The 373 gastric cancer patients were recruited from the Huashan Hospital and Huadong Hospital Affiliated to Fudan University (Shanghai, China), Renji Hospital and Tongren Hospital Affiliated to Shanghai Jiao Tong University School of Medicine (Shanghai, China) and PLA 175 Hospital (Xiamen, China), between January 2011 and December 2014. The mean age was 62.13 years and the age distribution was 27–90 years. The patients were diagnosed by pathology or imaging by enhanced computed tomography (CT) scan or positron emission tomography-CT scan. A total of 402 cancer-free controls were collected from the Taizhou longitudinal study without a history of other types of cancer or restriction of age and sex (17). All subjects were unrelated Han Chinese. All materials from patients, including peripheral blood samples and epidemiological investigations, were obtained with informed consent, and the whole study was approved by the Ethic Review Committees of Huashan Hospital and School of Life Science, Fudan University (Shanghai, China).

The target genes of the miRNA were obtained according to the miRNA target gene database (TargetScan 6.2 http://www.targetscan.org/vert_62/).

GO analysis was applied in order to organize genes into hierarchical categories and uncover the target genes of miRNA on the basis of biological process (18). In detail, two-sided Fisher's exact test and χ² test were used to classify the GO category, and the false discovery rate (FDR) (19) was calculated to correct the P-value. The present study selected only GOs that had a P-value of <0.01 and a FDR of <0.05.

Based on KEGG, pathway analysis was performed using Fisher's exact test and χ² test, and the threshold of significance was defined by P-values and FDRs. The screening criteria were P<0.05 (20–22).

Genomic (g)DNA of each subject was isolated by using the AxyPrep™ Blood Genomic DNA Miniprep kit (Axygen Biosciences; Corning Incorporated, Corning, NY, USA). Concentration of gDNA was determined using NanoDrop (C723 ND-1000 UV/Vis; NanoDrop Technologies; Thermo Fisher Scientific, Inc., Waltham, MA, USA). DNA samples were detected by agarose gel electrophoresis to ensure the DNA quality and a concentration >20 ng/µl were diluted into a working dilution of 10 ng/µl and added to 96-well plates for subsequent genotyping.

The SNPs were genotyped using matrix-assisted laser desorption/ionization-time of flight-based assay (MALDI-TOF) (23). The primers were designed using MassARRAY Assay Design software (Table I) and synthetized by Shanghai Generay Biotech Co., Ltd (Shanghai, China). The primer sequences are listed in Table I. Genotyping was performed using the MassARRAY Compact system (Sequenom Inc., CA, USA), and the data were analyzed using TyperAnalyzer software version 4.0 (Sequenom Inc.).

A total of two human gastric cell lines (HGC-27 and MGC-803) used in the present study, and the cell lines were purchased from Cell Bank of the Chinese Academy of Sciences (Shanghai, China), which were cultured in Dulbecco's modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc,) with 10% FBS (Biological Industries Israel, Kibbutz Beit Haemek, Israel). The cells were all maintained in a humidified incubator containing 5% CO₂ at 37°C.

miRNA was quantified using the miRcute miRNA qPCR Detection kit (SYBR green; Tiangen Biotech Co., Ltd., Shanghai, China) and analyzed using the 7500 Fast Real-Time Sequence detection system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The primers used for stem-loop reverse-transcription PCR for miR-1269a and 5S were purchased from Tiangen Biotech Co., Ltd. The relative expression levels of the miRNA were normalized to the expression of small nuclear RNA 5S and calculated using 2^{−[(Cq of miRNA) - (Cq of 5S)]} (24).

The wild-type and variant miR-1269a expression plasmids were generated by cloning the wild-type and variant pre-miR-1269a sequence into the retroviral transfer plasmid CMV-MCS-SV40-neomycin (Shanghai GeneChem Co., Ltd., Shanghai, China), respectively. The concentration of DNA plasmids transfected was 1 µg/ml in DMEM with Penicillin-Streptomycin (Gibco; Thermo Fisher Scientific, Inc.). The miRNA-1269a mimics and miRNA-1269a inhibitor of the wild-type and variant type as well as the miR-negative control (NC) were purchased from GenePharma (Shanghai GenePharma Co., Ltd., Shanghai, China). Zinc-finger protein 70 (ZNF70) small interfering (si)RNA and the siRNA NC were purchased from Biotend (Shanghai, China). Transfections were performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol.

