Effects of miR‑106b‑3p on cell proliferation and epithelial‑mesenchymal transition, and targeting of ZNRF3 in esophageal squamous cell carcinoma.

Previous studies have demonstrated that the dysregulation of microRNAs (miRs) is frequently associated with cancer progression. Deregulation of miR‑106b‑3p has been observed in various types of human cancer. However, the biological function of miR‑106b‑3p in esophageal squamous cell carcinoma (ESCC) remains unclear. Thus, the aim of this study was to investigate the role of miR‑106b‑3p in ESCC. In the current study, the results indicated that miR‑106b‑3p was upregulated in ESCC cell lines and tissues. An increase in miR‑106b‑3p using miR mimics significantly promoted the proliferation of ESCC cells in vitro. Furthermore, the results demonstrated that miR‑106b‑3p overexpression promoted migration, invasion and epithelial‑mesenchymal transition (EMT) of ESCC cells. In addition, zinc and ring finger 3 (ZNRF3) was identified as a target of miR‑106b‑3p in ESCC cells, and the ZNRF3 expression level was inversely associated with miR‑106b‑3p. It was also demonstrated that miR‑106b‑3p has a role in EMT by regulating Wnt/β‑catenin signaling pathway in ESCC. In conclusion, these data suggested that miR‑106b‑3p promotes cell proliferation and invasion, partially by downregulating ZNRF3 and inducing EMT via Wnt/β‑catenin signaling in ESCC cells. Thus, miR‑106b‑3p and ZNRF3 may be novel molecular targets for the future treatment of ESCC.

Introduction

Esophageal cancer (EC) is one of the most common malignant tumors worldwide. It consists of two histological types, adenocarcinoma (EAC) and esophageal squamous cell carcinoma (ESCC), each with distinct etiological and pathological characteristics. ESCC is the major histological type of EC in China (1). Despite advances in the treatment of ESCC, including surgery, chemotherapy, radiation or a combination of these options, the prognosis of patients with ESCC remains poor, with an overall 5-year survival rate of 20% following surgery (2). To improve the overall outcome for patients with ESCC, using molecular targets to treat cancer has brought promising results and attracted increasing attention (3-5). The molecular mechanisms may lead to the identification of new biomarkers and novel therapeutic targets.

MicroRNAs (miRNAs) are ~22-nucleotide, single-stranded, small non-coding RNAs that modulate gene expression by binding to the 3' untranslated region (3'-UTR) of their target mRNAs to repress protein translation or promote mRNA degradation (6). It serves key roles in various physiological processes, including cell proliferation, differentiation, apoptosis, invasion, metastasis and maturation (7). miRNA (miR)-106b-3p has been reported to be deregulated in some types of cancer, including increased in laryngeal carcinoma (8), bladder cancer (9), hepatocellular carcinoma (10) and renal cell carcinoma (11). However, the role of miR-106b-3p in ESCC is unclear.

Epithelial-mesenchymal transition (EMT) is a complex biological process. Specifically, following EMT, cells have increased expression of mesenchymal molecules, including Snail, Slug and Vimentin, and decreased expression of the epithelial adhesion marker E-cadherin, leading to enhanced motility and metastasis (12). The activation of Wnt/β-catenin is a key step in the process of tumorigenesis (13). Increasing evidence has demonstrated that aberrant activation of the Wnt/β-catenin signaling pathway promotes tumor progression in various types of human cancer (14-17). Zinc and ring finger 3 (ZNRF3) is part of the E3 ubiquitin ligase family, which negatively regulates Wnt/β-catenin signaling (18). ZNRF3 inhibits Wnt signaling by promoting the turnover of Frizzled and Low-density lipoprotein receptor-related protein 6 (18). Accumulating data have strongly demonstrated that ZNRF3 is involved in the process of tumorigenesis (19-21). β-catenin is a dual function protein that regulates coordinated cell-cell adhesion and gene transcription. β-catenin has two sub-cellular locations, either bound to E-cadherin on the cell membrane in adherens junctions in intercalated disc structures, or free in the cytoplasm. β-catenin regulates gene expression via the Wnt/β-catenin signaling pathway, inducing transition from an epithelial phenotype to a mesenchymal one.

