This article is mentioned in:

Introduction

Liver cancer ranks sixth for incidence and third for mortality among the most common cancers worldwide (1). In 2014, it was reported that among 87,988 malignant liver and intrahepatic cancer diagnoses reported during 2000–2010 in SEER 18 registries, 63,735 (72%) were classified as HCCs in USA. Between 2006 and 2010, US liver cancer mortality rates increased with age in all racial/ethnic groups (2). A significant variance in the incidence rate of liver cancer has been observed among different nations and regions, and 55% of new cases of liver cancer worldwide are reported to occur in China (3). In the USA, the incidence of liver cancer has been rising faster than that of other malignant cancers (4). The occurrence of liver cancer is closely correlated with infection by hepatitis B virus (HBV), and 50% of cases result from HBV infection, the infection rate of which is high (5,6). Although great advances in the surgical therapy and other treatments for liver cancer have been achieved, the 5-year survival rate is only 5–6% and the poor prognosis is a cause of severe economic loss and a heavy burden for society (7–9).

Liver cancer is usually occult in onset and no effective screening measure is able to screen for the disease validly and reliably; therefore, two-thirds of cases are not diagnosed until an advanced stage (10,11). Furthermore, liver cancer is often resistant to chemotherapeutic drugs because it usually originates from pre-existing liver diseases (12,13). These factors largely contribute to the high mortality rate of liver cancer. The initiation and progression of liver cancer is an interactive process involving multiple genes and a variety of environmental factors, which include multiple pathological stages and a variety of molecular events. These factors induce normal liver cells to transform into liver cancer cells and may even cause the occurrence of metastasis (14,15).

Recent studies of the molecular mechanism of liver cancer have mainly focused on the expression of specific tumor genes, differentially expressed genes and their proteomics (16,17). Numerous non-coding sequences in the human genome have been detected and their proportion is far greater than that of protein-encoding sequences (>97%). These sequences may be transcribed into multiple types of RNA that serve an important role in the development of diseases (18–20). Non-coding small RNA (length from 18 to 200 nucleotides), particularly microRNA, has attracted considerable attention (21,22). Studies on the expression profile and functions of microRNA are advancing the understanding of liver cancer. The association between microRNA and the incidence of liver cancer and its potential value for the diagnosis and treatment are being increasingly highlighted (23–25). MicroRNAs are single stranded, non-coding small molecular weight endogenous RNAs ~22 nucleotides in length. They reduce the expression of mRNA by complementary pairing with the 3′-untranslated region (3′-UTR) of targeted mRNA, which participates in multiple physiological processes, including cell proliferation, differentiation, apoptosis, metabolism and development, as well as multiple pathological processes such as cardiovascular disease, nervous system diseases and tumor diseases (22,26–28). More than one-third of human genes are considered to be the targets of conservative microRNAs and these microRNAs have received growing attention in the development of human diseases, particularly in cancers (29,30).

A number of studies have shown that miRNAs are closely associated with the occurrence, development and invasion of tumors by regulating the cell cycle, cell apoptosis, cell migration and the angiogenesis of tumors (31–33). The epidermal growth factor receptor has a tumor-promoting role on cell proliferation, survival, metastasis and tumor-induced vascularization) during hepatocellular carcinoma (HCC) formation, which is regulated by miR-302b (34–36). Wang et al (37) reported that hsa-miR-613 prohibits the proliferation and invasion of HCC by regulating the expression of doublecortin like kinase 1. In addition, the proliferation of liver cancer cells is accelerated by the reduction in NFAIP3 interacting protein 2 expression induced by interference with miR-1180 (38). In gastric cancer, hsa-microRNA-493 has been shown to reduce cell proliferation and invasion (39). In addition, Sakai et al (40) substantiated that binding of mitogen-activated protein kinase kinase 7 by miR-493 inhibits the hepatic metastasis of colon cancer cells. However, the role that miR-493-5p serves in liver cancer remains unclear.

The present study investigated the expression of miR-493-5p in liver cancer tissues and cell lines, and explored the effect of miR-493-5p on the biological behavior of the HepG2 cell line. Furthermore, whether the expression of vesicle associated membrane protein 2 (VAMP2) is regulated by miR-493-5p was also explored. The results of these investigations may provide a new method or strategy for the therapy of liver cancer.

