Samples of surgically resected gastric tumors and corresponding normal tissues from 42 patients with gastric cancer were collected from the Department of Gastrointestinal Surgery, The First Affiliated Hospital of Chongqing Medical University, Chongqing, China, after obtaining written informed consent from each patient. These specimens were momentarily stored in liquid nitrogen for further research. All the gastric cancer patients were admitted to our hospital from February 2012 to April 2013. All 42 patients were determinately diagnosed by a pathological technique and had not received any pre-operative chemotherapy, radiotherapy or immunotherapy. The tumors obtained were divided into 6 groups according to the age (>65 or ≤65 years) and gender (male or female) of the patients, the differentiation grade (low, moderate or high), tumor size (>3cm or ≤3cm), the lymphatic metastasis (positive or negative) and the TNM classification (I + II or III + IV) of tumors. The study was approved by the Ethics Committee of The First Affiliated Hospital of Chongqing Medical University. Total RNA was isolated from the tissues using RNAiso reagent (Takara Bio, Inc., Shiga, Japan). The concentration of the RNA was determined using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).

Three gastric cancer cell lines, SGC-7901, MKN-28, MGC-803, were acquired from the Molecular Medicine and Cancer Research Center of Chongqing Medical University. All the cell lines were maintained in RPMI-1640 medium (Invitrogen, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Invitrogen), 1% penicillin-streptomycin and 2.0 g/l sodium bicarbonate at 36.8°C. The cells were used for the extraction of total RNA and protein when they reached 80–90% confluent. The siRNA vectors targeting FEN1 in the human gastric cells were designed and constructed by Invitrogen. A negative control (NC) siRNA was also used. The sequences of the siRNA were as follows: sense strand, 5′-GGACUUGUAGUCCUGCGAUTT-3′ and antisense strand, 5′-AUCGCAGGACUACAAGUCCTT-3′. The cells were plated in 6-well plates and each well had 3×10⁵ cells. After 24 h, the cells were cultured in an incubator containing CO₂ at 36.7°C, the mixture of siRNA and Lipofectamine™ 2000 (Invitrogen) was added to each well containing cells and according to the manufacturer’s instructions. Fresh medium was used to replace the transfection mixture after 6 h. Forty-eight hours after transfection, the cells were used for further experiments.

The mRNA levels of FEN1 in the tumor tissues and corresponding normal tissues were examined by RT-PCR. Total mRNA from all the tissues was reverse transcribed into complementary DNA using a reverse transcription kit (Takara Bio, Inc.) according to the manufacturer’s instructions. The specific oligonucleotide primers used for FEN1 and those of the internal reference gene, β-actin, were designed using Primer 5.0 software (Premier Biosoft, Palo Alto, CA, USA) and synthesized by Takara Bio, Inc.. The primers for FEN1 were as follows: forward, 5′-AGCCCGTGTATGTCTTTG-3′ and reverse, 5′-AGTCAGGTGTCGCATTAG-3′ and for β-actin forward, 5′-CCTTCTACAATGAGCTGCGT-3′ and reverse, 5′-CCTGGATAGCAACGTACATG-3′. The PCR products were electrophoresed on 1.5% agarose gels. The expression of FEN1 relative to β-actin was measured using a Bio-Rad gel imaging analysis system and analyzed using Quantity One software (Bio-Rad, Hercules, CA, USA). The RT-PCR experiments were repeated independently 3 times.

Immunohistochemical staining was performed on paraffin-embedded tissues from 42 gastric cancer patients. The tissue sections were dewaxed with xylene and rehydrated with alcohol after baking in the 60°C thermostat for 1 h. After rinsing in PBS, the sections were heated with citrate buffer in a microwave oven for antigen retrieval. Normal goat serum was then used to block out non-specific binding after washing in PBS, the endogenous peroxidase activity of the tissues had been already blocked by 3% peroxide. The cells were incubated with primary anti-FEN1 antibody (Epitomics, Burlingame, CA, USA) overnight at a dilution of 1:300 at 4°C. The sections, after a thorough cleaning in PBS, were subjected to 1-h incubation with anti-rabbit secondary antibodies and were then incubated with horseradish peroxidase (HRP)-labeled streptavidin for 30 min. In addition, the tissue sections were stained with diaminobenzidine (DAB) and then redyed by hematoxylin counterstain. Ultimately, the treated sections were observed under a microscope and estimated by the standard described in a previous study (24). The staining was scored according to two standards of FEN1 expression: the staining intensity scored from grade 0 to 3 (0, negative; 1, pale yellow staining; 2, moderate yellow staining; 3, brown staining) and the percentage of positively stained cells (0, no FEN1-positive cells, 1, <10% positive cells; 2, 11–15% positive cells; 3, 51–80% positive cells and 4, >80% positive cells). The final result of FEN1 expression was the product of scores of the staining intensity and the percentage of positively stained cells (negative, 0–1; weakly positive, 1–2; moderately positive, 2–3; strongly positive, ≥3).

