Study The Expression Of AQP8 In Pancreatic Ductal Adenocarcinoma And Its Mechanism Of Action

Objective To study the expression and clinical significance of the aquaporins 8(AQP8)in pancreatic duct adenocarcinoma And Using AQP8 gene transfected to pancreatic cancer cell line MIA-PACA-2,the AQP8 overexpression was used to explore the mechanism of AQP8 in the development,invasion and metastasis of pancreatic cancer.Methods Immunohistochemistry(IHC)was used to detect the expression of AQP8 in paired paraffin-embedded tissues including pancreatic duct adenocarcinoma tissue(PC)and the non-carcinoma adjacent tissues of pancreatic carcinoma(Non-PC)from 87 patient with pancreatic duct adenocarcinoma and to investigate the relationship between the clinical and pathological features of pancreatic duct adenocarcinoma with AQP8.Meanwhile,Real-time Quantitative PCR(q RT-PCR)and Western blot(Wb)were used to detect the expression of AQP8 m RNA and the protein in 10 paired frozen specimens.In addition,AQP8 gene transfection was carried out in the pancreatic cancer cell line MIA paca-2 in which AQP8 was low expression.The experiment was divided into three groups which respectively were untransfected group(blank control group),the group of transfection air plasmid(negative control)and the group of AQP8 gene transfected(experimental group).q RT-PCR and Western blot were respectively used to detect the expression levels of AQP8 and the tumor cell migration associated proteins(MMP2,MMP9)and the epithelial mesenchymal transtion(EMT)related proteins(E-cadherin,Vimentin)in the three groups.At the same time,cell wound healing assay and cell invasion assay were performed to evaluate the migration and invasion ability of the cells in three experiments.Results AQP8 was mainly expressed in the cytoplasm of the ductal cells in PC.The positive expression rate of AQP8 protein was significantly higher in PC 79.31%(69/87)than in Non-PC 18.39%(16/87)by immunohistochemical method,difference was considered statistically significant(P<0.001).The high expression of AQP8 was correlated with pancreatic tumor size(P<0.001),differentiation(P<0.001),lymph node metastasis(P<0.001)and TNM stage(?/?)(P<0.001).q RT-PCR and Western blot indicated that AQP8 m RNA and AQP8 were significantly unregulated in PC than in Non-PC(P<0.01,P<0.001).QRT-PCR experiment result shows that there were significant statistical difference in m RNA expression of each protein in the group of in AQP8 gene transfected compared with the untransfected group and the group of transfection air plasmid(P < 0.001)in the cytology of pancreatic cancer cell line and there was no statistical difference in the untransfected group and the group of transfection air plasmid(P > 0.05).The results of Wb and q RT-PCR agree with each other.Cell wound healing assay experiment result shows that there was significant statistical difference in the group of AQP8 gene transfected compared with the untransfected group and the group of transfection air plasmid(P < 0.001)and no statistical difference in the untransfected group and the group of transfection air plasmid(P > 0.05).The number of invasive cells were respectively(65.20±6.91),(65.40±5.94),(109.20±11.3)in three groups(blank control group,negative control,experimental group).Similarly,the results of cell invasion assay agree with Cell wound healing assay.Conclusion The expression of AQP8 is correlated with pancreatic tumor size,differentiation,lymph node metastasis and TNM stage(?/?),The detection of AQP8 helps to assess the clinical condition of pancreatic cancer.AQP8,AQP8 m RNA,the tumor cell migration associated proteins(MMP2,MMP9)and the epithelial mesenchymal transtion(EMT)related proteins(Vimentin)were all high expression in the cytology of pancreatic cancer cell line,but the E-cadherin was low expression.meanwhile,cell wound healing assay and cell invasion assay also proved that the overexpression of AQP8 gene can increase the migration and invasion ability of pancreatic cancer cell line MIA-PACA-2.All the experiments suggest that AQP8 was closely related to the occurrence,development,migration and the invasion of pancreatic cancer.AQP8 may be the tumor marker of early diagnosis and the target of biological treatment.