Materials and methods

Samples

Tissue samples, comprising 15 fresh HCC tissues and 6 adjacent normal tissues from patients (age, 40–70 years old; 12 male and 3 female; 10 at cancer stage I–II and 5 at cancer stage III–IV) with HCC were collected in Anhui Provincial Hospital Affiliated to Anhui Medical University (Hefei, China) from 2012 to 2015. The samples were immediately placed in liquid nitrogen for analysis. All patients had not undergone radiotherapy, chemotherapy or other treatments and had no other complications such as inflammatory diseases. The study was approved by the Biomedical Ethics Committee of Anhui Medical University (approval no. 20140272). The patients provided written informed consent to participate in this study and cooperated fully with the researchers.

Reagents

The TaqMan miRNA isolation kit and TaqMan microRNA assay kit were purchased from Applied Biosystems (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The primers for miR-493-5p were: 5′-TCC TAC GGA GAG GCT CAG-3′ and 5′-TCC TCG TAG TCC AAC ACG-3′. The miR-493-5p mimic and negative control (mimic control) were synthesized by Shanghai GenePharma Co., Ltd. (Shanghai, China).

Fetal bovine serum (FBS), Dulbecco's modified Eagle's medium (DMEM) culture medium, RPMI-1640 culture medium and Lipofectamine® 2000 were all purchased from Invitrogen (Thermo Fisher Scientific, Inc.). The culture dishes, plates and Transwell chambers were from Corning Incorporated (Corning, NY, USA).

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and radioimmunoprecipitation assay (RIPA) protein lysis buffer were obtained from Beyotime Institute of Biotechnology (Shanghai, China). Trypsin and Hoechst 33342 were obtained from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Matrigel was from BD Biosciences (Franklin Lakes, NJ, USA). Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) were from Roche Diagnostics (Basel, Switzerland). The rabbit anti-human VAMP2 polyclonal antibody (ab3347) and mouse anti-human β-actin monoclonal antibody (ab8226) were from Abcam (Cambridge, UK). The horseradish peroxidase (HRP)-conjugated affinity purified goat anti-mouse (A1146) and goat anti-rabbit (A0545) IgG secondary antibodies were from Sigma-Aldrich (Merck KGaA). The bicinchoninic acid (BCA) protein quantification kit was from Bio-Rad Laboratories, Inc. (Hercules, CA, USA).

The vectors including pcDNA3.1 (Thermo Fisher Scientific, Inc.), pGEM-T (Promega Corporation, Madison, WI, USA) and pGL3-Basic (Promega Corporation) were preserved in the present authors' laboratory. The competent cells (DH5α) and TRIzol reagent were purchased from Invitrogen Thermo Fisher Scientific, Inc.). The DNA marker and restriction endonucleases BamHI and XhoI were purchased from Takara Bio, Inc. (Otsu, Japan). The T4 DNA ligase was acquired from Promega Corporation. The QIAGEN OneStep RT-PCR kit (cat. no. 210210) was acquired from Qiagen, Inc. (Valencia, CA, USA).

Methods

Cell culture

The HCCC-9810 cell line (an intrahepatic cholangiocarcinoma cell line) was cultured in RPMI-1640 medium containing 10% FBS. The HepG2 cell line (a hepatoblastoma cell line) was cultured in DMEM containing 1.0 mM sodium pyruvate and 10% FBS. The HL-7702 cell line (a normal human liver cell line) was cultured in RPMI-1640 medium containing 20% FBS. The HuH-7 cell line (a HCC cell line) was cultured in DMEM containing 10% FBS.

The four cell lines were incubated at 37°C with 5% CO2 and saturated humidity. The status of the cells was observed under an inverted phase microscope and the passage of the cells was conducted by 0.25% trypsinization when their confluence reached 70–80%. The culture medium was changed every other day.

The 293 cells were seeded in a 6-well plate at a density of 3×105/ml with a volume of 1,000 μl in each well. When the cell confluence reached 80%, the cells were transfected with the miR-493-5p mimic or negative control (final concentration, 50 nM; synthesized by Shanghai GenePharma Co., Ltd.), with or without the recombinant plasmid pcDNA3.1-VAMP2 (500 ng/well) using Lipofectamine 2000 according to the manufacturer's protocol. The cells transfected with the negative control and pcDNA3.1-blank plasmid served as a control. Untransfected cells served as the normal control.