Our previous studies have found that miR-106b-3p is upregulated in ESCC tissues. Furthermore, miR-106b-3p has been reported to be deregulated in certain types of cancer, including in laryngeal carcinoma, bladder cancer, hepatocellular carcinoma and renal cell carcinoma (8-11). However, the role of miR-106b-3p in ESCC is unclear. Therefore, the aim of the current study was to explore the potential mechanism of miR-106b-3p in cell proliferation and EMT of ESCC via ZNRF3 and the Wnt/β-catenin signaling pathway, and to determine its utility in ESCC diagnosis and therapy.

Patients and methods

Patient samples

A total of 50 paired ESCC tumor tissues and matched normal adjacent tissues were collected at the First Affiliated Hospital of Soochow University (Suzhou, China) between July 2016 and August 2017 (mean age 57±12 years; 21 female patients and 29 male patients). All the samples were histologically confirmed to be ESCCs esophageal hyperplasia, dysplasia or non-malignant tissues by the pathologist (22). All samples were immediately stored at -80°C. This study was approved by the Ethics Committee of Nanjing Medical University and each patient provided written informed consent.

Cell lines and cell culture

The three human ESCC cell lines (KYSE150, ECA-109 and EC9706) and normal epithelial cell line (HET-1A) were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). All cell lines were maintained in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) at 37°C in a humidified chamber with 5% CO2.

Cell transfection

Cells were transfected with miR-106b-3p mimics, miR-106b-3p inhibitors and NC using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) at a final concentration of 50 nM according to the manufacturer's instructions. The sequences were as follows: miR-106b-3p mimics, 5'-TAA AGT GCT GAC AGT GCA GAT-3'; miR-106b-3p inhibitors, 5'-AUC UGC ACU GUC AGC ACU UUA-3'; NC, 5'-CAG UAC UUU UGU GUA GUA CAA-3'. Subsequently, cells were cultured with fresh medium containing 10% FBS for 48 h. Transfection efficiencies were validated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR).

Total RNA extraction and RT-qPCR

Isolation of total RNA from samples was conducted using TRIzol reagent (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. cDNA was synthesized using the SYBR® PrimeScript™ RT-PCR kit (Takara Biotechnology Co., Ltd., Dalian, China) for 30 min at 37°C according to the manufacturer's instructions. ZNRF3 and miR-106b-3p expression was determined by RT-qPCR using SYBR Premix ExTaq (Takara Biotechnology Co., Ltd., Dalian, China) reagents. The primers used were as follows: Forward, 5'-CAT CGT CAA CAA GCA GAA AGT G-3' and reverse, 5'-GGA GAC CAC GAC GAA-GAA AG-3' for ZNRF3; forward, 5'-TTT TCG CCC TTA GCG TGA AGA-3' and reverse, 5'-GAG GCA GTC GAA TTT GCG T-3' for β-actin. The thermocycling conditions for qPCR were as follows: 95°C for 5 min followed by 40 cycles of 95°C for 15 sec and 60°C for 60 sec. All of the samples were normalized with small nuclear RNA U6, the relative quantification and statistical analysis was performed using the 2-ΔΔCq method (23). The experiments were repeated at least three times.

