| Backgroud Due to nonspecific symptoms,as well as highly invasive and metastatic properties at early stage,pancreatic cancer is a type of solid tumor with very low resection rate.Therefore,many patients with unresectable pancreatic cancer have become a major problem faced by clinicians.So far,pancreatic cancer is still lack of effective treatment,because of its high degree of malignancy,as well as chemical and radiation resistance.Generally,the median survival period of advanced pancreatic cancer is about 6-8 months with the gemcitabine-based chemotherapy.However,at research point of view,the chemical treatment is still the most potential treatment with the progress of the study about the mechanism maintaining cell survival in pancreatic cancer.In recent years,abnormal activation of hedgehog(Hh)signaling pathway was found to be a major event during pancreatic cancer development.Studies showed that the activation of Hh signaling pathway in pancreatic cancer was caused by upregulation of SHh gene and the ultimate effect was achieved through mediating the downstream target genes upon nuclear transcription factors Gli1.we have performed a systematic research about the target gene profiles upon Gli1 in a high-metastatic pancreatic cancer cell line through c DNA microarray and found that Hh signaling pathway regulated cell survival through a downstream complex molecular network.In this molecular network,Baculoviral IAP Repeat-containing 3(BIRC3)gene may be one of the key nodes,due to its strong suppression of apoptosis.It is important to study and clarify that Shh-Gli1 signaling might maintain cell survival through mediating transcription of BIRC3 gene in pancreatic cancer cells.the Gli1-dependent BIRC3 pathway might be a potential therapeutic target for pancreatic cancer.Objectives 1.To study the expression of BIRC3 with the overexpression and inhibition of key components in Hh signling pathway in pancreatic cancer cells,and to investigate the effect of Hh sighaling pathway on the transcription of BIRC3 gene in pancreatic cancer cells.2.To study the possible binding sites of Gli1 and BIRC3 promoters in Hh signaling pathway,and to elucidate the specific mechanism of Gli1 in regulating BIRC3 gene transcription.3.To investigate the relationship between Gli1-dependent BIRC3 and the survival of pancreatic cancer cells in vitro.Methods 1.To construct the lentiviral vector of SHh overexpression / Gli1-shRNA interference,transfected into pancreatic cancer cell lines As PC-1,Bx PC3,Panc-1,The expressions of SHh,Gli1 and BIRC3 mRNA in pancreatic cancer cells were detected by quantitative PCR in SHh overexpression and Gli1 interference in Hh pathway.2.The human BIRC3 promoter sequence was searched and downloaded from the eukaryotic promoter database,Bioinformatic methods were used to analyze the possible binding sites of Gli1 enhancer and BIRC3 promoter,pancreatic cancer cells As PC-1 wered transfected with SHh overexpression / Gli1-shRNA interference lentiviral vector,The binding sites between Gli1 enhancer and BIRC3 promoter were analyzed by chromosomal co-immunoprecipitation and dual luciferase reporter gene analysis.3.After pancreatic cancer cell line As PC-1 was overexpressed by SHh / Gli1-shRNA / si BIRC3,The relative expression of SHh,Gli1,BIRC3 and PNCA mRNA were detected by quantitative PCR and The proteins level of SHh,Gli1,BIRC3 and PNCA were detected by western blot.The cell proliferation was tested by MTT.Cell apoptosis was detected by flow cytometry with V-FITC/PI Annexin double staining method,and caspase-3 activity was detected in each group.Results 1.The expression of SHh,Gli1,BIRC3 mRNA were simultaneously increased with the overexpression of SHh in three pancreatic cancer cells,however,with the decline of Gli1 mRNA expression by the Gli1 interference,BIRC3 mRNA level synchronization decreased,indicating that the level of BIRC3 mRNA was positively correlated with the activation of Gli1 SHh signaling pathway.2.Bioinformatics analysis showed that the BIRC3 promoter region of 3 loci with standard Gli1 enhancer sequence "GACCACCCA" matching rate of 89%,chromatin immunoprecipitation showed that Gli1 antibody can enrich the promoter of BIRC3 co precipitation,suggesting that Gli1 and BIRC3 promoter in cell specific binding;Luciferase activity in a dose-dependent manner increased with SHh overexpression and luciferase activity had no significant change with Gli1 interference in all sites,while the 2 and 3 sites are significantly increased with SHh overexpression suggesting that sites 2 and 3 are Gli1 and BIRC3 promoter binding site.3.The results of q RT-PCR and western blotting showed that expression levels of BIRC3 and PCNA genes were increased significantly by SHh/Gli1-expression increasing.Moreover,the BIRC3 knocked-down significantly reversed upregulated PCNA induced by L-SHh transduction.The MTT data showed that cell proliferation were decreased in L-Gli1 i group and increased in L-SHh group.Moreover,si BIRC3 significantly reversed the response of PC cells induced by L-SHh transduction.The FACS data showed that the apoptosis ratio of L-Gli1 i group significantly increased,while the reduce of L-SHh group was not significant.It was impressive that the apoptosis ratio of the L-SHh + si BIRC3 group was increased significantly.The data showed that caspase-3 activity of L-Gli1 i group increased and that of L-SHh group decreased And caspase-3 activity of L-SHh+si BIRC3 group increased.Conclusions 1.The Gli1 promoted transcription of BIRC3 gene via cis-acting elements.2.SHh-Gli1 signaling pathway maintained cell survival partly through this Gli1-dependent BIRC3 modal in pancreatic cancer cells. |