Construction of recombinant plasmid pcDNA3.1-VAMP2

According to the mRNA sequence of VAMP2 (GenBank no. NM-014232) and restriction site analysis, primers were designed using Primer Premier 5 software (Premier Biosoft International, Palo Alto, CA, USA) for a location flanking the open reading frame of the VAMP2 gene. The primer sequences were: Forward, 5′-CG GGATCC ATG TCT GCT ACC GCT GCC AC-3′ (containing a BamHI site) and reverse, 5′-CC CTCGAG AGT GCT GAA GTA AAC TAT GA-3′ (containing a XhoI site) (the underlined sections of the primer sequences indicate the restriction endonuclease site). The primers were synthesized by Shanghai Invitrogen (Thermo Fisher Scientific, Inc.).

RNA extraction from the HepG2 cell line was performed using TRIzol reagent and first-strand cDNA synthesis was conducted using the QIAGEN OneStep RT-PCR kit. Amplification of the VAMP2 gene was conducted with the above primers and the PCR products were inserted into the pGEM-T vector. The positive clone was then sequenced and subcloned into the vector pcDNA3.1 for the establishment of the pcDNA3.1-VAMP2 recombinant plasmid.

Expression of miR-493-5p and VAMP2 in the liver cancer tissues and cell lines

The expression of miR-493-5p and VAMP2 was examined by RT-qPCR (temperature protocol: 16°C for 30 min, 42°C for 30 min, 85°C for 5 min, in the end hold in 4°C) in the cell lines and tissue samples. The thermocycling conditions were as follows: 95°C for 10 min, 95°C for 10 sec, 60°C for 60 sec, 40 cycles. Quantification was conducted using the 2−ΔΔCq method (41). The microRNA was extracted using the TaqMan miRNA isolation kit and the expression of miR-493-5p was detected using a TaqMan microRNA assay kit with U6 as an internal reference. RNA was extracted from the tissues and cell lines using TRIzol and first-strand synthesis was conducted using the RT kit. The expression of VAMP2 mRNA was detected by qPCR and GAPDH served as an internal reference. All the experiments were replicated three times.

Effect of miR-493-5p on VAMP2 expression

The miR-493-5p mimic and negative control were transfected into the HepG2 cells and 48 h later, the microRNA was extracted using a TaqMan miRNA isolation kit. The expression of miR-493-5p was detected by RT-qPCR and the expression of VAMP2 was confirmed by western blot analysis. For the latter, protein extraction was conducted by lysis using RIPA buffer solution and the total protein concentration was measured by the BCA kit. The proteins (50 μg per lane) were subjected to electrophoresis using 5% SDS-PAGE, transferred onto a polyvinylidene difluoride membrane (Bio-Rad Laboratories, Inc.) and blocked at 37°C in 5% skimmed milk TBS with Tween 20 (TBST) solution for 1 h. The VAMP2 polyclonal rabbit anti-human antibody was then added at a dilution of 1:500 and β-actin rabbit anti-human monoclonal antibody was used as internal reference (dilution 1:1,000). The membrane was incubated at 4°C overnight, washed with TBST three times, and then probed with HRP-conjugated goat anti-rabbit IgG (1:2,000) at 37°C for 1 h. Following washing with TBST, the membrane was treated with an ECL chemiluminescence reagent (Thermo Fisher Scientific, Inc.) and autoradiography was conducted. The relative expression of the target protein was evaluated as the gray value ratio of the target protein to β-actin (target protein/β-actin) content. The software used for quantification of the western blotting results was Quantity One (Bio-Rad Laboratories, Inc.).

Effect of miR-493-5p on cell viability

The miR-493-5p mimic and negative control were transfected into HepG2 cells and 48 h later, 100 μl MTT (0.5 mg/ml) solution was added to each well. The plate was then incubated at 37°C, 5% CO2 and saturated humidity for another 4 h. Following this, 100 μl 20% sodium dodecyl sulfate (containing 50% dimethylformamide) was added to dissolve the formazan crystals. The optical density value was measured using a microplate reader (BioTek Instruments, Inc., Winooski, VT, USA) at a wavelength of 570 nm. The experiments were conducted in triplicate.

