Visfatin facilitates esophageal cancer migration by suppressing miR‑3613‑5p expression and promoting VEZF1/VCAN production

Introduction

In 2020, esophageal cancer, a lethal type of cancer, was responsible for ~5.5% of all cancer-associated fatalities globally (1). Compared with other regions globally, the incidence and mortality rates of esophageal cancer are greater in Asian countries (2). According to the Taiwan Cancer Registry Annual Report, esophageal cancer ranks ninth for cancer-related mortality; squamous cell carcinoma (SCC) is the most frequent histological subtype, accounting for up to 91.4% of all cases (3). The prognosis is poor for esophageal SCC (ESCC) due to its aggressive nature, which includes early distant organ metastases and regional tracheal invasion (4). Typically, metastasis affects the liver, lung and lymph nodes (3,5). A thorough understanding of the intricate processes underlying the spread of ESCC and distant metastases is essential for future advancements in early prevention and intervention.

Small non-coding RNA molecules known as microRNAs (miRNAs/miRs) functionally control gene expression by degrading or suppressing the translation of mRNA targets (6,7). These compounds exert key regulatory effects on cellular functions such as apoptosis, differentiation and cell cycle entry and progression (8–10). miRNAs typically function as oncogenes or tumor suppressors in different types of cancer. In cancer, tumor development, aggressiveness and treatment evasion are associated with dysregulation of miRNAs (6,11). miRNAs primarily regulate intricate signaling pathways and networks that regulate gene expression to regulate the growth and progression of a tumor with metastases and treatment sensitivity (12). Targeting oncogenic miRNAs or boosting tumor suppressor miRNAs in cancer is considered to be a unique form of cancer treatment (12).

Previous studies have demonstrated that obesity enhances the risk of cancer progression and metastases, particularly for malignancy of the kidney, prostate, endometrium, breast, colon and esophagus (13,14). Adipocytes secrete bioactive chemicals known as adipokines, which are key in the advancement of cancer, metabolic disorder, cardiovascular disease, inflammation and metastasis (15–17). In the tumor microenvironment, adipokines may trigger the epithelial-to-mesenchymal transition and enhance metastasis (18). Visfatin was originally revealed in visceral adipose tissue and is considered a multifunctional adipokine and an extracellular nicotinamide phosphoribosyltransferase enzyme (19). Patients with numerous types of cancer have elevated serum levels of visfatin (20,21). Visfatin is key for the invasion and metastasis of cancer (19,22). In esophageal cancer, visfatin levels have been documented to be upregulated compared with those of healthy controls and promote VEGF-C-regulated lymphangiogenesis (3). However, the regulatory roles of visfatin in miRNA synthesis and cell motility in esophageal cancer remain unclear. The aim of the present study was to investigate the regulatory role of visfatin in miRNA synthesis and to elucidate the underlying mechanisms by which miRNA influences cell motility in esophageal cancer.

Materials and methods

Materials

Vascular endothelial zinc finger 1 (VEZF1; cat. no SC-365560; Santa Cruz Biotechnology, Inc.) β-actin (cat. no GT5512; Genetex International Corporation), and versican (VCAN) antibody (cat. no SAB1408906; MilliporeSigma) were used. Recombinant human visfatin (cat. no. 130-09-25UG) was obtained from PeproTech, Inc. The small interfering (si)RNAs targeting VEZF1 (cat no. sc-94046) and VCAN (cat no. sc-41903) were purchased from Santa Cruz Biotechnology, Inc. A non-targeting negative control siRNA (cat no. D-001810-10-05) was purchased from Thermo Fisher Scientific, Inc. PI3K (Ly294002) inhibitor (cat. no. ALX-270-038) was obtained from Enzo Life Sciences, Inc. AKT (cat. no. A6730) and mTOR (rapamycin) inhibitors (cat. no. R0395) were obtained from Sigma-Aldrich (Merck KGaA). The miR-3613-5p mimic (5′-UGUUGUACUUUUUUUUUUGUUC-3′) and miR mimic negative control (5′-UUGUACUACACAAAAGUACUG-3′) were purchased from AllBio. All other reagents and chemicals were obtained from MilliporeSigma, unless otherwise specified.

