Expression And Biological Function Of FXYD6 In Pancreatic Carcinoma

Pancreatic carcinoma has a very poor prognosis because there is no specific tumor marker and effective therapy,which makes diagnosis rather difficult.It is necessary to find the specific tumor markers with diagnostic value and therapy target for improving the diagnosis and prognosis of pancreatic carcinoma.During the research on oncomolecularbiology of cholangiocarcinoma,we have found that FXYD6(FXYD domain-containing ion transport regulator 6,FXYD6)promotes cell multiplication in cholangiocarcinoma.Pancreas and biliary ducts have the same embryological genesis and histologic characteristics.Tumors of these tissues are similar in pathological typing,metastasis and clinical prognosis.FXYD6 also plays an important role in the development and progression of pancreatic cancer possibly.Objective:(1)To prepare and identify anti-human FXYD6 McAb and FXYD6 shRNA expression vector.(2)To investigate expression of FXYD6 and its correlationship with clinicopathologic characteristics in pancreatic carcinoma. (3)To evaluate the therapeutic efficacy of target gene FXYD6 for gene therapy in pancreatic carcinoma.Methods:(1)Hybridoma cell lines which could secrete anti-human FXYD6 McAb were established by hybridoma technique.Anti-human FXYD6 McAb was produced by ascites revulsion.Purified McAbs were prepared by protein A chromatography.Titer and specificity of McAbs were assessed by ELISA.(2)Immunohistochemistry was used to detect the expression of FXYD6 in pancreatic carcinoma.The relationship between the expression of FXYD6 and clinical pathological factor was analyzed.(3)Small hairpin RNA targeting FXYD6 gene was designed and synthesized based on the microRNA mir-30.The complement form was obtained by annealing and cloned into pCGM30 vector. Recombinant plasmid was identified by using bacterial colonies PCR and sequencing of nucleic acid.(4)Pancreatic cancer cells sw1990 were divided into 7 groups:un-transfected group,blank group,non-vector group and FXYD6 shRNA experimental groups(pCGM30-FXYD6 shRNA-1～4).ShRNA expression plasmid was transfected into sw1990 cells by lipofectamine 2000.Growth condition was detected by MTT.Expression of FXYD6 protein was measured by western blot.Results:(1)One hybridoma cell that can secrete anti-human FXYD6 McAb was established.2.86 mg anti-human FXYD6 McAb with high specificity was prepared with titer of 1:5400.(2)42 of 47 patients with pancreatic carcinoma showed FXYD6 positive expression.The positive rate of FXYD6 in 47 cases of pancreatic cancer tissue was 89.34%(42/47).Expression of FXYD6 in pancreatic carcinoma is higher than in normal pancreas tissue(P＜0.05).According to the degree of FXYD6 expression,47 patients were divided into two groups:high expression group and low expression group.There was no statistically significant difference in age,gender,tumor size and distant metastasis between the two groups(P＞0.05),while there were statistical significances in tumor differentiated degree,lymphatic metastasis,TNM staging(P＜0.05).Spearman's rank correlation analysis showed expression of FXYD6 protein had a present relation to tumor size, differentiated degree,lymphatic metastasis and distant metastasis(P＜0.05).(3) Four small hairpin RNAs targeting FXYD6 gene based on the microRNA mir-30 were designed,synthesized and cloned into pCGM30 vector successfully.FXYD6 gene sequence in the recombinant plasmid was proved by sequencing of nucleic acid.(4)Four shRNA expression plasmids pCGM30-FXYD6 shRNA was transfected into sw1990 cells by lipofectamine 2000 respectively.Compared to each control group,Cell proliferation and expression levels of FXYD6 protein in experimental groups was decreased(P＜0.05).There was no statistically significant difference in cell proliferation and expression levels of FXYD6 protein between experimental groups(P＞0.05).Cell proliferation and expression level of FXYD6 protein was decreased gradually at 24 h,48 h and 72 h(P＜0.05,0.01).Conclusions:(1)FXYD6 McAb and pCGM30-FXYD6 shRNA are prepared successfully,which will be helpful to study the tissue distribution and biological function of FXYD6.(2)FXYD6 may promote proliferation and metastasis of pancreatic cancer,and affect the tumour differentiation.(3)Detection of FXYD6 protein can be used to guide for operation approach and scope in pancreatic cancer. (4)Cell proliferation can be decreased by inhibiting the expression of FXYD6 protein in pancreatic cancer cell sw1990,which shows FXYD6 must be a target of potential therapeutic value for gene therapy.