br Cells were seeded in six well
Cells (5 × 105) were seeded in six-well plates and allowed to form a monolayer at 80–90% confluence. The cell monolayer was then scrat-ched using a 10-μl pipette tip. After the detached SPDP were removed by washing, the cells were incubated at 37 °C. The initial scratch area and the remigration of cells to the wound were photographed for 36 h under serum starvation conditions. Each set of conditions was repeated in triplicate.
2.7. Cell invasion assay
Cell invasion assays were performed using Matrigel-coated upper chambers (BD Bioscience). Briefly, cells (2 × 105) suspended in 200 μl of serum-free medium were seeded into the upper chambers. Then, 1 ml of the medium containing 10% serum as a chemo-attractant was added to the lower chamber. After incubation for 24 h at 37 °C, the cells on the upper sides of the filter were gently removed with cotton swabs. The cells that had migrated or invaded the lower surfaces of the filter were fixed in 4% paraformaldehyde for 30 min and stained with 1% crystal violet for another 30 min. Each set of conditions was repeated in tri-plicate.
2.8. Western blotting
Western blotting analysis was performed as previously described (Liu et al., 2016). Anti-human CDH17 was obtained from Abcam (Abcam, Cambridge, UK), and an anti-human phospho-inhibitor of nuclear factor kappa-B (IκB) α, anti-human NF-κB (p65), and anti-human IκBα were purchased from Cell Signalling Technology (Danvers, MA, USA).
The concentration of MMPs in the cell culture medium was de-termined using the commercial Human MMPs Quantikine ELISA Kit (R &D Systems, Minneapolis, MN, USA). The absorbance was measured at 450 nm and corrected using the reading at 540 nm obtained using a Benchmark microplate reader (Bio-Rad, Model 680, Hercules, CA, USA).
2.10. Statistical analyses
The results are presented as the means ± SDs and were analysed using SPSS software (version 17.0). An unpaired student's t-test was
Fig. 1. CDH17 is significantly increased in gastric cancer. (a) High expression of CDH17 observed in GC via IHC (400×). (b) Moderate expression of CDH17 observed in GC via IHC (400×). (c) Low expression of CDH17 observed in GC via IHC (400×). (d) Negative CDH17 expression in para-carcinoma tissue observed via IHC (400×). (e) A graphical quantification of weak, mild and high expression of CDH17 in tumour and para-tumour tissues. (f) Mean and standard deviation of CDH17 mRNA expression levels in GC and para-carcinoma tissues determined through qRT-QCR. The data are shown as 2− Ct values (**P < 0.01). (g) The blood and lymph vessels in the human GC specimens were visualized with the D2-40 antibody using IHC. The brown tubular structures (black arrow) are blood and lymphatic vessels (200×). (h) A significant correlation was found between the CDH17 protein level and the number of blood vessels and lymph vessels in the human GC specimens (**P < 0.01). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
used for comparisons of two groups. One-way analysis of variance was applied for comparisons of multiple groups. P < 0.05 was considered statistically significant.
We analysed CDH17 protein expression in 160 pairs of GC tissues and para-carcinoma tissues via IHC. CDH17 was mainly observed in the cell membrane and cytoplasm of the diﬀerentiated GC tissues (Fig. 1a–c). However, negative or low levels of the CDH17 protein were
IHC of CDH17 expression in GC tissues and para-carcinoma tissues.
Group Number of CDH17 expression Positive rate
detected in para-carcinoma gastric tissues (Fig. 1d). As shown in Table 1, CDH17 was expressed in 68.13% (109/160) of the GC tissues, and this percentage was significantly higher compared with that of the para-carcinoma gastric tissues (21.88%, 35/160) (P < 0.05). A gra-phical quantification of weak, mild and high expression of CDH17 in tumour and para-tumour tissues is shown in Fig. 1e. To determine whether the level of CDH17 mRNA was also upregulated in GC, we employed qRT-PCR using RNA extracted from GC and paired para-carcinoma tissue samples from 84 patients with GC. The data are shown in Fig. 1f. The level of CDH17 mRNA was significantly higher in the GC tissues (6.30 ± 1.65) than in the matched para-carcinoma tissues (3.10 ± 1.05, P < 0.001). This result indicates that the average ex-pression levels of the CDH17 protein and mRNA are significantly in-creased in GC tissues compared with matched para-carcinoma gastric tissues.