Effect of miR-493-5p on cell proliferation

The miR-493-5p mimic and negative control were transfected into HepG2 cells and 48 h later, these cells were collected by trypsinization. The cells were then fixed using 75% ethanol and dyed with PI solution for 30 min in the absence of light. Afterwards, these cells were analyzed by flow cytometry (BD Biosciences), using FCM CellQuest software (BD Biosciences) for cell counting and FACsuite software for data analysis.

Effect of miR-493-5p on cell apoptosis

The miR-493-5p mimic and negative control were transfected into HepG2 cells and 48 h later, the cells were collected by trypsinization. The cells were then dyed with Annexin V-FITC and PI solution for 15 min in the dark. Finally, the cells were subjected to flow cytometric analysis for the detection of apoptosis.

Effect of miR-493-5p on cell invasion

The cell treatment and transfection were conducted as described above. At 24 h after transfection, the cells were seeded in a Transwell chamber and incubated for another 24 h. The cells were then washed with phosphate-buffered saline three times and stained with Hoechst 33342. The stained cells that had migrated through the Transwell polycarbonate membrane were counted under a microscope in six randomly selected fields. Matrigel was used in invasion assay.

Identification and confirmation of the targeting of VAMP2 by miR-493-5p

TargetScan (http://www.targetscan.org/) was used to predict the gene targeted by miR-493-5p, and VAMP2 was predicted as a potential target. To establish the fluorescent reporter vector, the 3′-UTR of VAMP2 and the 3′-UTR of VAMP2 containing a mutation in the seed region were synthesized and inserted into the pGL3-Basic vector at the XbaI restriction site. The HepG2 cells were seeded in a 6-well plate at a density of 3×105/ml in 1,000 μl culture medium. The cells were transfected with plasmid VAMP2 3′-UTR (wild-type) or mutant VAMP2 3′-UTR, in combination with either miR-493-5p mimic or negative control. At 48 h following transfection, the cells were lysed and detected using a Dual-Luciferase® Reporter Assay system (Promega Corporation).

Evaluation of the VAMP2-dependence of miR-493-5p

In order to verify that the effect of miR-493-5p on cell biological behavior is mediated via VAMP2, the overexpression vector pcDNA3.1-VAMP2 was established. HepG2 cells were divided into five groups: Control, pcDNA3.1 control, pcDNA3.1-VAMP2 transfection, miR-493-5p mimic + pcDNA3.1 and miR-493-5p mimic + pcDNA3.1-VAMP2 groups. The transfection procedure was conducted as described above. Cell behavior characteristics, including cell viability, cell proliferation and cell migration, were then measured by MTT, flow cytometry and Transwell chamber assays.

Statistical analysis

Data are presented as the means ± SD (3 replicates). SPSS 17.0 statistical software (SPSS, Inc., Chicago, IL, USA) was used to analyze the data with one-way analysis of variance. Multiple comparison between the groups was performed using Student-Newman-Keuls method. All P-values were two-sided and P<0.05 was considered to indicate a statistically significant result.

Results

Expression of miR-493-5p and VAMP2 in liver cancer tissues and cell lines

The results of RT-qPCR analysis indicated that the expression of miR-493-5p in the HCC tissue samples was significantly lower than that in the adjacent normal tissues (P<0.01). The expression of miR-493-5p in the liver cancer cell lines HCCC-9810, HuH-7 and HepG2 was reduced significantly compared with that in the HL-7702 hepatic cells (P<0.01; Fig. 1A and B). These results indicate that the expression of miR-493-5p was inhibited in the liver cancer tissues and cell lines. However, in contrast to miR-493-5p, the expression of VAMP2 was observed to be increased in the liver cancer tissues and cell lines compared with those in the adjacent normal tissues and HL-7702 cells, respectively (P<0.01; Fig. 1C and D). These results demonstrate that the expression of VAMP2 is elevated in liver cancer tissues and cell lines.