Cell culture

The invasive ESCC cell line KYSE-410 (cat no. 94072023) was obtained from the European Collection of Cell Cultures and human ESCC cell line CE81T (cat. no. 60166) was purchased from Bioresource Collection and Research Centre. Cells were cultured in either RPMI-1640 or DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% heat-inactivated fetal calf serum (Corning, Inc), 2 mM L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin. All cells were maintained at 37°C in a humidified atmosphere containing 5% CO2.

miRNA sequencing and bioinformatics analysis

For miRNA sequencing (NEXTflex small RNA sequencing kit v3, cat no. NOVA-5132-06, PerkinElmer) was utilized, high-quality total RNA samples from the visfatin (30 ng/ml)-treated for 24 h at 37°C and untreated control KYSE-410 cells were utilized. The experimental workflow was performed by Azenta Life Sciences. To prepare the library, 1 µg total RNA was used quantified using both Qubit and qPCR methods. The qualified library were sequenced pair end PE150 (150 pair-End) Small RNA sequencing was conducted using the Illumina, Inc. HiSeq/Novaseq or MGI2000 platform. Total RNA was extracted using the TRIzol (Thermo Fisher Scientific, Inc.) and RNA integrity was confirmed with an Agilent 2100 Bioanalyzer (RNA integrity number >7). To prevent adaptor-dimer formation, an excess of 3′ SR Adaptor for Illumina was hybridized with the SR RT Primer. Subsequently, the 5′ SR Adaptor for Illumina was ligated to the small RNA using a 5′ Ligation Enzyme. First-strand cDNA synthesis was performed using ProtoScript II Reverse Transcriptase (New England Biolabs, US). Each sample was then amplified by PCR kit (Bioo Scientific, PerkinElmer) using thermocycling conditions: initial denaturation 95°C for 2 min, 20–25 cycles of denaturation 95°C for 20 sec, annealing 60°C for 30 sec, Extension 72°C for 15 sec, final extension 95°C for 2 minusing P5 (Forward, 5′-GTTCAGAGTTCTACAGTCCGACGATC-3′) and P7 (Reverse, 5′-AGATCGGAAGAGCACACGTCT-3′) primers and the PCR product was purified by DNA Clean Beads (Bioo Scientific, PerkinElmer). The purified products of 140–160 bp were recovered and cleaned using PAGE (6%) and validated using an Agilent 2100 Bioanalyzer. The raw readings underwent quality control before processing, which included removing adapter sequences and contaminants. To guarantee data quality, analysis of the lengths and counts of the filtered reads was performed by Trimmomatic (V0.30), Cutadapt (V1.3) and FastQC (V0.10.1), along with an evaluation of the data volume (9). DEseq2 (V1.6.3), DEseq (V1.18.0), EdgeR (V3.4.6) and bowtie2 (V2.1.0), software was used for Heatmap and volcano plot to analyzed differentially expressed miRNA.

The levels of visfatin in patients with primary and metastatic esophageal cancer were analyzed using a UALCAN, dataset and cell motility genes associated with VEZF1 were obtained from The Cancer Genome Atlas (TCGA) database (3,11). Target genes were predicted using TargetScan (targetscan.org/vert_80/), miRTarBase (https://mirtarbase.cuhk.edu.cn), miRDB (https://mirdb.org/) and ENCORI (https://rnasysu.com/encori/index.php) databases (24). Spearman correlation was used to analyze gene correlation. Gene expression levels in ESCC patients were analyzed using the GEO dataset GSE161533. Kaplan-Meier analysis was performed to assess the VCAN levels in ESCC. Expression levels of Visfatin, VEZF1, and VCAN were evaluated using GSE77861, while miR-3613-5p levels were examined using GSE97051 in ESCC patient tissues.

