Study Of Mutual Regulation Between Fascin And HIF-1α And Its Mechanism In The Induction Of Invasion Of Pancreatic Cancer Cells

ObjectivePancreatic cancer is a malignant solid tumor which mainly comes from pancreatic ducts. In recent years, the incidence of pancreatic cancer is increasing stable at home and abroad, and the prognosis is the worst in the digestive system tumor and the survival rate of5years is only3%.Many dates reveal HIF-1α in pancreatic cancer organization has closely relations with prognosis and lymph node metastasis and clinical stages. Fascin, a cytoskeletal protein coing gene, plays an important role in cell migration and adhesion. In priliminary study, we found an over expression of Fascin in pancreatic cancer cells as well as an abscent expression in normal pancreatic cells. Recently, we also found a positive correlation in expression of Fascin and hypoxia inducible factor-1α (HEF-1α) in pancreatic cancer tissue. HIF-1and Fascin may regulate the expression of each other respectively.In this study, we propose to study the mechanism of positive feedback loop between HIF-1and Fascin and the role of Fascin in pancreatic cell invasion and metastasis based on previous research from cellular level.Methods1. Fascin and HIF-1α expression in pancreatic cancer tissue1) The expression of HIF-la and Fascin were examined by immunohistochemistry in tumor tissues of79pancreatic cancer patients, and preliminary analysis the relations between them.2. To explore the effect of HIF-1α on expression of Fascin under hypoxia state and its regulation mechanism.1) Mixed gas (1%2,5%CO2,94%N2) was used for the hypoxia incubator. Knockdown of HIF-1α by RNA interference; Fascin, HIF-1α, VEGF and β-actin mRNA and Protein levels were evaluated by realtime PCR and Western blot, VEGF is the positive control.2) HIF-1α expression plasmids were transfected.3) Chromatin inununoprecipitationassay (ChIP) was performed to detect the binding of HIF-1α to Fascin promoter.4) HIF-1α plasmids and firefly luciferase reporters carrying Fascin promoters were co-transfected and regul ation of Fascin gene was examined by luciferase assay.3. To explore the affect of Fascin on expression of HIF-1α and its regulation mechanism.1) Knockdown of Fascin by RNA interference or transfect Fascin expression plasmids, Fascin, HIF-1α and β-actin Protein levels were evaluated by Western blot, several cell signaling pathways located at upstream of HIF-la were evaluated.2) Add inhibitors of different cell signaling pathways to inhibit.4. To explore the effect of Fascin on invasion ability of pancreatic cancer cells and its regulation mechanism.1) The invasion ability of pancreatic cancer cells were evaluated by Transwell, the proteins related to invasion (MMP-2and MMP-9) and EMT were evaluated by Western blot.ResultsImmunohistochemical analysis showed that Fascin was overexpressed in pancreatic cancer tissues and correlated to the expression of HIF-1α. Hypoxia upregulated the expression of Fascin at mRNA and protein levels.Knockdown of HIF-1α decreased the expression of Fascin. Furthermore, HIF-1α could directly bind to the hypoxia-response element located in the promoter region of Fascin and upregulate the transcription of this promoter. After Knockdowning of Fascin or transfecting Fascin expression plasmids, there will be a same change on the expression of HIF-1α. Furthermore, overexpression of Fascin induced upgrade of HIF-1α through activation of PKC and ERK, because the inhibitors of them could block the effect of Fascin on level of HIF-la expression. After Knockdowning of Fascin by RNA interference or transfecting Fascin expression plasmids, there will be a same change on the invasion ability of pancreatic cancer cells too. And overexpression of Fascin could increase MMP-2expression. The inhibitors of ERK could block the effect of Fascin on level of HIF-la expression and invasion ability.ConclusionHIF-la and Fascin may regulate the expression of each other respectively: HIF-la activated the transcription of Fascin through directly binding to the promoter; Fascin increased the expression of HIF-1α through activation of PKC and ERK. The positive feedback loop between HIF-1and Fascin lead to rising of MMP2which located to downstream, and induced the high aggressive ability of pancreatic cancer cells.