Figure 1 Expression of miR-493-5p and VAMP2 mRNA in HCC tissues, adjacent normal tissues, and the normal and cancerous hepatic cell lines HCCC-9810, HuH-7, HepG2 and HL-7702. Expression of miR-493-5p in (A) HCC and adjacent tissues and (B) hepatic cell lines. Expression of VAMP2 mRNA in (C) HCC and adjacent tissues and (D) hepatic cell lines. (A and B) **P<0.01 vs. adjacent normal tissues. (C and D) **P<0.01 vs. HL-7702. VAMP2, vesicle associated membrane protein 2; HCC, hepatocellular carcinoma..

Effect of miR-493-5p mimic on the expression of miR-493-5p in HepG2 cells

The expression of miR-493-5p in HepG2 cells was detected following their transfection with miR-493-5p mimic. The results of RT-qPCR analysis demonstrated that the expression of miR-493-5p in the miR-493-5p mimic transfection group was significantly higher than that in the negative control and normal control groups, indicating that the transfection was effective in inducing the overexpression of miR-493-5p (P<0.01; Fig. 2A).

Figure 2 Effect of miR-493-5p on cell viability. (A) Expression of miR-493-5p in the miR-493-5p mimic transfection, normal ctrl and mimic ctrl groups. (B) Effect of miR-493-5p on cell viability. **p<0.01 vs. normal ctrl and negative ctrl. ctrl, control.

Effect of miR-493-5p on cell viability

In an MTT assay conducted 2 days after transfection of the HepG2 cells with the miR-493-5p mimic, the cell viability of the miR-493-5p mimic transfection group was significantly lower than that of the normal control and negative control groups (both P<0.01; Fig. 2B). These results indicate that the overexpression of miR-493-5p reduced the viability of the HepG2 cells.

Effect of miR-493-5p on the cell cycle and apoptosis

The HepG2 cell line was transfected with miR-493-5p mimic or negative control and 48 h later, the cells were subjected to flow cytometric analysis of the cell cycle and apoptosis. The percentage of cells in the S phase was determined relative to the total number of cells in the G0/G1, S and G2M phases. The flow cytometric analysis demonstrated that the percentage of cells in the S phase in the miR-493-5p mimic transfection group was significantly smaller compared with that in the normal control and negative control groups (both P<0.01; Fig. 3A). On the basis of these results, it may be speculated that miR-493-5p is an inhibitor of HepG2 cell replication. Analysis of cell apoptosis indicated that the proportion of apoptotic cells in the miR-493-5p mimic transfection group was significantly greater than that in the normal control and negative control groups (both P<0.01; Fig. 3B). This assay indicates that miR-493-5p promoted cell apoptosis.

Figure 3 Effect of miR-493-5p on cell proliferation and apoptosis. Flow cytometric results for (A) cell cycle analysis and (B) apoptosis analysis. **P<0.01 vs. the normal control group and the negative control group.

Effect of miR-493-5p on the invasion of HepG2 cells

The HepG2 cells were transfected with miR-493-5p mimic or negative control and 48 h later, they were subjected to Transwell invasion experiments. The results of Hoechst 33342 staining revealed that the number of cells migrating through the polycarbonate membrane in the miR-493-5p mimic transfection group was significantly reduced compared with that in the mimic and normal control groups (both P<0.01). These results suggest that miR-493-5p inhibits the migration of HepG2 cells (Fig. 4).

Figure 4 Effect of miR-493-5p on cell invasion evaluated using a Transwell chamber experiment. Hoechst stained cells in the (A) normal ctrl, (B) mimic ctrl and (C) miR-493 mimic groups. (D) Quantitative data for cell invasion. **P<0.01 vs. the normal ctrl and mimic ctrl groups. ctrl, control; magnification, ×200.

Confirmation that the VAMP2 gene is targeted by miR-493-5p

In order to confirm that the target site of miR-493-5p is located in the 3′-UTR of the VAMP2 gene, recombinant vectors containing wild-type and mutant VAMP2 3′-UTR in the seed region were established (Fig. 5A). These plasmids were transfected together with miR-493-5p mimic or negative control into the 293 cell line. In the cells transfected with wild-type VAMP2, the luciferase activity in the miR-493-5p mimic transfection group was significantly decreased compared with that in the negative control group (P<0.01; Fig. 5B). This indicates that the predicted site of VAMP2 was directly bound by miR-493-5p.