Migration and invasion assay

Transwell inserts (Costar, Inc.; 8-µm pores) were used in 24-well plates for the migration experiments and pre-coated with a layer of Matrigel at 37°C for 30 min before the invasion assay. KYSE410 and CE81T Cells were pretreated with or without (10 µM) of PI3K (Ly294002), AKT inhibitor (AKTi), mTOR (Rapamycin) inhibitors. KYSE410 and CE81T cells were transfected using (Lipofectamine 2000, Invitrogen, Thermo Fisher Scientific, Inc.) with or without siVEZF1, siVCAN siRNAs and miRNA mimics (50 nM) and incubated at 37°C for 24 h, immediately followed by migration and invasion assay. The upper chamber contained 1×104 KYSE410 and CE81T cells in 200 µl serum-free medium (DMEM or RPMI), whereas the lower chamber contained 300 µl 10% FBS and medium with various concentrations (1,3,10, 30) ng/ml of visfatin and incubated for 24 h at 37°C in 5% CO2. Cotton-tipped swabs were used to remove the Matrigel from the upper side of the filters, and PBS was used to wash the filters (23,24). Cells were fixed in 3.7% formaldehyde for 5 min at room temperature, incubated for 24 h at 37°C in 5% CO2 and stained with 0.05% crystal violet in PBS for 20 min at room temperature. Stained cells were observed under an Olympus CKX53 inverted light microscope, and quantified by ImageJ (V1.52a; imagej.net/ij/) and GraphPad prism (V8.0; graphpad.com/guides/prism/8/user-guide/index.htm).

Reverse transcription-quantitative (RT-q)PCR

TRIzol (Thermo Fisher Scientific) cat no. 12183555) was used to extract total RNA from the esophageal cancer KYSE410 cells. In summary, oligo-dT primers (Thermo Fisher Scientific, Inc.), 10 mM dNTP (Cyrusbioscience), 5× standard buffer (Invitrogen; Thermo Fisher Scientific) and 0.1 M DTT (Invitrogen; Thermo Fisher Scientific) were used to reverse-transcribe 1 µg RNA into cDNA in compliance with the manufacturer's instructions. The KAPA SYBER FAST qPCR kit (Applied Biosystems; Thermo Fisher Scientific, Inc.) was used to mix 100 ng cDNA sample with specific primers. qPCR was performed using a Senso Quest Labcycler thermal cycler. The thermocycling conditions were as follows: Initial denaturation at 95°C for 6 min, followed by 40 cycles of denaturation at 95°C for 30 sec, annealing at 60°C for 30 sec and extension at 72°C for 1 min, with a final extension at 72°C for 10 min. The Mir-XTM miRNA First Strand Synthesis kit (Takara Bio Inc.) was used to create cDNA from 100 ng total RNA for the miRNA assay. The endogenous control GAPDH was used to achieve relative quantification of gene expression. The comparative Cq approach was performed to calculate the relative expression (25,26). The sequences of primers were as follows: miR-3613-5p, 5′-TGTTGTACTTTTTTTTTTGTTC-3′ (melting temperature (Tm), 43.7°C; length, 22 bases); VEZF1: Forward, 5′-GGTTCTGCAGCATTTCACCC-3′ and reverse, 5′-TGATGGGAAGCTTCATGGGC-3′ (Tm, 53.8°C; length, 20 bases each); VCAN: Forward, 5′-GTAACCCATGCGCTACATAAAGT-3′ (Tm, 53.5°C; length, 23 bases) and reverse, 5′-GGCAAAGTAGGCATCGTTGAAA-3′ (Tm, 53.0°C; length, 22 bases) and GAPDH: Forward 5′-ACCACAGTCCATGCCATCAC-3′ (Tm, 53.5°C, length, 20 bases) and reverse, 5′-TCCACCACCCTGTTGCTGTA-3′ (Tm, 53.8, length, 20 bases). Relative gene expression levels were calculated using the 2−ΔΔCq method (27).

