The gastric epithelial-derived cancer cell lines AGS and BGC-823 and human embryonic kidney cell line HEK-293T were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). AGS and BGC-823 cells were grown in RPMI-1640 medium (Gibco, Rockville, MD, USA) and HEK-293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco). All cultured media were supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin-streptomycin mix (Sigma, St. Louis, MO, USA) in a humidified atmosphere containing 5% CO₂ at 37°C.

Standard strain H. pylori 43504, was purchased from the ATCC and cultured on rain-heart infusion plates containing 5% goat blood and incubated for 3–4 days in a humidified CO₂ incubator containing 10% CO₂, 5% O₂, and 85% N₂ at 37°C. Thereafter, H. pylori was collected and re-suspended in RPMI-1640 for a concentration of 3×10⁸ colony forming units/ml. Gastric cancer cells were then infected with H. pylori at a multiplicity of infection (MOI) of 1:25, 1:50, and 1:100 and incubated for 6 and 12 h.

Total RNA from cells was harvested with a miRNeasy kit (Qiagen, Dusseldorf, Germany). For analysis of FZD7 mRNA expression, RNA was reverse transcribed into cDNA using M-MLV Reverse Transcriptase (BioTeke, Beijing, China) and RT-PCR was performed using SYBR Green PCR Master Mix (Applied Biosystems, Carlsbad, CA, USA). For analysis of miR-27b expression, RNA was converted into cDNA using miScript reverse transcription kit (Qiagen), and real-time quantitative PCR (RT-qPCR) was conducted using TaqMan miRNA Reverse Transcription kit (Applied Biosystems). GAPDH or U6 small nuclear RNA was used as an internal reference for relative gene expression quantitation. The RT-qPCR reactions were performed in triplicate, and relative gene expression level was determined by using the 2^−ΔΔCt method.

An equal amount of protein from different samples was isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto a PVDF membrane (Bio-Rad, Hercules, CA, USA). The transferred protein on the membrane was confirmed by Ponceau staining solution. Then, the membrane was blocked in 3% nonfat milk for 1 h at 37°C. Primary antibodies, including anti-FZD7 and anti-GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, USA), were added and incubated at 4°C overnight. After three washes with Tris-buffered saline with Tween-20, horseradish peroxidase-conjugated secondary antibodies (1:5,000; Bioss Inc., Beijing, China) were added and incubated for 1 h. The membrane was then developed by use of enhanced chemiluminescence (Pierce, Rockford, IL, USA). Relative protein expression was quantitated by using Image-Pro Plus 6.0 software.

FZD7 was knocked down by transfection of FZD7 siRNA (Santa Cruz Biotechnology), according to the manufacturer's instruction. Briefly, FZD7 siRNA and transfection reagent were mixed in transfection medium for 45 min. Cells were washed with transfection medium and then incubated with the FZD7 siRNA mixture for 6 h. Normal growth media were added and incubated for 24 h. The old media were then replaced with fresh normal-growth media and further incubated for 24–72 h. Negative control (NC) siRNA was used as a control. For miRNA transfection, miR-27b mimics and NC miRNA (GenePharma, Shanghai, China) were transfected into cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) at a concentration of 20 nM and incubated for 48 h. For FZD7 overexpression, pcDNA3.1/FZD7 vectors were transiently transfected into cells using Lipofectamine 2000 (Invitrogen) for 48 h. The transfection efficiency was finally detected by RT-qPCR or western blot analysis.

For the MTT assay, cells were seeded into 96-well plates and transfected with FZD7 siRNA, miR-27b mimics, or pcDNA3.1/FZD7 vectors for 48 h, followed by H. pylori infection for 12 h. MTT solution (5 mg/ml) was added at 20 µl/well. Dimethyl sulfoxide was added at 200 µl/well to dissolve the formazan products. The optical density of the solution was detected using a microplate reader (Bio-Tek Instruments, Winooski, VT, USA) at a wavelength of 490 nm. For the colony formation assay, cells transfected with FZD7 siRNA, miR-27b mimics, or pcDNA3.1/FZD7 vectors followed by H. pylori infection were seeded into a 6-well plate in a growth medium containing 0.3% noble agar (200 cells/well) to form natural colonies for 14 days. The cells were washed with phosphate buffer saline and fixed with 4% paraformaldehyde. The plates were stained with crystal violet (Sigma), and the number of colonies was counted and averaged.

The targeted relationship between miR-27b and FZD7 3′-UTR was detected using a dual-luciferase reporter assay. Briefly, the cDNA fragments of FZD7 3′-UTR containing the miR-27b targeted site were inserted into pmirGLO vectors (Promega, Madison, WI, USA). HEK-293T cells were transfected with pmirGLO-FZD7 3′-UTR and miR-27b mimics or NC miRNA and incubated for 48 h. Cells were harvested and lysed. The activity of firefly and Renilla luciferase was tested with a Dual-Luciferase reporter assay kit (Promega). The WNT signaling activity was examined using Tcf luciferase reporter assays. Briefly, gastric cells were co-transfected with a TOPFlash firefly luciferase reporter vector (Addgene, Cambridge, MA, USA) and phRL-TK Renilla luciferase vectors (Promega) together with FZD7 siRNA, miR-27b mimics, or pcDNA3.1/FZD7 vectors, followed by H. pylori infection. After 48 h of transfection, cells were lysed and the activity of firefly and Renilla luciferase was detected, respectively.

