The Effect TCP1 On Proliferation,Cell Cycle And Metastasis Of Pancreatic Cancer Cells And Its Mechanism

Objective: Pancreatic cancer is a fatal disease with poor prognosis.The annual mortality rates of pancreatic cancer is almost equal to its incidence rates.The purpose of this research is to find out the effect of TCP1 on proliferation,cell cycle and metastasis of pancreatic cancer cells and revealing its relevant signaling molecules.At last,our study explored the expression of TCP1 in pancreatic carcinoma tissues and adjacent noncancerous tissue,and we evaluate the correlation of TCP1 expression with the clinical pathological features.Methods:1,Western blot was used to detect the expression of TCP1 in three kinds of pancreatic cancer cell lines,PANC-1?Bx PC-3?c FPAC-1.2,Construct Tet-on system mediated sh RNA expression vector targeting TCP1 and package the lentivirus.Use the lentivirus to establish stable pancreatic cancer cell lines.We use Western blot to find out the most suitable concentration of doxycycline to induce knockdown of TCP1.3,To observe the effect of TCP1 on the proliferation of pancreatic cancer cells,MTT assay and clone formation ratio methods were used.Flow cytometry was used to detect the changes of cell cycle after knocking down TCP1 in PANC-1 cell line.The influence of TCP1 on migration were observed using Transwell assay.Western blot were used to detect the possible mechanism of TCP1,the protein expression of c-MYc,m TOR,NF-?B,Raf-1,E-cadherin,Vimentin,ZEB1 and Akt were detected.4,Xenografts in immune deficiency(SCID)BALB/c mice was carried out to evaluate the effect of knockdown of TCP1 on tumorigenicity.5,Expression of TCP1 were examined by immunohistochemistry in all pancreatic carcinoma tissues and adjacent noncancerous tissue.Results:1,The TCP1 expression level of PANC-1 cell lines was reduced significantly after lentivirus transdusion,this means stable transfected pancreatic cancer cell line were established.2,Knockdown TCP1 could repress cells proliferation by 55.8% and reduce the number of colony by 67% in PANC-1 cell lines compared to control group.The silimar result was found in Bx PC-1 cell lines.3,Flow cytometric analysis revealed that knockdown TCP1 block the cell cycle at G0/G1-phase in PANC-1 cell lines.4,The number of migration and invasion cells of PANC-1 cell lines in TCP1 RNAi group were significantly lower than those in negative control group(p < 0.05)by 83.6% and 83.1%.The silimar result was found in Bx PC-1 cell lines.5,Knockdown TCP1 reduce the expression level of p Akt-473,p Akt-308,c-MYc,m TOR,CDK2,CDK6,NF-?B,Raf-1,Vimentin,ZEB1,increase the expression level of E-cadherin.6,The result of xenografts show that knockdown TCP1 inhibit the tumor growth by 78.7%,the average volumes and weight of tumors was significantly reduced when TCP1 RNAi was induced to express.Conclusions:1,Knockdown TCP1 significantly inhibit cells proliferation,colony formation and migration in vitro,block the cell cycle at G0/G1-phase.2,Knockdown TCP1 markedly block multiple cancer-driving pathways,include PI3K/Akt,c-MYc,m TOR,CDK2,CDK6,NF-?B,Raf-1,Vimentin,ZEB1 and increase the expression level of E-cadherin.3,Knockdown TCP1 inhibit the tumor growth in vivo,TCP1 may become a new target for gene therapy of pancreatic cancer.