Western blot analysis

Proteins from KYSE410 cells were extracted using RIPA lysis buffer (cat. No. P0012, Beyotime Institute of Biotechnology). The total protein concentration was determined using a BCA Protein Assay Kit and 30 µg of protein per lane was used for analysis. Proteins were separated by SDS-PAGE using a 10% gel and transferred onto PVDF membranes (Merck KGaA). Membranes were blocked with 5% non-fat milk at room temperature for 1 h and incubated with primary antibodies VEZF1 (1:1,000), VCAN (1:1,000) as target proteins and β-actin (1:3,000) for an entire night (18–20 h) at 4°C. Membranes were incubated for 1 h at room temperature with horseradish peroxidase-conjugated secondary antibodies (1:5,000) goat anti-mouse IgG (cat. no. sc-516102), Santa Cruz Biotechnology, Inc. An ECL kit (MilliporeSigma) was used to detect the expression of the target protein and an ImageQuant LAS 4000 biomolecular imager was used for visualization (28,29).

Luciferase reporter assay

A luciferase assay kit was utilized to track the luciferase activity in order to measure the 3′-untranslated region (UTR). After transfecting the cells with either the wild-type (wt)- or mutant (mt)-VEZF1-3′-UTR luciferase plasmid (Stratagene; Agilent Technologies, Inc.), using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific) the cells were transfected for 24 h at 37°C using miR-3613-5p mimic (sequence: 5′-ACAAAAAAAAAAGUACAACAUU-3′; (AllBio). Following 24 h transfection, cells were lysis and instantly the luciferase activity was measured using the Dual-Luciferase® Reporter Assay System (Promega Corporation). Firefly luciferase activity was normalized to Renilla luciferase activity to control for transfection efficiency.

Statistical analysis

Data were analyzed using ImageJ software (V1.52a) (https://imagej.net/ij/) and GraphPad Prism software (V8.0) (graphpad.com/guides/prism/8/user-guide/index.htm). All data are presented as the mean ± SD from 3 independent experiments. Statistical significance between two groups was assessed using the unpaired Student's t-test. Comparisons involving >2 groups with a single variable were performed using one-way ANOVA followed by Bonferroni's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

Results

Visfatin promotes esophageal cancer migration by inhibiting miR-3613-5p

Visfatin is key for the development of numerous types of cancer (3,30). However, the mechanisms through which visfatin affects esophageal cancer metastases remain unknown. The UALCAN data revealed that the levels of visfatin were higher in patients with metastatic esophageal cancer than in those with primary esophageal cancer (Fig. 1A). Transwell migration assay was used to examine the effects of visfatin on the migration of esophageal cancer cells. Visfatin promoted the migration of KYSE-410 esophageal cancer cells in a concentration-dependent manner (Fig. 1B). Additionally, visfatin enhanced the invasive ability of esophageal cancer cells (Fig. 1C).

Figure 1. Visfatin promotes the migration and invasion of esophageal cancer cells. (A) Visfatin mRNA levels in normal, primary and metastatic esophageal cancer tissues from TCGA dataset (UALCAN). Expression increased with lymph node metastasis (N0-N1), with significant upregulation in metastatic tissue (P=0.0194). Transwell (B) migration and (C) invasion assays in KYSE410 cells treated with visfatin. Crystal violet-stained images and quantification show dose-dependent increases in cell migration and invasion. *P<0.05 vs. Control. TCGA, The Cancer Genome Atlas; UALCAN, The University of Alabama at Birmingham Cancer data analysis Portal; ns, not significant.

The metastasis of esophageal cancer is associated with dysregulated miRNAs (31). To investigate whether miRNAs mediate visfatin-induced esophageal cancer cell migration, miRNA sequencing was performed to assess the differential expression of miRNAs in KYSE-410 cells treated with visfatin. The resulting heatmap and volcano plot illustrated differentially expressed miRNAs (Fig. 2A and B); among these, miR-3613-5p was the most downregulated (Fig. 2B). Visfatin (≥3 ng/ml) inhibited miR-3613-5p synthesis in a concentration-dependent manner (Fig. 2C). Transfection with a miR-3613-5p mimic increased the miR-3613-5p expression compared with control (Fig. 2D). miR-3613-5p mimic antagonized the visfatin-induced promotion of cell migration and invasion (Fig. 2E and F). Consistent with the KYSE-410 results, miR-3613-5p mimic treatment inhibited visfatin-induced migration and invasion in CE81T cells (Fig. 2G and H). Thus, these findings demonstrate that visfatin promoted esophageal cancer cell migration by decreasing miR-3613-5p production.

