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  • G1 br Knockdown and overexpression of CDH in AGS cells br

    2020-08-30


    3.5. Knockdown and overexpression of CDH17 in AGS cells
    First, we silenced CDH17 expression in AGS G1 using two in-dependent CDH17-specific small interfering RNAs (siCDH17 #1 and siCDH17 #2). As shown in Fig. 2c and d, the western blot results re-vealed that both siCDH17 #1 and siCDH17 #2 could effectively knock down CDH17 expression compared with the control cells. We then transfected the pcDNA3.1(−)/CDH17 plasmid into AGS cells to in-crease CDH17 expression. The results of the western blot analysis shown in Fig. 2e and f indicated that 72 h after transfection, CDH17 protein expression was sharply increased in AGS cells.
    3.6. Aberrant expression of CDH17 influences the proliferation, migration and invasion of AGS cells
    First, to determine whether CDH17 affected AGS cell proliferation, we measured proliferation using CCK-8 assays and observed that cell proliferation was slower in the siCDH17 #1 and siCDH17 #2 groups than in the control group (Fig. 3a). Furthermore, cell proliferation was higher in the pcDNA3.1(−)/CDH17 group than in the control group (Fig. 3b). To investigate whether CDH17 affected AGS cell migration, we performed cell scratch assays and observed that AGS cell migration was significantly inhibited after infection with siCDH17 #1 or siCDH17 #2 (Fig. 3c and d), whereas the cells in the scratched area quickly re-covered from the wound in the pcDNA3.1(−)/CDH17 group (Fig. 3e and f). Furthermore, Transwell chamber assays were performed to ex-amine alterations to the invasive ability of AGS cells after treatment. As shown in Fig. 3g and h, the number of invaded cells was significantly lower in the siCDH17 #1 and siCDH17 #2 groups than in the control group, whereas the number in the pcDNA3.1(−)/CDH17 group was higher than that in the control group (Fig. 3i and j). Taken together, these data demonstrate that aberrant expression of CDH17 can influ-ence the proliferation, migration and invasion abilities of AGS cells in vitro.
    Fig. 2. Knockdown and overexpression of CDH17 in AGS cells. (a) CDH17 protein expression in five distinct differentiated gastric cancer cell lines (MKN28, SGC7901, AGS, KATOIII and HGC27) and the human immortalized gastric epithelial cell line GES-1 was detected via western blotting. (b) The mRNA levels of CDH17 in MKN28, SGC7901, AGS, KATOIII, HGC27 and GES-1 were detected through quantitative PCR. (c, d) The CDH17 protein level in the siRNA groups (siCDH17#1 and siCDH17#2) was significantly decreased (*P < 0.05). (e, f) The CDH17 protein level in the pcDNA3.1(−)/CDH17 group was significantly increased (*P < 0.05).
    3.7. CDH17 significantly influences MMP-2 expression in AGS cells
    To determine the possible molecular mechanisms underlying CDH17-induced AGS cell proliferation, migration and invasion, the members of the matrix metalloproteinase (MMP) family were examined in AGS cells via qRT-PCR. The results showed that the mRNA expression levels of MMP-2 and MMP-9 were significantly decreased in the siCDH17 #1 and siCDH17 #2 groups (Fig. 4a), whereas the mRNA expression levels of MMP1, MMP3, MMP7, MMP8, MMP10, MMP11 MMP13 and MT1-MMP were not noticeably changed compared with the control group (Supplementary Fig. S4a). In the pcDNA(−)3.1/ CDH17 group, only the mRNA levels of MMP-2 and MT1-MMP were significantly increased compared with the control group (Fig. 4b), whereas the mRNA expression levels of MMP1, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11 and MMP13 were not noticeably changed (Supplementary Fig. S4b). A western blotting analysis was subsequently performed to measure the protein levels of MMP-2, MMP-9 and MT1-MMP in AGS cells, and the results revealed that only the MMP-2 protein level in the cell lysate was significantly changed in response to CDH17 knockdown and overexpression (Fig. 4c, d, e and f). Moreover, we performed Quantikine ELISAs to measure the concentrations of MMP-2, MMP-9 and MT1-MMP in the cell culture media, and the results de-monstrated that CDH17 knockdown or overexpression significantly decreased or increased the MMT-2 levels, respectively, strongly sug-gesting a role for CDH17 in modulating the MMP-2 levels (Fig. 4g and h); however, the levels of MMP-9 and MT1-MMP were unchanged compared with the control group (Supplementary Fig. S4c, d, e, f).
    3.8. CDH17 affects the activity of the canonical NF-κB pathway
    The gene that encodes MMP2 is a key target of the NF-κB pathway, and NF-κB signalling is critical for tumour development (Adya et al., 2008; Su et al., 2013). Thus, we sought to examine whether CDH17 acted through NF-κB signalling to influence MMP-2 in GC cells. We performed a western blot analysis to measure the IκBα and NF-κB (p65) 
    protein levels in the cytosolic and nuclear fractions of GC cells. As shown in Fig. 5a and b, CDH17 knockdown significantly increased the IκBα and NF-κB (p65) levels in the cytoplasmic fraction compared with the control group. However, in the nuclear fraction, the NF-κB (p65) levels were decreased in the CDH17 knockdown groups compared with the control group (Fig. 5b). In the pcDNA(−)3.1-CDH17 group, the IκBα and NF-κB (p65) levels in the cytoplasmic fraction were sig-nificantly decreased compared with the control group, whereas the NF-κB (p65) level in the nuclear fraction was increased (Fig. 5c and d). Additionally, the total cellular level of NF-κB (p65) presented no dif-ference between the CDH17-knockdown groups and the control group, as shown in Supplementary Fig. S5a. The same result was found in the comparison of the CDH17-overexpressing groups and the control group, as shown in Supplementary Fig. S5b. These data suggest that the changes in CDH17 levels did not influence the total cellular level of NF-κB (p65) but did affect its subcellular localization, which is consistent with the activation mechanism of the canonical NF-κB pathway. Taken together, these data suggest that CDH17 might function through NF-κB signalling to influence MMP-2.