Gastric cancer tumor tissues with matched adjacent non-tumor tissues were obtained from 27 patients that underwent surgical treatment at Zhejiang Provincial People's Hospital (Hangzhou, China). All patients provided a signed informed consent. None of them had a history of chemotherapy or radiotherapy before sampling, and the diagnosis of gastric cancer was pathologically confirmed. This study was approved by the Institutional Ethics Committee of Zhejiang Provincial People's Hospital.

RPMI-1640 was acquired from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Fetal bovine serum (FBS) was purchased from Gibco (Rockville, MD, USA). Inhibitors AST-1306 and LY294002 were obtained from Selleck Chemicals (Houston, TX, USA). A BCA Protein Assay kit was purchased from Beyotime Institute of Biotechnology (Shanghai, China). Antibodies against β-actin (cat. no. 4970S), ERBB4 (cat. no. 4795S), PI3K (cat. no. 4249S), Akt (cat. no. 9272S), and p70S6K (cat. no. 2708S) and secondary antibodies (cat. no. 7054S) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). All antibody dilution ratio was 1:1,000. MTT was purchased from Beyotime Institute of Biotechnology. Other chemicals were obtained from commercial sources. An IHC staining kit was purchased from Thermo Fisher Scientific, Inc.

Gastric cancer cell lines MNK-45 and SGC-7901 were acquired from the Cell Bank of the Chinese Academy of Sciences (Shanghai Institute of Cell Biology, Shanghai, China). MNK-45 and SGC-7901 cells were cultured in RPMI-1640 containing 10% FBS and maintained at 37°C in an atmosphere of 5% CO₂ in a humidified incubator. MNK-45 and SGC-7901 cells (1.5×10⁵) were seeded in 6-well plates, and incubated for 12 h, then transfected with a lentiviral vector encoding small interfering RNA targeting ERBB4 [lentiviral vector siRNA-ERBB4 (Lv-siRNA-ERBB4)] and negative control lentiviral vector (Lv-NC). Lv-siRNA-ERBB4 and Lv-NC were synthesized by Shanghai GeneChem, Co., Ltd. (Shanghai, China). ViralPlus Transduction Enhancer and Polybrene were used for lentiviral vector transfection.

Cells or tissues were lysed using RIPA buffer containing 1 mM PMSF protease inhibitor mixture. The total protein concentration was assessed using the BCA assay and was equalized with the extraction reagent. Protein (10 µg/lane) samples were separated by 10% SDS-PAGE, and electrotransferred onto PVDF nitrocellulose membranes The membranes were blocked with 5% skim milk for 2 h at 37°C. Then, the membranes were incubated with primary antibodies against β-actin (cat. no. 4970S), ERBB4 (cat. no. 4795S), PI3K (cat. no. 4249S) Akt (cat. no. 9272S) and secondary antibody anti-rabbit IgG (cat. no. 7054S; all from Cell Signaling Technology, Danvers, MA, USA) and diluted 1:1,000. Subsequently, a Gel imaging analysis system and Pierce Western Blot Signal Enhancer (cat. no. 21050; Thermo Fisher Scientific, Inc.) were used to detect the protein bands.

The cell proliferation of MNK-45 and SGC-7901 cells was analyzed using flow cytometry. Briefly, the cells were plated at a density of 1,000-10,000 cells/well in a 96-well plate, and incubated at 37°C for 12, 24, 36, 48 and 60 h. At 12, 24, 48 or 60 h, the cells were incubated with RPMI-1640 medium containing 0.5 mg/ml MTT at 37°C for 4 h. Formazan crystals were dissolved with 150 µl DMSO. The absorbance of each well, including blanks, was measured at 490 nm using an automatic microplate reader subsequent to 10 min of oscillation. The formula of the cell growth inhibition rate was as follows: Inhibition rate = (1 - absorbance of the COE group/absorbance of the blank control group) ×100%.

In order to further examine the effect of ERBB4 on the growth of gastric cancer cells in vivo, the animal imaging technique was used to perform the assay in vivo. Four-week-old nude mice were purchased from the Laboratory Animal Center of Zhejiang University. All animal experiments were performed in accordance with the guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Human gastric cancer cell lines MNK-45 were transfected with Lv-siRNA-ERBB4-GFP and Lv-NC-GFP and then transplanted subcutaneously in nude mice. Tumor cells labeled with green fluorescence were used for in vivo fluorescence imaging after 7 days. The initial inoculum concentration of MNK-45 was increased to 1×10⁷ cells/ml. Tumors were formed ~7 days after the injection. Tumors sizes were measured every 7 days using an in vivo imaging system (PerkinElmer, Inc., Waltham, MA, USA). The fluorescence signal reflecting the tumor sizes of the mice were collected and analyzed using the in vivo imaging system. This assay was approved by the Institutional Ethics Committee of Zhejiang Provincial People's Hospital.