Figure 2. Visfatin enhances cell migration by inhibiting mir-3613-5p. (A) Heatmap and (B) volcano plot of differentially expressed mirs in KYSE-410 cells treated with visfatin. Cells were (C) stimulated with visfatin and (D) transfected with mir-3613-5p mimic and mir expression was verified by reverse transcription-quantitative PCR. (E) KYSE410 cells transfected with miR-3613-5p mimic with or without visfatin treatment and cell migration and invasion was checked. (F) quantification of cell migration and cell invasion assay. (G) CE81T cells were transfected with mir-3613-5p mimic and treated with or without visfatin; cell migration and invasion were examined. (H) quantification of cell migration and cell invasion assay. *P<0.05 vs. Control; #P<0.05 vs. Visfatin. Mir, microRNA.

miR-3613-5p inhibits the VEZF1/VCAN axis and mediates visfatin-promoted cell migration

A total of four publicly available miRNA databases (TargetScan, miRTarBase, miRDB and ENCORI) predicted that miR-3613-5p targets seven potential candidates (Fig. 3A). Among these, VEZF1 was markedly upregulated compared with ANP32B in patients with esophageal cancer (Fig. 3B). Additionally, VEZF1 was more highly upregulated in patients with metastatic than in those with primary esophageal cancer, however this was not significant (Fig. 3C). The direct application of visfatin to esophageal cancer cells enhanced VEZF1 mRNA and protein expression (Fig. 3D and E). Transfection of the cells with VEZF1 siRNA, effectively suppressed VEZF1 protein levels, as confirmed by western blot analysis (Fig. 3F), and inhibited the visfatin-induced promotion of cell migration and invasion (Fig. 3G-I). These findings were validated in CE81T cells, where VEZF1 siRNA also reversed visfatin-induced cell motility, confirming a consistent effect across ESCC cell lines (Fig. 3J-L). Subsequently, wt- and mt-VEZF1-3′-UTR luciferase plasmids were generated to examine the direct binding effects of miR-3613-5p on VEZF1 (Fig. 4A). The miR-3613-5p mimic decreased wt-VEZF1-3′-UTR, but not mt-VEZF1-3′-UTR luciferase activity (Fig. 4B). Furthermore, the miR-3613-5p mimic blocked visfatin-induced VEZF1 mRNA expression (Fig. 4C) and protein levels (Fig. 4D).

Figure 3. VEZF1, regulated by miR-3613-5p, is involved in visfatin-induced esophageal cancer cell migration. (A) miR databases (TargetScan, miRTarBase, miRDB and ENCORI) predicted that miR-3613-5p targets seven potential candidates. (B) Gene levels in patients with esophageal cancer retrieved from TCGA. (C) VEZF1 gene levels in normal tissue and tissues from patients with primary and metastatic esophageal cancer retrieved from TCGA. (D) KYSE 410 cells were stimulated with visfatin, and VEZF1 expression was examined using reverse transcription-quantitative PCR and (E) western blot analysis. (F) VEZF1 siRNA transfection efficiency examined by western blot. (G) KYSE410 cells were transfected with VEZF1 siRNA and treated with visfatin, then cell (H) migration and (I) cell invasion was examined. (J) CE81T cells were transfected with VEZF1 siRNA followed by visfatin treatment and then (K) Migration and (L) invasion was examined. *P<0.05 vs. control; #P<0.05 vs. visfatin. miR, microRNA; TCGA, The Cancer Genome Atlas; VEZF1, Vascular endothelial Zinc Finger 1; si, small interfering; FC, fold change; ANP32B, Acidic Leucine-Rich Nuclear Phosphoprotein 32 Family Member B; CDK, Cyclin-dependent kinase; F11R, F11 receptor; LMNB2, Lamin B2; FLVCR, Feline Leukemia Virus subgroup C Receptor 1; QSER, Glutamine and Serine-rich protein 1; ns, not significant.