Western blot analysis

Total protein was extracted using radioimmunoprecipitation assay lysis buffer supplemented with protease inhibitors (Gibco; Thermo Fisher Scientific, Inc.). The protein concentration was measured using the bicinchoninic acid method. Protein (50 µg/well) was separated on 10% SDS-polyacrylamide gels and then blotted onto polyvinylidene difluoride membranes (EMD Millipore, Bedford, MA, USA). Subsequently, membranes were blocked with 5% skimmed milk at room temperature for 1 h, and then incubated with primary antibodies at 4°C overnight. The specific primary antibodies used were as follows: ZNRF3 (cat. no. ab122353; 1:1,000 dilution), p21 (cat. no. ab109520; 1:1,000 dilution), p27 (cat. no. ab32034; 1:1,000 dilution), cyclin D1 (cat. no. ab16663; 1:1,000 dilution), Slug (cat. no. Ab27568; 1:1,000 dilution), Snail (cat. no. Ab53519; 1:1,000 dilution), N-cadherin (cat. no. Ab76057; 1:1,000 dilution), E-cadherin (cat. no. Ab1416; 1:1,000 dilution), Vimentin (cat. no. Ab8978; 1:1,000 dilution) and GAPDH (cat. no. ab9485; 1:1,000 dilution; all Abcam, Cambridge, MA, USA). Then, the membranes were washed with PBS three times and incubated with horseradish peroxidase-conjugated secondary antibody (cat. no. ab6721; 1:2,000; Abcam) at room temperature for 1 h. GAPDH used as the internal reference protein. Signals were detected using an enhanced chemiluminescence kit (GE Healthcare, Chicago, IL, USA).

Cell proliferation and colony formation assays

Cell proliferation ability was detected using an MTT assay. Briefly, 1×106 cells (KYSE150 and ECA-109) were seeded into 96-well plates and cultured. MTT solution (10 µl) was added to each well, followed by incubation for 4 h. The culture medium discarded and 150 µl dimethyl sulfoxide (DMSO; Gibco; Thermo Fisher Scientific, Inc.) was added. Then the mixture was gently shaken for 15 min and the cells were evaluated at 24, 48, 72 and 96 h, after seeding. The absorbance was measured at 490 nm using a microplate reader. For the colony formation assay, 500 cells per well were seeded into 6-well plates. After incubation for 3 weeks, cells were fixed with 4% paraformaldehyde at room temperature for 20 min. Finally, the cells were stained with 1% crystal violet for 4 h at room temperature and counted.

Flow cytometry

The effects of miR-106b-3p on cell cycle were explored by flow cytometry in ESCC. After transfection for 48 h, 1×106 cells were seeded into 6-well plates and washed twice with PBS. Subsequently, cells were digested with 0.25% trypsin and fixed with 70% ethanol at -4°C overnight. Finally, 500 ml PI/RNase was added to cells and incubated in the dark for 45 min at 37°C. The multiplication cycle was detected using flow cytometry (FACSCalibur; BD Biosciences, San Jose, CA, USA).

Cell adhesion assay

Approximately 1×105 cells were harvested and resuspended in culture medium and then transferred to 96-well plates were pre-coated with collagen I, collagen IV and fibronectin (Sigma-Aldrich; Merck KGaA). Following incubation for 2 h at room temperature, the non-adherent cells were removed by washed twice with PBS. The adhesive cells were fixed in 4% paraformaldehyde and then stained with 0.5% crystal violet. The cells were examined under a light microscopy.

Wound healing assays

Cells which were transfected with miR-106b-3p mimics, miR-106b-3p mimics NC, miR-106b-3p inhibitors or miR-106b-3p inhibitors NC were cultured to 80-90% confluence in 24-well plates in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS. Linear scratch wounds were created on the confluent cell monolayers with a pipette tip after transfection for 24 h. Images were captured at 0, 24 and 48 h under an inverted microscope (magnification, ×100).

Transwell assay

Cell migration and invasion assays were performed using Transwell chambers. Cells were seeded in 24-well plates and harvested. For invasion or migration assays, the upper chamber was first coated with or without Matrigel, respectively, then 1×105 KYSE150 or ECA-109 cells were seeded in the upper chamber containing fresh serum-free culture media and supplemented medium was added to the lower chamber containing 20% FBS. The cells on the upper surface were gently removed after culturing for 24 h. Migrant cells attached to the lower surface were fixed with 4% paraformaldehyde for 30 min at room temperature and stained with 0.1% crystal violet for another 30 min at room temperature. Cells were counted under a microscope in four randomly selected fields (magnification, ×200). All experiments were conducted three times.

