| Objective The study aimed to learn the expression of Sp1 in pancreatic cancer.Meanwhile,the correlation between Sp1 expression and the parameters of the patients was also investigated.Finally,the biological significance as well as the relative pro-tumoral mechanisms of Sp1 in pancreatic cancer also was examined.Materialss and Methods 1.To examine the expression and the biological significance of Sp1 in pancreatic cancer.To this end,Sp1 expression was detected in the clinical samples of pancreatic cancer.Subsequently,the correlation between Sp1 and the parameters of the patients were also analyzed.Finally,Sp1 expression and its role in pancreatic cancer cells were examined using western blot,invasion assay and migration assay.2.To examine the role of Sp1 in angiogenesis of pancreatic cancer.To test this,COX-2 expression,together with it correlation with Sp1,was investigated using immunohistochemistry.Then luciferase reporter gene assay and Ch IP assay were conducted to confirm the relationship between them.After that,the si RNA strategies,invasion assay,colony formation assay and tube formation assay were used to evaluate the role of COX-2 in angiogenesis of pancreatic cancer.Finally,western blot,sh RNA and pharmacological inhibition strategies were used to learn the role of EGFR-p38/MAPK signaling in Sp1 resultant COX-2,VEGF expression and finally angiogenesis in pancreatic cancer.3.To explore the role of M2 macrophage and the relationship with Sp1.In addition,the mechanism whereby Sp1 promotes M2 macrophage polarization in pancreatic cancer was also examined.To obtain this,we initially examined the correlation between Sp1 and M2 macrophage in the disease using immunohistochemistry as well as Elisa assay.Then,the mechanisms whereby Sp1-regulated bio-factors promote M2 macrophage polarization were investigated using western blot and Elisa assay.Finally,Spl regulated factors that associat with M2 macrophage polarozation were investigated using human quantitative antibody chip-900.Results 1.Base on the immunohistochemical dada,we found that Sp1 was elevated and responsible for nodal stage in pancreatic cancer.The survival analysis in combine with COX regression assay indicated that elevated Sp1 confers a poor prognosis for the patients,and it also functions as an independent prognostic factor for the disease.Finally,we showed that Sp1 was also upregulated in pancreatic cancer lines associating with enhanced proliferation and invasion.2.The data from western blot and TCGA indicated that Sp1 was positively correlated with COX-2 in pancreatic cancer.As revealed by the luciferase reporter gene assay and Ch IP assay,transcription activation was the dominant machinery to link Sp1 and COX-2 in pancreatic cancer.In addition,colony formation,invasion and tube formation assay indicated that elevated COX-2 in pancreatic cancer could facilitate angiogenesis in a manner dependent on VEGF secretion of the cancerous cells.Finally,si RNA and pharmacological inhibition strategies indicated that EGFR-p38/MAPK functions upstream of Sp1 resultant COX-2 and VEGF expression,and finally angiogenesis in pancreatic cancer.3.The immunohistochemical data together with Elisa assay showed that Sp1 expression was positively correlated with M2 macrophage infiltration within the stroma.The mechanistic study showed that Sp1 regulated bio-factors could activate p38/MAPK signaling of macrophage,leading to M2 macrophage in phenotype.Finally,we obtained 52-elevated and 26-decreased factors upon Sp1 knockdown that associat with M2 macrophage polarization.Conclusion Elevated Sp1 was positive correlated with the aggressive phenotype of pancreatic cancer.For instance,Sp1 could promote the proliferation and migration of pancreatic cancer cells;promote the angiogenic process and an unfavorable macrophage in phenotype.Overall,these data indicated that targeted inhibition of Sp1 in combine with other chemo agent was an alternative approach against pancreatic cancer. |
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