Figure 4. miR-3613-5p directly binds the 3′-UTR of the VEZF1 gene. (A) Binding site of miR-3613-5p on the VEZF1 3′-UTR. (B) Cells were transfected with luciferase plasmids and miR-3613-5p mimic, and luciferase activity was examined. Cells were transfected with a miR-3613-5p mimic and treated with visfatin; VEZF1 expression was examined using (C) reverse transcription-quantitative PCR and (D) western blot assay. *P<0.05 vs. control; #P<0.05 vs. visfatin. miR, microRNA; UTR, untranslated region; VEZF1, Vascular Endothelial Zinc Finger 1; wt, wild Type; mt, mutant; ns, not significant.

To investigate whether the visfatin-induced expression of VEZF1 regulates cell motility genes, TCGA database was searched for genes associated with VEZF1 (Fig. 5A). Among 300 genes exhibiting a positive correlation, 13 genes were also associated with cell adhesion functions (Fig. 5B). Data from the GSE161533 database indicated that VCAN was the most upregulated gene in patients with esophageal cancer (Fig. 5C). Kaplan-Meier analysis confirmed that the VCAN levels were higher in patients with esophageal cancer than in healthy controls (Fig. 5D). Visfatin promoted the mRNA and protein expression of VCAN (Fig. 6A and B). Transfection with VCAN siRNA effectively decreased VCAN protein levels, as confirmed by western blot analysis (Fig. 6C), and also attenuated visfatin-induced cell motility (Fig. 6D-F); this effect was validated in CE81T cells (Fig. 6G-I). miR-3613-5p mimic and VEZF1 siRNA also suppressed visfatin-induced VCAN expression (Fig. 6J and K), indicating that the inhibition of miR-3613-5p and the promotion of VEZF1 occurred upstream of visfatin-induced VCAN expression and cell motility. Mechanically, visfatin inhibits miR-1264 and promotes platelet derived growth factor C (PDGF-C) synthesis through activation of the PI3K/AKT/mTOR signaling pathway (19). To elucidate the molecular mechanism, the present study examined the role of the PI3K/AKT/mTOR pathway in visfatin-mediated ESCC migration and invasion Transwell assay results indicate that treatment with PI3K (Ly294002), AKTi and mTOR (rapamycin) pathway inhibitors effectively reversed the migration and invasion effects induced by visfatin, confirming the involvement of PI3K/AKT/mTOR signaling axis in visfatin-driven migration and invasion (Fig. 6L-Q). To explore the clinical relevance of visfatin-related genes, GSE77861 dataset was analyzed to compare gene expression between normal and esophageal cancer tissue. Visfatin (Fig. 6R), VEZF1 (Fig. 6T) and VCAN (Fig. 6U) were significantly upregulated in esophageal cancer tissues compared with normal controls (P<0.05). In addition, analysis of the GSE97051 dataset showed that miR-3613-5p expression was slightly decreased in cancerous tissue compared with normal samples (Fig. 6S). Together, these findings support the potential involvement of the visfatin/miR-3613-5p/VEZF1/VCAN axis in the progression of esophageal cancer.

Figure 5. VCAN is highly expressed in patients with esophageal cancer. (A) Spearman correlation analysis from TCGA. (B) A total of 13 genes correlated with VEZF1 and were associated with cell adhesion functions. (C) Gene levels in patients with esophageal cancer retrieved from the GSE161533 and TCGA database. (D) Kaplan-Meier analysis of VCAN level in normal and cancer stage in ESCC *P<0.05. VCAN, Versican; VEZF1, Vascular Endothelial Zinc Finger 1; TCGA, The Cancer Genome Atlas; FC, Fold Change; PLXNC1, Plexin C1; ERBIN, interacting protein; CLDN, claudin; ARAHGAP, Rho GTPase-activating protein 5; RGMB, Repulsive Guidance Molecule BMP Co-Receptor B; ITGBL, Integrin subunit beta 1; PTPRK, Protein Tyrosine Phosphatase Receptor Type Kappa; DDR, DNA Damage Response; PARD, Par-3 family cell polarity regulator; SPECC1L, sperm antigen with calponin homology and coiled-coil domains 1 like; CDON, Cell Adhesion Associated, Oncogene Regulated; PTPRT, protein tyrosine phosphatase, receptor type, T.