Immunofluorescence

Cells (1×105 cells/well) were seeded on 6-well plates and incubated for 48 h. Subsequently, KYSE150 and ECA-109 cells were fixed with 4% paraformaldehyde for 30 min at room temperature and incubated with the primary antibody (ZNRF3; cat. no. ab122353; 1:1,000 dilution; Abcam) at room temperature for 2 h after blocking cell with 3% bovine serum albumin (Gibco; Thermo Fisher Scientific, Inc.). Fluorescein isothiocyanate-conjugated secondary antibody (cat. no. ab205718; 1:2,000; Abcam) was incubated with cells for 1 h at room temperature. The nuclei were counted and stained with DAPI for 5 min at room temperature. The slides were examined under a fluorescence microscopy.

Dual-luciferase reporter assays

A search for putative targets of miR-183 was performed with TargetScan Human7.2 (www.targetscan.org/vert_72/) and miRanda (www.microrna.org/) software. The targeting gene (ZNRF3) of miR-106b-3p was predicted using microRNA.org (http://www.microrna.org) and verified in a dual-luciferase reporter gene assay. The wild-type 3'-UTR of human ZNRF3 mRNA was amplified from human genomic DNA containing miR-106b-3p binding site inserted into the SpeI/HindIII sites of pMIR-REPORT™ (Promega Corporation, Madison, WI, USA) luciferase reporter plasmid to generate pMIR-ZNRF3-WT plasmid which were designed and synthesized by Invitrogen (Thermo Fisher Scientific, Inc.) The complementary sequence for miR-106b-3p seed sequence in ZNRF3 3'-UTR was mutated and named as pMIR-ZNRF3-MUT plasmid. The 10 nM pMIR-ZNRF3-MUT or pMIR-ZNRF3-WT and PRL-TK-Renilla were co-transfected in KYSE150 and ECA-109 cells with 10 nM miR-106b-3p mimic and negative control using Lipofectamine® 3000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.). After 48 h, cells were lysed and assayed for luciferase activity using a dual luciferase reporter assay (Promega Corporation). Normalized luciferase activity was reported as luciferase activity/Renilla luciferase activity.

Statistical analysis

All data are presented as the mean ± standard deviation. SPSS 21.0 statistical software (IBM Corp., Armonk, NY, USA) was used to explore the statistical analysis. Comparisons between two groups were conducted using two-tailed Student's t-test and multiple group comparisons were conducted via one-way analysis of variance with Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

Results

miR-106b-3p is upregulated in ESCC tissues and cell lines

The expression of miR-106b-3p in 50 paired ESCC tissues and non-tumor tissues was detected by RT-qPCR (Fig. 1A). We found tThat the expression levels of miR-106b-3p were significantly up-regulated in ESCC tissues compared to with non-tumor tissues. Furthermore, the expression of miR-106b-3p in ESCC cell lines (KYSE150, ECA-109, EC9706) was significantly increased compared with the normal epithelial cell line HET-1A (Fig. 1B). ZNRF3 expression was determined by western blot analysis and immunofluorescence (Fig. 1C and D). The proliferation abilities of cell lines were performed by MTT and colony formation assays (Fig. 1E and F). These results suggested that miR-106b-3p may function as a regulator in the progression of ESCC.

