The gastric carcinoma cell lines MGC803, MKN45, HGC27 and BGC823 were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). The cells were cultured with Dulbecco’s modified Eagle’s medium (DMEM; HyClone Laboratories, Beijing, China) supplemented with 10% fetal bovine serum (FBS; Gibco, Carlsbad, CA, USA), in humidified 5% CO₂ incubator at 37°C.

Total RNA was isolated using RNAiso Plus (Takara Bio, Shiga, Japan). Reverse transcription was performed using RevertAid First Strand cDNA Synthesis kit (Thermo Fisher Scientific) according to the manufacturer’s recommendations. The SYBR-Green-based real-time PCR was then performed in triplicate using CFX-96 sequence detection system (Bio-Rad Laboratories) and gene expression was normalized by GAPDH. Primers are listed in Table I. The relative fold change in RNA expression was calculated using the 2^−ΔΔCt method.

The ADAM17 shRNA sequence was obtained from Sigma Company official website, which was produced by Sangon Biotech, Co., Ltd. (Shanghai, China). The oligo sequence of ADAM17 shRNA included: ADAM17 shRNA (F): 5′-CCG GCC TAT GTC GAT GCT GAA CAA ACT CGA GTT TGT TCA GCA TCG ACA TAGG TTT TTG-3′ and ADAM17 shRNA (R): 5′-AAT TCA AAA ACC TAT GTC GAT GCT GAA CAA ACT CGA GTT TGT TCA GCA TCG ACA TAG G-3′. The ADAM17 shRNA sequence was inserted into the EcoRI and AgeI site of the pLKO.1-TRC plasmid and ligated into the vector (Sigma-Aldrich, St. Louis, MO, USA).

The packaging plasmid psPAX2 and the envelope plasmid pMD2.G were purchased from Sigma-Aldrich. PLKO.1-sh-ADAM17 was cotransfected with psPAX2 and pMD2.G into HEK293T cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). Viruses were harvested 48 h after transfection and viral titers were determined. Cells were infected with 1×10⁶ recombinant lentivirus transduction units in the presence of 8 mg/ml polybrene Sigma-Aldrich. Puromycin (1:10,000 dilutions) was added to cells until the cells in the blank group were non-viable. Cells which survived were stable infected cells.

Cells were seeded in 6-well plates at a density of 4×10⁵ cells/well. After 24 h of culture, the medium was replaced by Opti-MEM (Invitrogen) and cultured. In total, 2 μg plasmid was transfected using 6 μl Lipofectamine 2000 transfection reagent (Invitrogen). After incubation for another 48 h, the treated cells were used to investigate the effect of gene rescue using western blot analysis or Transwell and Cell Counting kit-8 assay.

The cultured cells were rinsed with cold phosphate-buffered saline (PBS) before treated with RIPA lysis buffer at 4°C for 10 min. Then the mixture was centrifuged under 4°C at 12,000 r/min for 15 min. The supernatant was removed and the protein concentration was measured with the BCA method. Approximately 40 μg of protein was loaded in each lane, and separated by 10% SDS-PAGE and then transferred to the PVDF membrane. The membrane was blocked by 5% non-fat milk powder for 1 h at room temperature before overnight incubation with primary antibodies 4°C, followed by the secondary antibody. The antibodies were rabbit anti-ADAM17 (cat. no. 3976), mouse anti-β-tubulin (cat. no. 6181), rabbit anti-N-cadherin (cat. no. 13116), rabbit anti-E-cadherin (cat. no. 3195), rabbit anti-vimentin (cat. no. 5741), rabbit anti-Snail (cat. no. 3879), rabbit anti-TGF-β (cat. no. 3711), rabbit anti-Smad2 (cat. no. 5339), rabbit anti-p-Smad2 (cat. no. 3108), rabbit anti-Smad3 (cat. no. 9523), rabbit anti-p-Smad3 (cat. no. 9520) (all from Cell Signaling Technology, Danvers, MA, USA).