HGC-27 and MGC-803 cell lines were seeded in 6-well plate at a suitable density (60–70%) and after 24, 48 and 72 h of transfection. The cells were washed in PBS, harvested by trypsin, and subsequently treated with 100 µl Muse™ Annexin V Dead Cell reagent (EMD Millipore; Billerica, MA, USA) at room temperature in the dark for 20 min. This was followed by detection of apoptosis using Muse™ Cell Analyzer (EMD Millipore).

Proliferation was measured using the cell-counting kit-8 (CCK-8 kit; Dojindo Molecular Technologies, Inc., Shanghai, China). According to the manufacturer's instructions, 2×10³ cells were seeded into 96-well culture plates and allowed to adhere for 24 h at 37°C. The cells were subsequently transfected with 50 nM negative control or siRNA and incubated at 37°C for 24, 48, 72 or 96 h. At the endpoint, 20 µl CCK-8 (5 g/l) was added for a further 4 h at 37°C. A Multiscan GO spectrophotometer (Thermo Fisher Scientific, Inc.) was used to measure the absorbance at 450 nm.

Treated cells were washed twice with ice-cold PBS and then treated with RIPA lysis buffer supplemented with 1 mM phenylmethylsulfonyl fluoride (both from Beyotime Institute of Biotechnology, Haimen, China). Following centrifugation at 10,000 × g for 15 min at 4°C, concentration of the proteins was measured using BCA protein assay kit (Beyotime Institute of Biotechnology). Cell protein lysates were separated on 12% SDS denatured polyacrylamide gel and electro-transferred onto polyvinylidene fluoride membranes. The membranes were blocked with 5% non-fat milk and were incubated with primary antibodies against ZNF70 (zinc-finger protein 70; cat. no. ab49339; 5:1,000; Abcam, Cambridge, UK), GAPDH (cat. no. 5174S, Cell Signaling Technology Inc., Danvers, MA, USA), Bik (cat. no. 4592S, Cell Signaling Technology Inc., Danvers, MA, USA), Bim (cat. no. 2933S, Cell Signaling Technology Inc., Danvers, MA, USA) and Bak (cat. no. 12105S, Cell Signaling Technology Inc., Danvers, MA, USA) at 4°C overnight, which were all diluted to 1:1,000. The membranes were then washed 3 times with TBST (Bioscience Shanghai, Inc., China) and incubated with anti-rabbit immunoglobulin G at room temperature ~1 h, horseradish peroxidase-linked antibody (cat. no. 7074; 1:2,000; Cell Signaling Technology Inc.), according to the manufacturer's instructions. Finally, the images were captured using a gel imaging analysis system (Tanon 4100; Tanon Science and Technology Co., Ltd., Shanghai, China).

All statistical tests were performed using the software SPSS (version 19. 0; IBM Corp., Armonk, NY, USA) and Excel (Microsoft Corporation, Redmond, WA, USA). Using Pearson's chi-squared test of goodness of fit, genotype frequencies were evaluated for Hardy-Weinberg equilibrium. Binary logistic regression was applied to analyze the association between the genotypes of miRNA SNP and the susceptibility to gastric cancer following adjustments for age, sex, smoking status and drinking status. Odds ratios and 95% confidence intervals (CI) were also calculated. Pearson's chi-squared test of independence was then used to investigate the association between the SNPs and qualitative clinical indexes, including age, sex, smoking and alcohol consumption status, and tumor size in gastric cancer patients. A two-sided P<0.05 was considered to indicate a statistically significant difference.

The data from cellular experiments are expressed as the mean ± standard deviation for three independent experiments. Subsequently, ANOVA and multiple comparisons test were used to evaluate the significant difference between three groups of data, and the student's t-test was used to evaluate the significant difference between two groups of data. Statistics was performed with the use of GraphPad Prism (version 6.0; GraphPad Software, Inc., La Jolla, CA, USA). P<0.05 was considered to indicate a statistically significant difference.

The present study included 373 patients with gastric cancer and 402 healthy subjects, and the demographic characteristics were summarized in Table II. It was indicated that there were significant differences between the patients with gastric cancer and the healthy controls in terms of age, sex and smoking status. However, there were no differences between the two groups for alcohol consumption.

