Mechanisms Of MiR-200 Regulating VASH2 Gene To Promote Epithelial Mesenchymal Transformation (EMT) In Pancreatic Cancer Cells

Background:Pancreatic cancer is one of the most malignant tumors, the survival of5years is less than5%. Surgery is the only way to be cured, which is applicable for only20%patients. But the prognosis is still discouraging. So postoperative adjuvant therapy is needed. Vasohibin2(VASH2) was first described as a pro-angiogenic factor, which promoted angiogenesis in the sprouting front in a mouse model. But the effect of VASH2in the tumorigenesis and progression is not clear.Methods:Quantitative real time PCR and western blot was used to measure the expression of VASH2in pancreatic cancer tissues and tumor cell lines. microRNAs targeting VASH2were predicted by bioinformatics, among the results, miR-200a/b/c was the star microRNA and had the most relationship with VASH2. The expression of miR-200a/b/c was measured by qPCR and the correlation between VASH2and miR-200a/b/c was analyzed by statistics. Transfection in vitro with mimics or inhibitors of miR-200a/b/c was used to observe the effect of miR-200a/b/c on VASH2transcription and expression.3’UTR luciferase reporter assay was used to confirm the correlation between miR-200a/b/c and VASH2. Besides, promoter luciferase assay was used to confirm the effect of VASH2on the transcription of miR-200a/b/c.Results:we discovered the differential expression of VASH2in pancreatic cancer tissues, which showed VASH2was higher in panceatic cancers than the adjacent non-cancerous tissues. Besides, miR-200a/b/c, as one of the most matching microRNAs of VASH2, miR-200a/b/c had a negative correlation with VASH2. While miR-200a/b/c mimics or inhibitors was transfected, VASH2was down-regulated in pancreatic cancer cells.3’UTR luciferase reporter assay confirmed the negative correlation between miR-200a/b/c and VASH2. Further promoter luciferase reporter assay also showed the negative regulation of VASH2on miR-200a/b/c transcription.Conclusion:VASH2was upregulated in pancreatic cancers. miR-200a/b/c, as a specific microRNA of VASH2, could regulate VASH2in postotranscriptional level. Besides, VASH2could negatively regulate miR-200a/b/c transcription. Background:previous studies confirmed that VASH2could promote angiogenesis in a model of wound healing. Our research showed that VASH2was more highly expressed in pancreatic cancer tissues than in the adjacent non-cancerous tissues. Significant differential expression of VASH2indicated that VASH2might not only contribute to pro-angiogenesis. Besides, bioinformatics and former experiment confirmed that there was a tight link between VASH2and miR-200family, which mainly took parts in regulating EMT. So we predicted that VASH2might take an important role in EMT.Objective:to measure the variation of EMT phenotype marker in pancreatic cancer cell lines after changing VASH2expression; to observe the effect of VASH2on malignant tranformation of pancreatic cancer cell lines; to explore the possible signal pathway, in which VASH2regulates EMT.Methods:after screening the stable VASH2-overexpressing and VASH2-knockdown cell lines by flowcytometry, to measure the change of EMT marker by qPCR, western blot and immunofluorescence. To observe the effect of VASH2on proliferative, invasive and anti-apoptotic ability and rate of stem cells by MTT, Transwell and flowcytometry.Results:qPCR and western blot confirmed the efficiency of VASH2overexpression and knockdown. Overexpression of VASH2made the epithelial pancreatic cell lines BxPc-3prone to mesenchymal phenotype, such as upregulating Vimentin and down-regulating E-cadherin. Knockdown of VASH2made the PANC-1cells prone to epithelial phenotype, such as lower Vimentin and higher E-cadherin. Functional test verified that VASH2had the ability to pro-proliferation, pro-invasion, anti-apoptosis and promotion of stem cell rate. Further research found that VASH2promoted MMP2and CXCR4experssion so that it increased the invasion ability of pancreatic cancer cells. While in apoptosis assay, VASH2upregulated Bcl2and ABCC1so as to resist Gemcitabine-induced apoptosis. Besides, our research primarily found VASH2promoted EMT probably via Shh signal pathway.Conclusions:VASH2could promote EMT of pancreatic cancer cell lines. Specificly, VASH2could promote proliferation, invasion, anti-apoptosis and stem cell rate. All these above might take place through Shh signal pathway.