Figure 5 Confirmation of VAMP2 gene targeting by miR-493-5p. (A) Schematic representation of the VAMP2 3′-UTR showing the putative miR-493-5p target site; (B) relative activity of luciferase in different groups; (C) verification by western blot analysis that VAMP2 is regulated by miR-493-5p. a, normal ctrl; b, mimic ctrl; c, miR-493 mimic. **P<0.01 vs. the negative ctrl and normal ctrl groups. VAMP2, vesicle associated membrane protein 2; UTR, untranslated region; ctrl, control..

The regulation of the VAMP2 gene by miR-493-5p was verified by western blot analysis. The results demonstrated that the protein level of VAMP2 in the miR-493-5p mimic transfection group was significantly lower than those in the mimic control and normal control groups (P<0.01; Fig. 5C). These results of the luciferase and western blot assays confirm that the overexpression of miR-493-5p resulted in a reduction of VAMP2 protein levels.

miR-493-5p affects cell behavior by decreasing the expression of VAMP2

HepG2 cells were cotransfected with miR-493-5p mimic and pcDNA3.1-VAMP2. Whether the effects of miR-493-5p on cell behavior are dependent upon the regulation of VAMP2 expression was evaluated by MTT, flow cytometric and Transwell chamber migration assays (Fig. 6).

Figure 6 Verification of the effect of miR-493-5p on cell behavior through the regulation of VAMP2 expression. (A) Expression of VAMP2 mRNA determined by reverse transcription-quantitative polymerase chain reaction; (B) cell viability analysis by MTT assay; (C) cell apoptosis analysis by flow cytometry; (D) invasion analysis by Transwell chamber assay. **P<0.01 vs. the normal ctrl and pcDNA3.1 ctrl groups, ##P<0.01 as indicated. VAMP2, vesicle associated membrane protein 2; ctrl, control.

The expression of VAMP2 mRNA was detected using RT-qPCR, and the results indicated that the expression of VAMP2 mRNA in the pcDNA3.1-VAMP2 transfection group was increased significantly compared with that in the normal and pcDNA3.1 control groups (P<0.01). However, the expression of VAMP2 mRNA in the miR-493-5p mimic + pcDNA3.1-VAMP2 transfection group was significantly reduced compared with that in the pcDNA3.1-VAMP2 transfection group (P<0.01), significantly increased compared with that in the miR-493-5p mimic + pcDNA3.1 transfection group (P<0.01), and slightly reduced compared with that in the normal control and pcDNA3.1 control group (Fig. 6A).

Analysis using MTT and Transwell experiments demonstrated that the cell viability and invasion ability of cells in the miR-493-5p mimic + pcDNA3.1-VAMP2 group was significantly lower than that in the pcDNA3.1-VAMP2 transfection group (P<0.01), significantly higher than that in the miR-493-5p mimic + pcDNA3.1 transfection group (P<0.01), and slightly lower than that in the normal control and pcDNA3.1 control groups (Fig. 6B and D). Flow cytometric analysis indicated that the percentage of apoptotic cells in the miR-493-5p mimic + pcDNA3.1-VAMP2 transfection group was significantly higher than that in the pcDNA3.1-VAMP2 transfection group (P<0.01), significantly lower than that in the miR-493-5p mimic + pcDNA3.1 transfection group (P<0.01) and slightly higher than that in the normal control and pcDNA3.1 control groups (Fig. 6C). These results indicate that the overexpression of VAMP2 reverses the effect of miR-493-5p as an inhibitor of proliferation and inducer of apoptosis.

