Effect Of B7-H1on Proliferation Of Human Pancreatic Cancer Cell And The Underlying Mechanism

Pancreatic cancer is a disease with a high mortality rate and short survival, as a result of the early metastases, the aggressive behaviour and the lack of effective therapeutic strategies. Complete surgical resection of the tumour is still a predominant treatment for cure or at least prolonged survival.The administration of chemotherapeutic agents for the treatment of advanced disease has failed and current research focuses on the understanding of molecular pathways in order to evaluate the role of targeted therapy.B7-H1(CD274, PD-L1) is a cell-surface glycoprotein belonging to the B7family of costimulatory molecules, which was first identified in1999. Many studies have confirmed that B7-H1is widely distributed in various human cancers.Cancer cell-associated B7-H1is closely correlated with poor prognosis and a higher malignancy grade. Moreover, B7-H1has been shown to be involved in the protection of cancer cells from activated tumour antigen-specific T lymphocytes through the induction of T lymphocyte anergy or apoptosis after ligation to the T lymphocyte receptor PD-1or other receptors. Recent studies indicate that B7-H1not only mediates the inhibition of the activated T cell response; but also accelerates carcinogenesis in solid tumour cells. ObjectiveAnalysis of B7-H1expression in pancreatic carcinoma and pancreas.To construct B7-H1gene-overexpression and RNA interference (RNAi) lentiviral vectors, and to investigate the effects of B7-H1gene expression on proliferation and migration of human pancreatic cancer cells.MethodsB7-H1expression of30pancreatic duct adenocarcinomas specimens were detected by immunohistochemical staining. PCR amplication technique was used to amplify the full-length open reading frame (ORF) sequence of B7-H1gene,and the single-strand RNAi fragment targeting the B7-H1gene was synthesized. Then the fragments of B7-H1-ORF and B7-H1-RNAi were packaged with lentiviral vector packaging system. The virus was infected into pancreatic cancer cells to establish the cell lines stably expressing B7-H1-ORF or B7-H1-RNAi. The mRNA and protein expressions of B7-H1were detected by RT-PCR and Western blotting, respectively. The growth of curve, cell cycle and the ability of colony formation were performed for detecting the proliferation of human pancreatic cancer cells. Further analyses of the cell cycle-related molecules by Western blotting to confirm this result. RT-PCR analyses the expression of several important EMT-related molecules in the B7-H1-overexpressing cell line.The analysis software SPSS17.0single factor variance (ANVOA) were used and the data are presented as the means±SD. The significance of the differences between the groups was judged using a two-tailed Student’s t-test. All of the tests were considered significant if the P value was less than0.05. ResultsThe immunohistochemical analysis showed that (62.4±9.1)%of the tumour samples expressed B7-H1and that this protein was primarily located in the cytoplasm of the tumour cells. The lentiviral vectors carrying B7-H1-ORF or B7-H1-RNAi were successfully constructed, and the human pancreatic cancer tumour cells with stably B7-H1overexpressing or depleting were established. Compared with the control cells, cell growth rate and colony formation numbers were bigger in B7-H1-ORF PANC-1cells. In contrast, the B7-H1-RNAi BxPC-3cells reduced their cell growth rate and colony formation numbers to those transfected with a negative control shRNA. Cell cycle analysis also confirmed this result.B7-H1overexpression in PANC-1cells caused the upregulation of cyclin D1, CDK4/6, p-Rb and p-JNK, which was reduced in B7-H1-RNAi BxPC-3cells.There was no distinct expression change in EMT-related molecules after the overexpression of B7-H1in PANC-1cells.ConclusionB7-H1expression level is up-regulated in pancreatic carcinoma tissues. The proliferation of pancreatic tumour cells was promoted in the B7-H1-overexpressing cell line, and the down-regulation of B7-H1by shRNA in a pancreatic cancer cell line with high constitutive B7-H1expression decreased cell growth. In addition, the upregulation of cyclin D1by B7-H1overexpression was, at least in part, due to an increase in the phosphorylation level of the JNK pathway. B7-H1overexpression did not contribute to EMT-related molecules expression change.