As shown in Table III, compared with the wild-type GG, the AA genotype was associated with a significant decreased risk of gastric cancer (P=0.045; OR, 0.610; 95% CI, 0.376–0.990). However, there was no statistically significant association between the GA genotype and gastric cancer risk (P=0.567; OR, 1.106; 95% CI, 1.106 (0.783–1.564). A similar association was identified in the recessive model (AA vs. GG + AG; P=0.014; OR, 0.576; 95% CI, 0.371–0.896). Collectively, the findings indicated that miR-1269a rs73239138 may have a role in decreasing the risk of gastric cancer.

Subsequently, the expression levels of miR-1269a in HGC-27 and MGC-803 gastric cancer cell lines transfected with pre-miR1269a and per-miR1269a variant plasmid were analyzed by RT-qPCR. As shown in Fig. 1, the expression level of miR-1269a was significantly downregulated in cells that were transfected with pre-miR1269a-variant compared with pre-miR1269a wild-type-transfected cells (P<0.0001), indicating that the decreased expression of miR-1269a by the SNP may have contributed to the development of gastric cancer.

To determine the functional alteration of miR-1269a variant in gastric cancer progression, the authors of the present study examined the rate of apoptosis of MGC-803 and HGC-27 cells transfected with wild-type miR-1269a and mimics of miR-1269a variant by flow cytometric (FCM) analysis. As shown in Fig. 2A and B, the proportion of apoptotic cells was significantly decreased in MGC-803 or HGC-27 cells transfected with wild-type miR-1269a mimics after 72 h compared with control miR mimics. However, the apoptotic rate of cells transfected with miR-1269a variant type was significantly increased compared with cells transfected with miR-1269a wild-type (miR-NC vs. miR-1269a vs. miR-1269a variant, 19.10 vs. 11.62 vs. 22.95% in HGC-27; 21.17 vs. 13.97 vs. 20.73% in MGC-803). These results suggested that wild-type miR-1269a may have promoted tumor genesis in gastric cancer through prohibiting the apoptosis of gastric cancer cells.

The authors further detected the expression levels of proteins involved in apoptosis (Bik, Bim and Bak) between cells transfected with wild-type and miR-1269a variant by western blotting. As expected, compared with cells transfected with miR-1269a variant, the expression of Bik, Bim and Bak was downregulated in gastric cancer cells transfected with wild-type miR-1269a. However, the expression of these proteins was upregulated in cells transfected with the wild-type inhibitor compared with cells transfected with inhibitor of miR-1269a variant (Fig. 2C). Collectively, these results indicated that wild-type miR-1269a may have a role in the proliferation of gastric cancer cells by inhibiting apoptosis. However, expression of miR-1269a variant increased the apoptosis of gastric cancer cells and reduced the tumor-promoting effect of miR-1269a.

It is important to investigate potential mRNAs, which are regulated by miR-1269a, which may further evaluate the function of miR-1269a. The potential target genes were obtained using the miRNA target gene database (TargetScan 6.2). Then, the authors used gene ontology (GO) analysis and pathway analysis to exclude any unlikely targets. The authors obtained a total of 23 predicted target genes of hsa-miR-1269a (Table IV), and excluded those by overexpressing miR-1269a in order to investigate which target did not respond (data not shown). As presented in Fig. 3A, a binding site in ZNF70 mRNA 3′-untranslated region (UTR) was identified. Using western blotting, it was demonstrated that ZNF70 expression was downregulated in MGC-803 and HGC-27 cells overexpressing miR-1269a compared with cells transfected with miR-control. However, this effect was reversed in cells overexpressing miR-1269a variant. Similarly, ZNF70 expression was upregulated in cells transfected with miR-1269a inhibitor and reversed in cells transfected with miR-1269a variant inhibitor compared with cells transfected with miR-inhibitor control (Fig. 3B). Therefore, these results suggest that miR-1269a targets and inhibits ZNF70 in gastric cancer cells.

To further confirm ZNF70, as a target of miR-1269a, is involved in the pathology of gastric cancer, the authors of the present study have used siRNA to inhibit the endogenous ZNF70, and the siRNA interference efficiency is presented as Fig. 3C. As shown in Fig. 3D, the CCK-8 analysis revealed that when ZNF70 was suppressed, the growth rates of gastric cancer cells were increased significantly compared with the negative control. This result suggested that ZNF70 may be a tumor suppressor gene and the suppression of ZNF70 may have an important role in miR-1269a-mediated gastric cancer progression.