Discussion

Liver cancer is a highly malignant tumor with an insidious onset, invasive properties, and high recurrence and fatality rates. Liver cancer is often detected at a late stage because it is difficult to diagnose at an early stage and the disease lacks sensitivity to radiation and chemotherapy, which makes surgical removal more challenging. This results in a poor prognosis and endangers human life (42,43). As with other cancers, multiple genes and environmental factors are involved in the initiation and progression of liver cancer. These are associated with numerous pathological processes and molecular events, and result in the transformation of normal liver cells into cancer cells and the occurrence of metastasis (44). Previous studies have shown that non-coding RNA is involved in tumorigenesis (45,46). While certain microRNAs, including miR-21 and miR-765 increase tumor invasion and metastasis and thus function as carcinogenic miRNAs (47), other microRNAs, such as miR-335, miR-146a and miR-543, inhibit tumor occurrence, development and invasion, and function as suppressor miRNAs (48). Notably, miR-493-5p has been reported to serve a tumor suppressor role and prohibit the proliferation and invasion of gastric carcinoma (39). In the present study, in HCC specimens, adjacent normal tissues and liver cancer cell lines, the expression of miRNA-493-5p was measured by RT-qPCR and the results revealed that the level of miR-493-5p in the HCC tissues was significantly lower than that in the adjacent normal tissues. Furthermore, the expression of miR-493-5p in the liver cancer cell lines HCCC-9810, HuH-7 and HepG2 was significantly reduced compared with that in the HL-7702 hepatic cell line. This suggests that miR-493-5p functions as a suppressor miRNA in liver cancer.

A recent study revealed that miR-493-5p was able to reduce the expression of mitotic arrest deficient-2 and was involved in the regulation of cancer cell mitosis as well as the response to paclitaxel chemotherapy (49). Okamoto et al (50) reported that the apoptosis of colon cancer cells induced by miR-493-5p blocked their metastasis from the colon to the liver. In the present study, the role of miR-493-5p in the cell behavior of HepG2 cells was analyzed using MTT, flow cytometry and Transwell assays. The results demonstrate that the overexpression of miR-493-5p decreased cell viability, attenuated cell proliferation and migration and increased apoptosis in HepG2 cells. These data suggest that miR-493-5p may act as a tumor suppressor miRNA in liver cancer.

VAMP2, a member of the soluble N-ethylmaleimide sensitive factor attachment protein receptor family, is responsible for the intracellular transportation and extracellular secretion of vesicle, for example, in the transport of membrane proteins and the secretion of cytokines and hormones (51,52). A number of events in vesicle transport, including tethering, anchoring and membrane fusion, involve the participation of VAMPs (53). A recent study demonstrated that extracellular vesicles and their contents are potential biomarkers for liver cancer (54). In the present study, the expression of VAMP2 was examined by RT-qPCR in HCC tissues and adjacent normal tissues as well as in liver cancer cell lines. The results revealed that the expression of VAMP2 in HCC tissue was significantly higher than that in the adjacent normal tissue and that the expression of VAMP2 in liver cancer cell lines (HCCC-9810, HuH-7 and HepG2) was significantly higher than that in the HL-7702 normal hepatic cell line. The initiation and progression of certain types of cancer is closely associated with compositional changes in the nervous system. For example, a study revealed that acetylcholine is involved in the occurrence and progression of hepatocarcinoma (55), and acetylcholine is transported by vesicles (56). The results of the present study demonstrate that VAMP2 expression is increased in liver cancer tissues, which suggests that VAMP2 serves an important role in the occurrence and development of liver cancer.

Using the online software TargetScan, it was predicted that the VAMP2 gene is a potential target of miR-493-5p. The luciferase reporter system was used to verify the interaction between miR-493-5p and the 3′-UTR of the VAMP2 gene. The interaction was further confirmed by western blot analysis, and the results indicated that the protein level of VAMP2 in the miR-493-5p mimic transfection group was significantly lower than that in the untransfected and negative control groups, which indicates that the expression of VAMP2 was regulated by miR-493-5p. In order to verify whether the effect exerted on cell behavior by miR-493-5p was dependent upon VAMP2 participation, the recombinant vector pcDNA3.1-VAMP2 was established. The results of MTT, flow cytometry and Transwell experiments supported the conclusion that miR-493-5p exerted its effects on cell behavior by regulating the expression of VAMP2. These results demonstrate that the overexpression of VAMP2 reversed the impact of miR-493-5p as an inhibitor of proliferation and accelerator of apoptosis in liver cancer cells.

In conclusion, the present study revealed that miR-493-5p expression was reduced in liver cancer tissues and cell lines, and the expression of VAMP2 was negatively regulated by miR-493-5p. In addition, it demonstrates that miR-493-5p suppresses cell proliferation and cell migration, and promotes cell apoptosis in liver cancer by decreasing the expression of VAMP2. Therefore, the induction of miR-493-5p may be a therapeutic option for patients with liver cancer.

References