Figure 6. VCAN is involved in visfatin-induced esophageal cancer migration. Cells were stimulated with visfatin and VCAN expression was examined using (A) RT-qPCR and (B) western blot analysis. (C) VCAN siRNA transfection efficiency confirmed by western blot analysis. (D) KYSE410 cells were transfected with or without VCAN siRNA followed by visfatin treatment and cell (E) migration and (F) invasion were examined. (G) CE81T cells were transfected with VCAN siRNA and treated with visfatin; cell (H) migration and (I) invasion were examined. Cells were transfected with miR-3613-5p mimic or VEZF-1 siRNA and treated with visfatin; VCAN expression was examined using (J) RT-qPCR and (K) western blotting. (L) KYSE 410 cells treated with PI3K (Ly294002), AKT and mTOR (rapamycin) inhibitors and visfatin treatment to assess effects on (M) migration and (N) invasion. (O) CE81T cells treated with PI3K (Ly294002), AKT and mTOR (rapamycin) inhibitors and visfatin treatment were assayed for (P) cell migration and (Q) invasion. Gene Expression Omnibus dataset GSE77861 shows significantly increased mRNA expression of (R) NAMPT, (S) miR-3613-5p, (T) VEZF1 and (U) VCAN in esophageal cancer compared with normal tissue. *P<0.05 vs. control; #P<0.05 vs. visfatin. miR, microRNA; VCAN, versican; RT-q, Reverse Transcriptase Quantitative; si, small interfering; VEZf-1, Vascular Endothelial Zinc Finger 1; NAMPT, Nicotinamide phosphoribosyltransferase; Akti, Akt inhibitor.

Discussion

Esophageal cancer is a relatively prevalent malignancy worldwide, marked by a poor prognosis and a strong tendency for metastasis. It is the eighth most commonly diagnosed cancer and the sixth leading cause of cancer-related deaths globally. Notably, over 80% of all cases and fatalities are reported in developing countries. In the United States alone, the National Cancer Institute estimated approximately 18,000 new cases and over 15,000 deaths due to esophageal cancer in 2013 (32). The ESCC subtype, which accounts for almost 90% of esophageal malignancies in Asia, has a high mortality rate and poor prognosis (33). Despite progress in detection and treatment, the 5-year survival rate of patients with esophageal cancer is relatively low (34). The high mortality rate from esophageal cancer may be decreased with improved treatment approaches (35). The present study demonstrated that the levels of visfatin were associated with metastasis in patients with esophageal cancer. The present study showed that inhibition of miR-3613-5p and the promotion of the VEZF1/VCAN axis mediated visfatin-facilitated esophageal cancer cell motility (Fig. 7). The present study also demonstrated the expression of visfatin, VEZF1, and VCAN in normal and cancer samples from the GEO database. However, a limitation is the lack of experimental validation using clinical samples from patients with ESCC, underscoring the need for further investigation.

Figure 7. Mechanisms underlying the roles of visfatin in esophageal cancer cell migration and invasion. Visfatin enhances cell migration and invasion in esophageal cancer cells. The inhibition of miR-3613-5p and the promotion of the VEZF1/VCAN axis mediate visfatin-induced esophageal cancer cell motility. miR, microRNA; VEZF1, Vascular Endothelial Zinc Finger 1; VCAN, Versican; UTR, Untranslated region.

Adipocytokines are associated with development, spread, recurrence and metastasis of numerous types of malignancy (36). Lower resistin mRNA levels are found in ESCC samples and blood compared with normal esophageal samples (37), Patients with ESCC have lower adiponectin levels compared with controls (38). Additionally, a significant association has been found between leptin levels and advanced tumor stage in ESCC, as well as lymph node involvement (39). Specifically, visfatin serves a key role in inflammation and cancer. Additionally, visfatin promotes the metastasis of chondrosarcoma (40). Visfatin is associated with a higher disease stage in ESCC tissue and promotes lymphangiogenesis. Our previous study demonstrated that visfatin is highly expressed in ESCC N1 and N2 stage samples compared with N0 and is associated with lymph node metastasis (3). In the present study, Transwell migration and Matrigel invasion assays revealed that visfatin facilitated the migration and invasion of esophageal cancer cells. To the best of our knowledge, the present study is the first to demonstrate that visfatin promotes cell motility in esophageal cancer.

