| Background:Because metastasis is closely correlated with the prognosis of patients with gastric cancer, gastric cancer remains a major cause of mortality and morbidity worldwide. The molecular mechanisms of gastric cancer metastasis have not been elucidated, and further understanding of the molecular mechanisms will provide a new target for effective prevention and treatment of gastric cancer.AQP5 is an isoform of AQP family. Ectopic overexpression of AQP5 was seen in a variety of tumors and correlated with the clinicopathological characteristics of patients with tumor. In vitro and in vivo studies showed that ectopic overexpression of AQP5 could play a role in the biological behavior of tumors, especially proliferation and metastasis, which suggests that AQP5 may take part in the metastasis of tumor. However, the role of AQP5 expression in human gastric cancer and its molecular mechanisms remain unclear. To investigate whether AQP5 plays a role in human gastric cancer and its molecular mechanisms, we carried out the study.Objective:1. To investigate the expression of AQP5 in human gastric cancer and the correlation of AQP5 expression with the clinicopathologic characteritstics of patients with gastric cancer.2. Gene recombination technology was used to construct the eukaryotic expression plasmid of pcDNA3.1-AQP5/myc-His. The empty vector plasmid of pcDNA3.1/myc-His (control group) and the recombined plasmid of pcDNA3.1-AQP5/myc-His (AQP5 group) were transfected into gastric cancer cell line AGS using Lipofectamine(?) 2000, respectively. RT-PCR and Western blot were applied to evaluate the expression of AQP5 mRNA and protein in the selected positive cells. Following ectopic overexpression of AQP5 or inhibition of AQP5 by its specific inhibitor, acetazolamide (AZA), the proliferation, apoptosis and migration of AGS cells were analyzed by MTT assay, colony formation assay, flow cytometry (FCM) and wound healing assay.3. Western blot was applied to evaluate the expression of MMP2, MMP9, AKT and p-AKT following ectopic overexpression of AQP5, inhibition of AQP5 by AZA or treatment with LY294002, a specific inhibitor of PI3K/AKT signaling pathway.Methods:1. RT-PCR and Western blot were used to detect the expression of AQP5 in human gastric cancer cell lines including MKN45, MKN28, AGS and SGC7901. Immunohistochemistry was applied to evaluate the expression of AQP5 in 40 specimens of human gastric cancer tissues and corresponding adjacent gastric tissues.2. Gene recombination technology was used to construct the eukaryotic expression plasmid of pcDNA3.1-AQP5/myc-His. The empty vector plasmid of pcDNA3.1/myc-His (control group) and the recombined plasmid of pcDNA3.1-AQP5/myc-His (AQP5 group) were transfected into gastric cancer cell line AGS using Lipofectamine(?) 2000, respectively. RT-PCR and Western blot were applied to evaluate the expression of AQP5 mRNA and protein in the selected positive cells. Following ectopic overexpression of AQP5 or inhibition of AQP5 by its specific inhibitor, acetazolamide (AZA), the proliferation, apoptosis and migration of AGS cells were analyzed by MTT assay, colony formation assay, flow cytometry (FCM) and wound healing assay.3. Western blot was applied to evaluate the expression of MMP2, MMP9, AKT and p-AKT following ectopic overexpression of AQP5, inhibition of AQP5 by AZA or treatment with LY294002, a specific inhibitor of PI3K/AKT signaling pathway.Results:1. Heterogeneous expression of AQP5 mRNA and protein was observed in the four gastric cancer cell lines. The relative intensity values to β-actin of AQP5 mRNA of MKN45, MKN28, AGS and SGC7901 cells was 0.25±0.04,0.19±0.03,0.46±0.05 and 0.59±0.04, and the relative intensity values to P-actin of AQP5 portein of MKN45, MKN28, AGS and SGC7901 cells was 0.30±0.05,0.11±0.06,0.42±0.04 and 0.53±0.06, respecially; The positive expression rate of AQP5 in gastric cancer tissues was 67.5% while corresponding adjacent gastric tissues was 37.5%. Therefore, AQP5 expression was up-regulated in gastric cancer tissues in comparison to corresponding adjacent gastric tissues (χ2=7.218, P<0.05); The positive expression rate of AQP5 in gastric cancer tissues with lymph node metastasis was 84% while the positive expression rate of AQP5 in gastric cancer tissues without lymph node metastasis was 40%(χ2=8.274, P<0.01). Hence, overexpression of AQP5 was correlated with enhanced lymph node metastasis (r=0.455, P<0.01), while there was no correlation of AQP5 expression with other clinicopathologic characteritstics including gender, age, tumor size, depth of invasion and cell differentiation (r=-0.062,0.039,0.232,0.088 and 0.165, respectively, P>0.05).2. Expression plasmid of AQP5 was constructed using pcDNA3.1/myc-His vector, and the authenticity of the construct was verified by DNA sequencing; The expression of AQP5 mRNA and protein in the AQP5 group were notably up-regulated in comparison to the control group (P<0.01); The proliferation and migration of AGS cells were enhanced in comparison to the control group (P<0.01), while there was no difference in the apoptotic rate of cells between the AQP5 group and the control group (P>0.05); The expression of AQP5 mRNA and protein were notably down-regulated in a dose-dependent manner when treated with 0,5,10 and 20 mM of AZA for 24 h (5 mM group and 10 mM group vs 0 mM group, P<0.05; 20 mM group vs 0 mM group, P<0.01; 10 mM group and 20 mM group vs 5 mM group, P<0.05; 20 mM group vs 10 mM group, P<0.05); The proliferation rate of AGS cells was decreased in a dose-dependent manner when treated with 0,5,10 and 20 mM of AZA for 24 h (P<0.01), and the apoptotic rate and relative migratory rate of AGS cells were enhanced in a dose-dependent manner when treated with 0,5,10 and 20 mM of AZA for 24 h (P<0.01).Conclusion:1. Our data suggest that overexpression of AQP5 may play a role in the metastasis of human gastric cancer and AQP5 is a potential therapeutic target against gastric cancer.2. Overexpression of AQP5 facilitated the proliferation and migration of AGS cells, while inhibition of AQP5 by AZA attenuated the proliferation and migration, and induced the apoptosis in gastric cancer AGS cell line, and the inhibitory roles of AZA in the proliferation and migration of AGS cells might be related to down-regulation of AQP5.3. The facilitated effects of AQP5 on the metastasis of gastric cancer might partly be correlated with the regulation of MMP2 and MMP9 expression via the PI3K/AKT signaling pathway.In summary, the facilitated effects of AQP5 on the metastasis of gastric cancer might partly be related to the regulation of MMP2 and MMP9 in gastric cancer through the PI3K/AKT signaling pathway and enhanced proliferation and migration by AQP5 overexpression... |
PDF Full Text Request