All specimens were fixed in neutral buffered formalin and embedded in paraffin. The specimens were cut into 5-µm sections, deparaffinized in xylene, and rehydrated in graded ethanol. After non-specific binding sites were blocked by exposing them to 10% normal goat serum in PBS for 20 min, the sections were incubated overnight at 4°C using a series of antibodies (ERBB4, 1:200; PI3K, 1:200; Akt, 1:200). Following this incubation, the slides were rinsed with PBS and incubated with biotinylated IgG for 20 min at 37°C. Finally, the sections were slightly counterstained with hematoxylin for 30 sec before coverslip mounting.

The data processing was carried out using SPSS 19.0 statistical software. One-way analysis of variance (ANOVA) with Dunnett's test was used to determine statistically significant differences. All data were expressed as the mean ± standard deviation (x±s). P<0.05 and P<0.01 indicated differences that were statistically significant. All experiments were repeated at least three times.

We analyzed 378 gastric cancer samples obtained from The Cancer Genome Atlas (TCGA) datasets to identify critical ERBB4 involved in gastric cancer. The analysis demonstrated that high ERBB4 levels in TCGA gastric cancer data were closely related to the poor survival rate of gastric cancer patients suggesting that ERBB4 was a prognostic indicator of gastric cancer (Fig. 1A). Quantification of the western blot analysis was performed. In addition, western blot analysis revealed that ERBB4 was significantly overexpressed in MNK-45 and SGC-7901 gastric cancer cell lines compared to immortalized stomach GES cells (Fig. 1B). Furthermore, western blot analysis also revealed that ERBB4 was markedly upregulated in tumor tissues compared to normal stomach tissues and paracancerous tissue (Fig. 1C).

To further study the function of ERBB4 in gastric cancer, lentiviral vectors encoding siRNAs targeting ERBB4 (GenBank accession number NC_000002.12) were constructed by Shanghai GeneChem, Co., Ltd. Three different siRNAs targeting ERBB4 (Lv-siRNA-ERBB4) were designed to ensure the interference effect. The three Lv-siRNA-ERBB4 vectors were transfected into MNK-45 and SGC-7901 cells with ViralPlus Transduction Enhancer and Polybrene so that their specificity for ERBB4 disruption could be determined. The validated Lv-siRNA-ERBB4 was selected for the construction of the lentiviral vector (Fig. 2A-C). A non-silencing sequence was used as a negative control (Lv-NC). Western blot analysis revealed that ERBB4 was markedly silenced by Lv-siRNA-ERBB4 (Fig. 2D and E).

The MTT assay revealed that after Lv-siRNA silenced ERBB4 expression, MNK-45 and SGC-7901 cell viability and cell proliferation was decreased. Compared with the control group, the cells after Lv-siRNA silenced ERBB4 expression exhibited a marked growth inhibition (Fig. 3A and B). Western blot analysis revealed that ERBB4 silencing, inhibited the downstream PI3K/Akt signaling. Furthermore, the same results were obtained with the ERBB4 inhibitor, AST-1306. In addition, the PI3K/Akt signaling inhibitor LY294002 also provided further evidence, constistent with the aforementioned results. (Fig. 3C-F).

To confirm the effects of ERBB4 in vivo, we used a xenograft mouse model. The analysis demonstrated that Lv-NC cells formed significantly larger tumors than the Lv-siRNA-ERBB4 group of cells in the nude mice (Fig. 4A). In addition, the body weight of nude mice exhibited no difference (Fig. 4B). Furthermore, the tumor volume was significantly diminished in the Lv-siRNA-ERBB4 group compared to the Lv-NC group (Fig. 4C). Notably, silencing of ERBB4 expression resulted in a significantly enhanced survival time of the tumor-bearing nude mice (Fig. 4D).

Lv-siRNA-ERBB4 significantly inhibited tumor growth in nude mice. The size of the transplanted tumors in nude mice treated with AST-1306 or transfected with the lentivirus was significantly lower than that in the Lv-NC group (Fig. 5).

To further ascertain the signal transduction pathway mediating the anti-proliferation effects in vivo, xenografts of nude mice were prepared as paraffin-embedded sections. The IHC analysis revealed that the inhibitor of ERBB4 (AST-1306) significantly downregulated the expression level of the proliferation-related proteins PI3K and Akt (Fig. 6).