Figure 1

miR-106b-3p is upregulated in ESCC tissues and cell lines. (A) Expression of miR-106b-3p in 50 paired ESCC tissues and adjacent non-tumor tissues were examined by reverse transcription-quantitative polymerase chain reaction. (B) Expression of miR-106b-3p in the ESCC cell lines. The expression of ZNRF3 was detected by (C) western blot and (D) immunofluorescence. Proliferation of cells was determined by (E) MTT and (F) clone formation assay. The results were presented as the mean ± standard deviation of triplicate experiments. **P<0.01 and ***P<0.001 vs. normal tissues and HET-1A. ESCC, esophageal squamous cell carcinoma; miR, microRNA; ZNRF3, zinc and ring finger 3.

miR-106b-3p promotes cell proliferation

To characterize the function of miR-106b-3p on cell proliferation in ESCC, miR-106b-3p mimics, inhibitors and corresponding negative controls were synthesized and transfected into KYSE150 and ECA-109 cells. The expression of miR-106b-3p was determined by RT-qPCR (Fig. 2A) and ZNRF3 expression was detected by RT-qPCR and western blot (Fig. 2B and C). MTT assay was used to examine the proliferation of KYSE150 and ECA-109 cells (Fig. 2D); the data demonstrated that the proliferation rate of cells was markedly increased by the transfection of miR-106b-3p mimics compared with the negative control, while that of cells in the miR-106b-3p inhibitors group was decreased. Colony formation assays further confirmed the proliferative function of miR-106b-3p in ESCC cells (Fig. 2E). These results indicated that miR-106b-3p silencing could suppress the proliferation of ESCC cells.

Figure 2

miR-106b-3p promoted cell proliferation. (A) KYSE150 and ECA-109 cells transfected with miR-106b-3p inhibitor exhibited a decrease in miR-106b-3p expression, KYSE150 and ECA-109 cells transfected with miR-106b-3p mimics showed a increase in miR-106b-3p expression. ZNRF3 (B) mRNA and (C) protein expression was increased in KYSE150 and ECA-109 cells transfected with miR-106b-3p inhibitor. (D) Viability of cells measured by MTT assay. (E) Colony formation assays were performed to test cell proliferation. The data are presented as the mean ± standard deviation of three independent experiments. **P<0.01 and ***P<0.001 vs. NC. NS, no statistical difference vs. control; miR, microRNA; NC, negative control; ZNRF3, zinc and ring finger 3; OD, optical density.

Flow cytometry was used to analyze cell cycle distribution in KYSE150 and ECA-109 cell lines following mimic and inhibitor transfection. Downregulation of miR-106b-3p induced G1 cell cycle arrest, which was demonstrated the by reduced percentage of S and G2/M and the increasing percentage of G1 (Fig. 3A). Additionally, p21 and p27 were increased by miR-106b-3p inhibitor, and cyclin D1 was decreased by miR-106b-3p inhibitor (Fig. 3B). Collectively, these data demonstrated that miR-106b-3p had a growth-stimulative function in ESCC.

Figure 3

Effect of miR-106b-3p on cell cycle in KYSE150 and ECA-109 cells. (A) Cell cycle progression was assayed in KYSE150 and ECA-109 cells by flow cytometry. (B) Western blot analysis in KYSE150 and ECA-109 cells for the protein levels of p27, cyclin D1 and p21 in cells transfected with miR-106b-3p mimics and miR-106b-3p inhibitor. GAPDH was used as an internal control. *P<0.05; **P<0.01 vs. control. miR, microRNA; NC, Control.

Downregulation of miR-106b-3p suppresses the adhesion and EMT of ESCC cells in vitro

To functionally investigate the biological role of miR-106b-3p in ESCC, gain-of-function experiments were performed. Considering the implication of miR-106b-3p in cell motility and adhesion, an adhesion assay was conducted. Cell morphology was observed using a microscope (Fig. 4A) and adhesion ability was detected by adhesion assay. The results showed that upregulation of miR-106b-3p exhibited a significant reduction in attachment to collagen I, collagen IV and fibronectin, which are essential components of the extracellular matrix (Fig. 4B).