The measurement of viable cell mass was performed with Cell Counting kit-8 (Beyotime Institute of Biotechnology, Shanghai China) according to the manufacturer’s instructions. Briefly, 3,000 cells/well were seeded in a 96-well plate, grown in an incubator (5% CO₂, at 37°C). Respectively in the first, second, third, fourth and fifth day, 10 μl CCK-8 was added to each well, and cells were incubated at 37°C for 2 h and the absorbance was finally determined at 490 nm.

Transfected MGC803 and MKN45 cells were harvested, resuspended in medium and transferred to the 6-well plate (500, 1,000 and 2,000 cells/well) for 10–14 days until large colonies were visible. Colonies were fixed and stained with 0.05% crystal violet for 30 min, and the number of colonies was counted or photomicrographs were taken under phase-contrast microscope.

Cells have grown to confluence in complete cell culture medium. At time 0 h, a scrape wound was created across the diameter with a 10-μl pipette tip followed by extensive washes with medium to remove dead and floating cells. The distance was recorded at 0 and 48 h. Images were captured using an inverted microscope equipped with a digital camera.

For assessing cell migration, 1×10⁵ cells in serum-free media were seeded into the Transwell inserts (Corning) containing 8-μm permeable pores and were allowed to migrate toward 10% FBS-containing medium. Twenty-four to 36 h later, the migrated cells on the bottom of the insert were fixed with 4% paraformaldehyde solution followed by crystal violet (1%) staining. Images were taken after washing the inserts three times with PBS. Five independent fields were counted for each Transwell and the average numbers of cells/field were represented as graphs. For assessing cell invasion, 1×10⁵ cells in serum-free medium were seeded in the Transwell inserts which had been covered with a layer of BD Matrigel basement membrane. The cells were later processed similarly to that of cell migration assay. Finally, invaded cells were counted and the relative number was calculated.

The data are presented as mean ± SD from at least three independent experiments. All statistical analyses were carried out using SPSS Statistics 19 software. Comparisons between the groups were analyzed using the Student’s t-test (two groups) or a one-way ANOVA (multiple groups). P<0.05 was considered statistically significant.

First, we analyzed the ADAM17 expression in MGC803, MKN45, HGC27 and BGC823 cells using real-time PCR and western blot analysis. We found that ADAM17 expression, at both mRNA and protein levels, was higher in MGC803 and MKN45 cells than that in HGC27 and BGC823 cells (Fig. 1A and B). Subsequently, we constructed plasmids sh-ADAM17 and pRK5M-ADAM17 to identify the role of ADAM17 in the development of gastric cancer, and then we examined the knockdown effect of sh-ADAM17 at both protein and mRNA levels using sh-EGFP as a control. The mRNA and protein levels of ADAM17 were significantly decreased in sh-ADAM17 group compared with sh-EGFP group (Fig. 1C and D). Similarly, pRK5M-vector or pRK5M-ADAM17 were transferred into HGC27 and BGC823 cells, then ADAM17 mRNA and protein levels were examined by real-time PCR and western blot analysis, and the results indicated that ADAM17 expression at both mRNA and protein levels was significantly increased in pRK5M-ADAM17 group compared with pRK5M-vector group (Fig. 1E and F).

To investigate the effect of ADAM17 on cell growth in gastric carcinoma cells, we first used the CCK-8 assay to determine the growth curves. The results indicated that ADAM17 knockdown significantly inhibited the proliferation of MGC803 and MKN45 cells (Fig. 2A). Expectedly, overexpression of ADAM17 promotes the ability of proliferation in HGC27 and BGC823 cells (Fig. 2B). To further confirm the effect of ADAM17 on proliferation of gastric carcinoma cells, we evaluated their ability of colony formation in the above mentioned cells. Colonies with strong, highly dense staining and at least 50 cells per colony were counted. We found that ADAM17 knockdown resulted in smaller colonies and lower colony density compared to the control group in both MGC803 and MKN45 cells. The colony formation rates were 202±11 and 50±9 in sh-EGFP and sh-ADAM17 MGC803 cells, and 331±8 and 114±7 in sh-EGFP and sh-ADAM17 MKN45 cells (Fig. 2C). To confirm the above results, the HGC27 and BGC823 cells were transfected with pRK5M-vector or pRK5M-ADAM17 plasmids, respectively. We found that ADAM17 overexpression increased the colony sizes and densities compare to control group, and the number of colonies (defined as ≥50 cells) was 150±10 and 300±13 in BGC823 cells, 77±12 and 153±9 in HGC27 cells, respectively (Fig. 2D). It was confirmed that ADAM17 promotes proliferation and colony formation in gastric carcinoma cells.