At the post-transcriptional level, small, non-coding miRNAs are key for regulating gene expression (41). This regulation controls physiological and pathological processes, including cancer, by destroying or inhibiting the translation of target mRNAs (42–44). A promising treatment strategy to combat tumor metastasis is to alter miRNA expression through pharmacological intervention, which may be utilized to inhibit cancer cells from migrating (45,46). In the present study, the miRNA sequencing analysis revealed that miR-3613-5p was the most downregulated miRNA following the use of visfatin. Subsequent experiments demonstrated that visfatin reduced miR-3613-5p expression and introducing a miR-3613-5p mimic into esophageal cancer cells reversed visfatin-induced cell motility. These findings indicated that visfatin promoted esophageal cancer cell migration and invasion by suppressing miR-3613-5p synthesis. Additionally, visfatin-induced inhibition of miR-1264 promotes PDGF-C synthesis via the PI3K/AKT/mTOR pathway (19). In the present study, Transwell assays demonstrated that inhibition of the PI3K/AKT/mTOR pathway effectively reversed the metastatic effects induced by visfatin. To the best of our knowledge, however, there is no direct evidence linking the PI3K/AKT/mTOR pathway to the regulation of miR-3613-5p. Absence of direct evidence limits understanding of the upstream regulatory network controlling miR-3613-5p expression in response to visfatin stimulation. Whether the PI3K/AKT/mTOR pathway is involved in visfatin-mediated regulation of miR-3613-5p expression needs further investigation.

With its six-type zinc finger motifs, poly glutamine domain and proline-rich region, VEZF1 is a potential zinc finger transcription factor that is key for angiogenesis (47). Initially, VEZF1 expression was found in both the embryo proper and the mesodermal components of the extraembryonic mesoderm (48). Subsequently, endothelial cells that emerge during angiogenesis were found to express VEZF1 (48). By targeting downstream genes, such as metallothionein 1 and stathmin, VEZF1 controls different phases of angiogenesis (49). VEZF1 transcriptional activity also controls the metastasis of hepatocellular carcinoma (50). According to four publicly accessible miRNA databases, miR-3613-5p targets seven possible candidates, and patients with esophageal cancer have significantly higher levels of VEZF1. Visfatin-induced cell migration and invasion were reduced by VEZF1 siRNA, suggesting that VEZF1 mediated the motility of esophageal cancer. The present study also identified VCAN as a downstream molecule of VEZF1. Therefore, the VEZF1/VCAN axis may mediates visfatin-induced esophageal cancer cell migration.

In conclusion, the present study demonstrated that visfatin facilitated the migration and invasion of esophageal cancer cells. The inhibition of miR-3613-5p and the promotion of the VEZF1/VCAN axis mediated visfatin-induced esophageal cancer cell motility.

Acknowledgements

Not applicable.

Funding

The present study was supported by the National Science and Technology Council (grant nos. 113-2320-B-039-049-MY3, 113-2320-B-371-002- and 112-2314-B-039-018-MY3), China Medical University (grant no. CMU111-ASIA-05) and China Medical University Hospital (grant nos. DMR-114-014, DMR-114-021, DMR-113-008 and DMR-114-069).

Availability of data and materials

The data generated in the present study may be found in the Gene Expression Omnibus under accession number GSE298998 or at the following URL: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE298998).

Authors' contributions

CLH, CHT and PIL wrote the manuscript. SSG, JHG, CLL, YHC and CLH performed experiments and analyzed data. HCT, PIL, YHC, MYL and CHT analyzed data. HCT, SSG and CHT edited the manuscript. All authors have read and approved the final manuscript. CLH, MYL and CHT confirm the authenticity of all the raw data.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References