Figure 4

miR-106b-3p promoted adhesion and epithelial-mesenchymal transition of ESCC cells. (A) Cell morphology was observed using a microscope. (B) miR-106b-3p mimics promoted cell adhesion. (C) Western blot analysis was performed to analyze the protein expressions of Slug, Snail, E-cadherin, N-cadherin and Vimentin in ESCC cells. GAPDH was used the internal control. The data are presented as the mean ± standard deviation of three independent experiments. *P<0.05; **P<0.01 vs. Control group. ESCC, esophageal squamous cell carcinoma; miR, microRNA; NC, negative control.

EMT can change cell invasion and migration ability. To identify whether miR-106b-3p can affect EMT, changes in the expressions of common EMT markers in miR-106b-3p-expressing cells were determined by western blot analysis (Fig. 4C). Cells transfected with miR-106b-3p mimics presented a mesenchymal-like phenotype, and the mesenchymal marker expressions (Vimentin and N-cadherin) were enhanced. By contrast, decreased protein expression of Vimentin and N-cadherin was observed in cells transfected with miR-106b-3p inhibitors compared with the control group. In addition, miR-106b-3p inhibitors markedly increased the expression of the epithelial marker E-cadherin (Fig. 4C). However, miR-106b-3p inhibitor did not affect Slug expression levels and had suppressive effect on Snail levels in KYSE150 and ECA-109 cell lines. Collectively, the results indicated that miR-106b-3p promotes the invasive characteristics of ESCC cells.

miR-106b-3p induces the migration and invasion of ESCC

Wound healing and Transwell assays were performed to further investigate the effects of miR-106b-3p on migration and invasion of ESCC cells. The wound healing assay demonstrated that miR-106b-3p expression was positively associated with the rates of wound healing, and silencing of miR-106b-3p expression with slower healing (Fig. 5A). In addition, in vitro migration and invasion assays demonstrated that miR-106b-3p inhibitor significantly attenuated the migration and invasion abilities of KYSE150 cells (Fig. 5B). Similar results were obtained using the ECA-109 cell line. These results demonstrated that miR-106b-3p promoted ESCC cell migration and invasion. To investigate cell invasive capacity, proteins associated with invasion were studied in KYSE150 and ECA-109 cells. MMP-2 and MMP-9 expression was increased in cells transfected with miR-106b-3p mimics, as detected by western blot analysis (Fig. 5C). The results indicated that miR-106b-3p may promote the migration and invasion of ESCC cells.

Figure 5

miR-106b-3p promoted migration and invasion capabilities of ESCC cells. (A) Wound-healing assay to explore the effect of miR-106b-3p on the motility of ESCC cells (magnification, x200). (B) Effects of miR-106b-3p on cell migration and invasion in cells were detected using Transwell assay in vitro (magnification, x200). (C) Proteins expression assayed in cancer cells with miR-106b-3p transfection. ESCC, esophageal squamous cell carcinoma; miR, microRNA; NC, negative control; MMP, matrix metalloproteinase.

ZNRF3 is a direct target of miR-106b-3p

Based on miR target analysis using the websites TargetScan and miRanda, ZNRF3 identified as a potential target gene of miR-106b-3p (Fig. 6A). The predicted binding of miR-106b-3p to the ZNRF3 3'UTR was validated in a luciferase reporter assay. The reporter plasmids were co-transfected with miR-106b-3p mimics or NC in KYSE150 and ECA-109 cells. The luciferase activity assay revealed that miR-106b-3p significantly decreased the luciferase activity of the wild type 3'UTR of ZNRF3, without effect on the mutant (Fig. 6B). Futhermore, immunofluorescence analysis was used to observe the expression of ZNRF3 regulated by miR-106b-3p in KYSE150 and ECA-109 cells. The results demonstrated that overexpression of miR-106b-3p reduced the expression of ZNRF3 in ESCC cells (Fig. 6C). These results suggested that ZNRF3 was a direct target gene of miR-106b-3p.