Western blot analysis was used to verify the inhibitory effects of FEN1 siRNA. The 3 gastric cell lines were lysed using cell lysis buffer for the isolation of total protein. The bicinchoninic acid (BCA) protein assay kit was used to measured the concentration of the extracted protein of which 30 μl was separated on SDS-PAGE for electrophoresis and electrophoretically transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were blocked in 5% fat-free milk at room temperature and incubated for 2 h with a 1:1,000 dilution of primary rabbit monoclonal antibodies, followed by HRP-conjugated secondary antibody. After rinsing, the blots were detected using a chemiluminescence western blotting detection system. The western blot analysis experiments were repeated independently 3 times.

The cells in the experimental group and control group were trypsinized and placed into 96-well culture plants in the logarithmic phase at a density of 7×10³ cells/well. The MTS kit (CellTiter 96 AQueous one solution reagent; Promega Corp., Madison, WI, USA) was then added to each well and the cells were incubated for a further 4 h before harvesting at 24, 48, 72 and 96 h according to the manufacture’s instructions. Optical density was measured at 490 nm for the absorbance values. These procedures were repeated independently 3 times.

The fluorescein-isothiocyanate-labeled enhanced AnnexinV/propidium iodide Apoptosis Detection kit (Invitrogen) was used to determine the apoptotic rate of the cells in the experimental group and control group according to the manufacture’s instructions. Methotrexate (MTX; Shanghai Yuanye Bio-Technology Co., Ltd, Shanghai, China) was used to induce cell apoptosis. The cells were then subjected to flow cytometry within 1 h.

All the data were analyzed with a paired t-test, Wilcoxon rank sum test and χ² test using IBM SPSS 19.0 software (SPSS Inc., Chicago, IL, USA). A value of P<0.05 was considered to indicate a statistically significant difference.

The FEN1 mRNA expression levels in the gastric tumor and corresponding normal tissues was determined by semiquantitative RT-PCR and the results are shown in Fig. 1. The FEN1 mRNA expression of 6 out of 42 paired gastric tumor tissues is shown in Fig. 1A. Of the 42 samples, 32 (76%) displayed a higher FEN1 expression in the tumor tissues compared to the corresponding normal tissues (Fig. 1B). The relative mRNA expression of FEN1 in the tumor tissues and matched normal tissues was 1.32±0.26 and 1.02±0.34, respectively. The difference between the 2 groups (corresponding to normal and tumor tissues) was statistically significant (P<0.01) as shown in Fig. 1C.

The relative protein expression of FEN1 in the 42 paired tissues was determined by immunohistochemistry. As shown in Fig. 2, FEN1 was primarily expressed in the nucleus and occasionally, in the cytoplasm. As shown in Table I, 32 samples of the 42 tumor tissues (76%) and 8 of the 42 matched normal tissues (19%) had a positive expression of FEN1. A higher expression of FEN1 was observed in the tumor tissues in comparison to the corresponding normal tissues and the difference was statistically significant as determined by the Wilcoxon rank sum test (P<0.01).

The results of immunohistochemical staining and the clinicopathological characteristics of all the patients are presented in Table II. We observed a significant difference between the groups (positive or negative for FEN1 expression) as regards the degree of differentiation (P=0.027), lymphatic metastasis (P=0.001), tumor size (P=0.026) and TNM stage (P=0.020). However, as regards age and gender, no significant differences were observed between the groups (P>0.05). The outcomes intimated that the expression of FEN1 positively correlates with the degree of differentiation, lymphatic metastasis, tumor size and TNM stage in gastric cancer, but has no significant correlation with age and gender, as shown in our 42 cases of gastric cancer.

In order to validate the inhibitory effects of FEN1 siRNA, the expression levels of FEN1 in the SGC-7901 cells were determined by western blot analysis. As shown in Fig. 3, the downregulation of FEN1 expression was observed in the SGC-7901 cells transfected with FEN1 siRNA compared to the cells in the negative control group (transfected with NC siRNA; P<0.01). These results demonstrated that FEN1 expression in the SGC-7901 cells was markedly decreased following transfection with FEN1 siRNA.

The proliferation of the SGC-7901 cells transfected with FEN1 siRNA was determined to be markedly suppressed at 24, 48, 72 and 96 h in comparison to the cells transfected with the negative control (NC siRNA). The data from MTS assay revealed that the proliferation of the SGC-7901 gastric cancer cells was suppressed following the downregulation of FEN1 gene expression by siRNA (Fig. 4).

The apoptotic rates of the 2 groups of cells were detected by flow cytometry, and the results demonstrated that the average apoptotis rate of the SGC-7901 cells transfected with FEN1 siRNA was markedly increased in comparison to the cells in the negative control group (transfected with NC siRNA; P<0.01). The outcomes intimated that the downregulation of FEN1 expression induced the apoptosis of SGC-7901 gastric cancer cells (Fig. 5).