Quantitative data are shown as mean ± standard deviation. Statistical analyses were processed with SPSS version 11.5 software (SPSS Inc., Chicago, IL, USA) with one-way analysis of variance. A p-value of <0.05 was regarded as statistically significant.

To explore whether FZD7 is involved in carcinogenesis related to H. pylori infection, we examined its expression level in two gastric cancer cell lines, AGS and BGC-823, infected with H. pylori. The results showed that both the mRNA (Fig. 1A) and protein (Fig. 1B and C) expression level of FZD7 were highly upregulated in AGS and BGC-823 cells by H. pylori infection in a dose-dependent manner. Further data showed that the mRNA (Fig. 1D) and protein (Fig. 1E and F) expression levels of FZD7 increased in a time-dependent manner. These results indicate that FZD7 was induced by H. pylori in gastric cancer cells.

To investigate the potential biological role of FZD7 in H. pylori-induced carcinogenesis, we silenced the expression of FZD7 by using FZD7 siRNA (Fig. 2A and B) and detected its effect on gastric cancer cell proliferation with H. pylori infection. MTT results showed that H. pylori infection significantly promoted gastric cancer cell proliferation and that FZD7 knockdown could reverse this promotion (Fig. 2C and D). Similarly, H. pylori infection markedly upregulated the colony formation of gastric cancer cells, and FZD7 knockdown reversed this upregulation (Fig. 2E and F). These results suggest that the increased expression of FZD7 induced by H. pylori infection might contribute to H. pylori-related gastric carcinogenesis.

miRNAs have emerged as novel tools for cancer treatment because of their regulatory function on gene expression (30). We sought to identify and characterize novel miRNA that could target and regulate FZD7 expression and were thus involved in gastric carcinogenesis related to H. pylori. Through bioinformatic analysis, we found that miR-27b harbored a putative binding site for FZD7 3′-UTR (Fig. 3A). This miRNA attracted our interests because of its critical role in tumorigenesis of various cancer types (29). To validate whether FZD7 is a bona fide target of miR-27b, we performed a dual-luciferase reporter assay using two luciferase reporter vectors containing the putative miR-27b binding sites in the wild-type FZD7 3′-UTR (WT) and mutant 3′-UTR (MT). The results showed that co-transfection of HEK-293T cells with miR-27b mimics and luciferase reporter vectors containing the WT FZD7 3′-UTR led to a significant decrease in luciferase activity, compared to NC miRNA transfection (Fig. 3B). In contrast, the luciferase activity of luciferase reporter vectors containing the MT FZD7 3′-UTR was not affected by miR-27b mimics transfection (Fig. 3B). The data imply that miR-27b could target the 3′-UTR of FZD7 directly. Next, we detected the expression of miR-27b in H. pylori-infected cells and found that miR-27b expression was significantly decreased by H. pylori infection in AGS and BGC-823 cells, which could be upregulated by transfection of miR-27b mimics (Fig. 4). To investigate whether miR-27b regulated FZD7 expression, we overexpressed miR-27b by transfection of miR-27b mimics in AGS and BGC-823 cells. The results showed that both the mRNA (Fig. 5A and B) and protein (Fig. 5C and D) expression of FZD7 that was upregulated by H. pylori infection was significantly decreased by miR-27b overexpression in AGS and BGC-823 cells. Taken together, these results demonstrated that miR-27b regulated FZD7 expression.

The regulation of FZD7 by miR-27b is involved in H. pylori-induced cell proliferation. As previously described, H. pylori infection significantly promoted the expression of FZD7, and FZD7 was verified as a direct target gene of miR-27b. We thus speculated that miR-27b may play an important role in H. pylori-induced cell proliferation by regulating FZD7 expression. To test the hypothesis, we overexpressed miR-27b in H. pylori-infected cells and detected its effect on cell proliferation. As expected, the results showed that the cell proliferation (Fig. 6A) and colony formation (Fig. 6B) of gastric cancer cells promoted by H. pylori infection could be significantly reversed by miR-27b overexpression. However, restoring expression of FZD7 (Fig. 7A and B) in H. pylori-infected cells significantly reversed the inhibitory effect of miR-27b overexpression on H. pylori-induced cell proliferation (Fig. 7C and D). In summary, these results indicate that miR-27b inhibited H. pylori-induced cell proliferation directly through targeting miR-27b.

A previous study has reported that H. pylori infection is associated with activation of the WNT signaling pathway that contributes to carcinogenesis (12). We speculated that the increased expression of FZD7 induced by H. pylori infection might contribute to the activated WNT signaling pathway. To test this hypothesis, we suppressed the expression of FZD7 by FZD7 siRNA or miR-27b overexpression and detected their effect on the WNT signaling pathway. The results showed that H. pylori infection significantly increased the activity of the WNT signaling pathway, but this promotion was significantly decreased by FZD7 knockdown or miR-27b overexpression in AGS (Fig. 8A) and BGC-823 (Fig. 8B) cells. Further data showed that the inhibitory effect of miR-27b on WNT activity was significantly reversed by restoration of FZD7 expression (Fig. 8C and D). Taken together, the promoted FZD7 expression induced by H. pylori infection contributed to the activated WNT signaling pathway, which could be directly inhibited by miR-27b overexpression.