Next, we examined the ability of migration by Transwell assays and wound scratch assays. First, we examined the migratory potential of MGC803 and MKN45 cells using Transwell assays. We found that the migration rates were 180±15 and 60±12 in sh-EGFP and sh-ADAM17 MGC803 cells, and 120±14 and 40±11 in sh-EGFP and sh-ADAM17 MKN45 cells (Fig. 3A). To test the effects of ADAM17 knockdown on cell motility, we performed wound scratch assays. For these assays, a scrape wound was created on confluent cultures of MGC803 and MKN45 cells expressing either sh-EGFP or sh-ADAM17. MGC803 and MKN45 cells expressing sh-ADAM17 displayed reduced motility in comparison to MGC803 and MKN45 cells expressing sh-EGFP (Fig. 3B). To confirm the above results, HGC27 cells and BGC823 cells were transfected with pRK5M-vector or pRK5M-ADAM17 plasmids. Conversely, overexpression of ADAM17 promoted the ability of migration in HGC27 and BGC823 cells (Fig. 3C and D). These data suggest that ADAM17 promotes the ability of migration in gastric carcinoma cells.

Then, we examined the effect of ADAM17 on the invasion ability of gastric carcinoma cells using BD Matrigel invasion assays. We transfected MGC803 and MKN45 cells with sh-EGFP or sh-ADAM17 plasmids, and HGC27 and BGC823 cells with pRK5M-vector or pRK5M-ADAM17 plasmids for 72 h. The number of invasive cells were 170±12 and 74±9 in sh-EGFP and sh-ADAM17 MKN45 cells, and 200±6 and 98±8 in sh-EGFP and sh-ADAM17 MGC803 cells (Fig. 4A), indicating that knockdown of ADAM17 obviously inhibited the invasion ability of MGC803 and MKN45 cells. Moreover, the number of invasive cells were 105±9 and 200±5 in vector and pRK5M-ADAM17 BGC823 cells, and 98±11 and 210±8 in vector and pRK5M-ADAM17 HGC27 cells, suggesting that upregulation of ADAM17 significantly enhanced the invasion ability of BGC823 and HGC27 cells (Fig. 4C). Also, ADAM17 knockdown was conduced to downregulate the MMP-2 and MMP-9 at both mRNA and protein levels in MGC803 and MKN45 cells (Fig. 4B and E), while ADAM17 overexpression upregulated the expression of MMP-2 and MMP-9 in BGC823 andHGC27 cells (Fig. 4D and F). The above data suggest that ADAM17 promotes the invasion ability of gastric cancer cells.

The EMT is deemed to be associated with the ability of migration and invasion in cancer cells. Therefore, we detected EMT markers at the protein level by western blotting. Our data suggested that ADAM17 knockdown resulted in downregulation of vimentin, Snail, N-cadherin and upregulation of E-cadherin in MGC803 and MKN45 cells (Fig. 5A). In contrast, ADAM17 overexpression led to upregulation of vimentin, Snail, N-cadherin and downregulation of E-cadherin in BGC823 and HGC27 cells (Fig. 5B). It is confirmed that ADAM17 promotes EMT in gastric carcinoma cells. As TGF-β/Smad signaling is closely related to EMT in cancer, we investigated the effects of ADAM17 on the classic TGF-β/Smad signaling. We found that ADAM17 knockdown downregulated TGF-β, p-Smad2/3 in MGC803 and MKN45 cells (Fig. 5C), while ADAM17 overexpression resulted in upregulation of TGF-β, p-Smad2/3 in BGC823 and HGC27 cells (Fig. 5D). Knocking down or overexpressing ADAM17 had no influence on total Smad2/3 protein. These data suggest that ADAM17 promotes EMT in gastric carcinoma cells via TGF-β/Smad signaling.