Study On Aberrant Promoter Methylation Of TFPI-2Gene In Pancreatic Cancer

Pancreatic carcinoma is one of the most lethal malignancies. Despite recent advancesin pathology, molecular basis and treatment, the overall survival rate remains4%forall stages and races. Though great efforts in primary prevention, combined therapywith operation, radiotherapy and drug treatment, the long term survival rate of thesepatients has not been substantially improved[1-2].The pancreatic cancer is as same asthe other malignant tumor which caused by the envioronmental factor and the heredityfactor.It was demonstrated that the susceptibility of pancreatic cancer heredity wasclosed to the oncogene and antioncogene. The function change of oncogene andantioncogene played the vital role in the pancreatic cancer occurrence. Antioncogeneplayed very important negativited regulated function on control of auxesis,cellproliferation and differentiation,in the meantime it could inhibit tumor growspotentially.If the abnormal phenomenon of antioncogene function deactivated, orantioncogene deletion and mutation happened, the vicious transformation would takeplace on cells in order to the development of the malignant tumor. The variation ofantioncogene included deletion,point mutation,low expression and methylation takedplace on development of the malignant tumor.The gene TFPI-2was first found in placenta by Jandial and Horne and was calledplacenta protein5.With the research to TFPI-2gene develop,TFPI-2not only couldinhibit serine protease,but also inhibit tumor invasion and metastasis. In the light ofthe study,TFPI-2gene was thought has the effecct of inhibit the occurrence anddevelopment of tumor, such as liver cancer, ovarian cancer, breast cancer, stomachcancer, pancreatic cancer, esophageal cancer, fibrosarcoma and glioma. The missingof TFPI-2gene often promoted the invasion and metastasis of tumor[3-6].On the otherhand,some studies show that the inactivation of TFPI-2gene may be due to the highmethylation of TFPI-2gene[7]. For that reason,pancreatic carcinoma and adjacentnormal tissues by ectome, pancreatic carcinoma cell line named Panc-1were regardedas object of study in this investigation. Cell culture, RT-PCR, Western-blot and MSP technique were used to detected expression of TFPI-2gene mRNA, change of TFPI-2gene function and state of methylation on the pancreatic carcinoma and adjacentnormal tissues by ectome,pancreatic carcinoma cell line Panc-1and Panc-1cell linetreated with5-Aza-dC.The result of this study could explain clearly that the closedrelationship between the expression change of TFPI-2gene on the pancreaticcarcinoma tissues and occurance and growth of pancreatic carcinoma.Furthermore, toinvestigate the mechanism of TFPI-2gene action in pancreatic carcinoma genetherapy as the candidacy antioncogene in order to provide the objective observationindex of pancreatic carcinoma patient’s early specificity diagnosis or judgment ofprognosis and establish the theory and the foundation of clinical application ofpancreatic carcinoma gene therapy.Part One： Study on Promoter methylation of TFPI-2Gene ofpancreatic cancer tissue and normal tissue.Objectives To investigate the aberrat methylation of TFPI-2gene in pancreaticcarcinoma and analyse the relationship between the methylation and pancreaticcancer.Methods Use Methylation-specific PCR to detect promoter methylation of theTFPI-2gene in pancreatic cancer tissues and their adjacent tissues from45patientsand normal pancreaitc tissue from8patients.Results Promoter methylation of TFPI-2was found in71.1%（32/45）and31.1%（14/45）of carcinoma adjacent tissues,and the different was significant(P<0.01).The8nomal pancreas tissues has not found aberrant methylation.The positive rate ofTFPI-2methylation was also related to clinical stage,tissue differentiation,tumor size（P<0.05）.Conclusion Aberrant methylation of TFPI-2gene in pancreatic cancer,which mayassociated with occurrence and progression of pancreatic cancer. Part Two: Effect of5-Aza-dC on the expression and methylation ofTFPI-2gene in Panc-1pancreatic cancer cell lineObjective To investigate the effects of5-aza-2,-deoxycytidine(5-Aza-dC), amethylation inhibitor, on the expression and methylation of TFPI-2gene in Panc-1cell lines of pancreatic cancer.Methods Panc-1cell lines was treated with different dosages of5-Aza-dC. TFPI-2gene DNA, mRNA and protein were determined by MSP, RT-PCR, Western blotrespectively.Results MSP detection showed that the TFPI-2gene hypermethylation haseffectively been reversed by5-Aza-dC.Moreover, the expression levels of TFPI-2mRNA treated with5-Aza-dC were increased (0.211±0.087,0.327±0.068and0.609±0.017,respectively).Western blot indicated that5-Aza-dC could recover theTFPI-2protein expression (0.429±0.121，0.675±0.044and1.132±0.124, respectively).These effects within certain extent were dose dependent with statisticalsignificance(P<0.05).Conclusion The hypermethylation of promoter region is a main cause for TFPI-2gene transcriptional inactivation in Panc-1cell lines.5-Aza-dC may effectively causedemethylation and reactivating the gene transcription silenced.