfection with miR-590-5p inhibitor and si-PTEN. Taken together, these data suggested that miR-590-5p overexpression contributes to cell proliferation by targeting PTEN in EEC.

In conclusion, our study demonstrated that miR-590-5p was overexpressed in EEC and acts as oncogene through targeting a tumor suppressor gene, PTEN. These findings suggested that knockdown of miR-590-5p alone or in conjunction with other antitumor treatments may represent a novel effective therapeutic intervention to prevent progression of EEC in the future.

Acknowledgements

The study was supported by the Medicine and Health Science and Technology Planning Project of Zhejiang Province (Grant No.: 2014KYB239).

Disclosure of conflict of interest

None.

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