Figure 6

ZNRF3 is a direct target of miR-106b-3p. (A) Computer prediction of the 3'-UTR of ZNRF3 mRNA contained a target site for miR-106b-3p. (B) The pMIR-ZNRF3-MUT or pMIR-ZNRF3-WT and PRL-TK-Renilla were co-transfected in KYSE150 and ECA-109 cells with miR-106b-3p mimic and negative control. Luciferase activity assay revealed that miR-106b-3p mimics suppressed WT ZNRF3 3'-UTR luciferase activity, while it had no effect on MUT ZNRF3 3'-UTR luciferase activity compared to control in KYSE150 and ECA-109 cells. (C) Effects of miR-106b-3p dysregulation on endogenous ZNRF3 expression, which was analyzed by immunofluorescence. Data are from three independent experiments and are presented as the mean ± standard deviation. ***P<0.001 vs. control. UTR, untranslated region; ZNRF3, zinc and ring finger 3; WT, wild type; miR, microRNA; MUT, mutant; Luc/Rluc, luciferase/Renilla luciferase; NS, no statistical difference vs. control; NC, negative control.

miR-106b-3p promoted EMT in ESCC cells by Wnt/β-catenin signaling pathway

To further elucidate the underlying molecular mechanisms of miR-106b-3p on promoting cell EMT, western blot and RT-qPCR assays were performed to explore mRNA and protein levels of factors associated with the Wnt/β-catenin signaling pathway. As shown in Fig. 7, the result of RT-qPCR assay demonstrated that the mRNA expressions of PI3K, AKT, Wnt and β-catenin were upregulated in the miR-106b-3p mimics group, while decreased in miR-106b-3p inhibitors group compared with control group. The result of western blot assay showed that p-PI3K, p-AKT, Wnt and β-catenin were all upregulated in the miR-106b-3p mimics group compared with the levels in the control group (Fig. 8). Inversely, silencing of miR-106b-3p increased p-GSK3 levels. In summary, the results demonstrated that miR-106b-3p activated Wnt/β-catenin signaling via targeting of ZNRF3 and subsequently promoted EMT.

Figure 7

miR-106b-3p activates the Wnt/β-catenin signaling pathway in ESCC. The gene expressions of, PI3K, AKT, Wnt, GSK3 and β-catenin in (A) ECA-109 and (B) KYSE150 cells transfected with miR-106b-3p mimics, miR-106b-3p mimics NC, miR-106b-3p inhibitors, miR-106b-3p inhibitors NC or control using RT-PCR analysis. The data are presented as the mean ± standard deviation of three independent experiments. *P<0.05 and **P<0.01 vs. control. miR, microRNA; NC, negative control; p-, phospho-; PI3K, phosphoinositide 3-kinase; AKT, protein kinase B; GSK3, glycogen synthase kinase-3.

Figure 8

Protein levels of p-PI3K, PI3K, p-AKT, AKT, Wnt, p-GSK3, GSK3 and β-catenin in (A) ECA-109 and (B) KYSE150 cells transfected with miR-106b-3p mimics, miR-106b-3p mimics NC, miR-106b-3p inhibitors, miR-106b-3p inhibitors NC or control using western blot analysis. GAPDH was used as the internal control. miR, microRNA; NC, negative control; p-, phospho-; PI3K, phosphoinositide 3-kinase; AKT, protein kinase B; GSK3, glycogen synthase kinase-3.

Discussion

Despite the advancements in treatment options, improvements in the survival of patients with ESCC have been limited due to lack of early detection. Recent evidence has indicated that alterations of specific miRNAs may be promising translational biomarker candidates in cancer. Furthermore, the identification of tumor-associated miRNAs and their direct target genes is critical for understanding the pathogenesis and biological significance of miRNAs in carcinogenesis and progression of ESCC, which may provide a novel prognostic and therapeutic targets for patients with ESCC.

