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  • br Fig CDH is significantly

    2020-08-30


    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. 
    3. Results
    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 14003-96-4 of the differentiated GC tissues (Fig. 1a–c). However, negative or low levels of the CDH17 protein were
    Table 1
    IHC of CDH17 expression in GC tissues and para-carcinoma tissues.
    Group Number of CDH17 expression Positive rate
    cases
    Positive Negative
    tissues
    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.
    3.2. Relationship between CDH17 and clinicopathological factors
    The relationship between CDH17 protein expression and selected clinicopathologic factors is shown in Table 2. High expression (+++ to ++++) of CDH17 was correlated with lymph node metastasis (P < 0.05) and the TNM stage (I–IIa vs. IIb–IIIc, P < 0.05) but not with sex, age, tumour size, invasion depth, Lauren type, or degree of differentiation.
    Table 2
    Relationship between CDH17 and the clinicopathological characteristics of GC tissues.
    Clinicopathologic n CDH17 expression X2 value P value
    variables
    Sex
    Age
    Tumour size
    Invasion depth
    Lauren type
    Lymph node metastasis
    TNM stage
    Degree of differentiation
    3.3. CDH17 expression is positively associated with the number of blood vessels and lymph vessels in GC tissues
    Because CDH17 expression was correlated with lymph node me-tastasis in the clinical samples, we further examined the relationship between CDH17 expression and the number of vessels (blood and lymph) in the clinical samples. The blood and lymph vessels in the human GC specimens were visualized with the D2-40 antibody through IHC (Fig. 1g). A significant correlation was observed between the CDH17 protein levels and the number of blood and lymph vessels in the human GC specimens (**P < 0.001, Fig. 1h).
    3.4. CDH17 expression in distinct differentiated GC cell lines
    We measured the CDH17 protein levels in five distinct differentiated GC cell lines (MKN28, SGC7901, AGS, KATOIII and HGC27) and the human immortalized gastric epithelial cell line GES-1 through a wes-tern blot analysis. The results showed that the intermediately differ-entiated cell lines SGC7901, AGS and KATOIII expressed approximately the same level of CDH17 protein, whereas the highly differentiated cell line MKN28 expressed a relatively high level of CDH17 protein; how-ever, low CDH17 protein expression was detected in the un-differentiated cell line HGC27 and the immortalized gastric epithelial cell line GES-1 (Fig. 2a). The results were corroborated by qPCR ana-lysis (Fig. 2b). In the present study, we aimed to explore the changes in the malignant biological behaviour of tumour cells in response to CDH17 knockdown and overexpression; therefore, we used the AGS cell line in the experiments described below.