The data of the current study demonstrated that the levels of miR-106b-3p were significantly upregulated in ESCC tissues and cells compared with adjacent non-tumor tissues and normal human epithelial cells, respectively. Overexpression of miR-106b-3p significantly promoted the cell proliferation and invasion of ESCC cells. Conversely, miR-106b-3p inhibited the proliferation and invasion of ESCC cells in other cancer types (24,25). The data indicated that decreased expression of miR-106b-3p may have a critical role in the progression of ESCC.

In order to explore the mechanisms of migration and invasion by miR-106b-3p of ESCC cells, the potential target genes of miR-106b-3p were predicted. In a previous study, DAB2 clathrin adaptor protein was confirmed as a target of miR-106b of cervical cancer (26,27). In addition, miR-106b was reported to be expressed at a lower level in osteosarcoma cells and target the high mobility group AT-hook 2 gene to inhibit the cell progression (28). Furthermore, Zhang et al (29) reported that miR-106b was upregulated in colorectal cancer and promoted cell invasion and migration by targeting DLC1 Rho GTPase activating protein. In the current study, ZNRF3 was identified as a direct target of miR-106b-3p in a luciferase reporter assay. This observation was validated by the increased ZNRF3 mRNA and protein expressions in ESCC cells transfected with miR-106b-3p inhibitor. ZNRF3 is part of the E3 ubiquitin ligase family, which negatively regulates Wnt signaling. ZNRF3 was associates with the Wnt receptor complex and inhibits Wnt signaling by promoting the turnover of Frizzled and LDL receptor related protein 6 receptors. Accumulating data have strongly suggested that ZNRF3 is involved in cancer development and exhibits low expression in various human cancer types (19-21).

The role of EMT in tumor metastasis has received increasing attention and is a critical process providing epithelial-derived tumor cells with the increased migration and invasive abilities, contributing to tumor metastasis (30-32). EMT is a critical process by which epithelial cells lose their epithelial morphology and acquire a mesenchymal phenotype, characterized by the decreased expression of epithelial proteins, including E-cadherin and tight junction protein ZO-1, and the increased expression of mesenchymal proteins, including Vimentin and fibronectin (22,30,31). Snail and Slug are members of zinc finger family and have a central transcriptional role in the regulation of EMT by directly binding to specific E-boxes on the E-cadherin promoter (33,34). Furthermore, miR-106b-3p promoted EMT by downregulating E-cadherin expression and inducing the expression levels of Snail, N-cadherin and Vimentin in ESCC cells.

Dysregulation of the Wnt/β-catenin pathway has been observed in various cancer typess and to regulate cancer metastasis (35,36). Furthermore, ZNRF3, which was was identified as a direct target of miR-106b-3p in the current study, is a negative regulator of Wnt/β-catenin signaling. The Wnt/β-catenin signaling pathway has an important role in the maintenance of stem cell properties and tumorigenesis (37,38). Additionally, the nuclear accumulation of β-catenin is a key step in the activated Wnt signaling pathway (39). The data of the current study revealed that overexpression of miR-106b-3p markedly upregulated the expression levels of Wnt, GSK3 and β-catenin, and reduced the p-GSK3 level. Thus, these results suggested for the first time that miR-106b-3p has a role in EMT via regulation of the Wnt/β-catenin signaling pathway in ESCC.

In conclusion, the findings of the present study indicated that miR-106b-3p was upregulated, while ZNRF3 was downregulated in ESCC. It was demonstrated that ZNRF3 was a direct target of miR-106b-3p and ectopic expression of miR-106b-3p promoted cell proliferation, migration and invasion capacity of ESCC cells by targeting ZNRF3. Further research provided evidence that miR-106b-3p promoted EMT and activated the Wnt/β-catenin signaling pathway. Taken together, these results suggested that miR-106b-3p has an important role in invasion and EMT of ESCC cells.

To the best of our knowledge, this is the first study to investigate miR-106b-3p in ESCC. These findings expand the understanding of the molecular mechanisms underlying miR-106b-3p function and proliferation, invasion and EMT of ESCC, and also suggest a potential prognostic marker and therapeutic target in human ESCC.