EGFL7 Promotes The Invasion And Metastasis Of Pancreatic Carcinoma By Modulating Epithelial-Mesenchymal Transition(EMT)

Part I The expression of EGFL7 in four human pancreatic cancer cell linesObjective To investigate the expression of EGFL7 in Patu8988, PANC-1, Bx PC-3, Capan-1 and lay the foundation for follow-up study.Methods Four different pancreatic cancer cell lines atu8988, PANC-1, Bx PC-3, Capan-1 were cultured. Detection the expression of EGFL7 in those cell lines above using Real time-polymerase chain reaction(PCR) and Western blot.Results Real-time PCR analysis showed that the m RNA expression of the 4 pancreatic cancer cell lines were:(1.73±0.15),(2.26±0.20),(1.53±0.10),(1.00±0.10) respectively, which confirmd that EGFL7 expressed in PANC-1 has a significant higher level than that in other cell lines.(P<0.05). Western blot analysis also showed that the protein relative quantity of the 4 pancreatic cancer cell lines were:( 0.26±0.03),(0.32±0.04),(0.23±0.02),(0.15±0.02) individually, which further confirmd that the expression of EGFL7 in PANC-1 reached a maximum at protein level.(P<0.05).Conclusions The expression of EGFL7 in PANC-1 was higher than that in the others(Bx PC-3, Capan-1 and Patu8988).Part II Construction and screening of plasmid vector-mediated EGFL7 gene targeted si RNA interference in PANC-1Objective To design and construct plasmid vector-mediated EGFL7 gene targeted si RNA interference in PANC-1 and detect the best inhibitory effectof constructed vectors.Methods According to the encoding sequence of EGFL7 in Gene Bank and principle of si RNA designing, four specific si RNA were designed and synthesized, then via annealing and ligating with GV248 in order. Constructed vectors were stably transfected into PANC-1 cells with LipofectamineTM2000. The expression of EGFL7 was confirmed by Real-time PCR and Western blot.Results The recombinant plasmid vector-mediated EGFL7 genewere successfully constructed, and the inhibitory effect of constructed vector with EGFL7-si RNA-1 was much more obviously than the others.Conclusions EGFL7-si RNA-1 interference plasmid has the best silencing efficiency of EGFL7 on PANC-1 cellsPart III Roles and mechanisms of EGFL7 in the invasion of PANC-1Objective To investigate the changes of invasion abilityof PANC-1 after RNA interference of EGFL7 and biomarkers of EMT.Methods PANC-1 cells were divided into three groups: PANC-1(Control); NCPANC-1(negative control); si-PANC-1(transfected with small interfering RNA of EGFL7). Methyl thiazol tetrazolium(MTT), Transwell assay were used to analyze the proliferation, migration and invasion of PANC-1 cell.Real-time PCR and Western blot were used to detect the expression of EMT markers E-Cadherin, N-Cadherin, Vimentin and Fibronectin in PANC-1, NC-PANC-1, and si-PANC-1 cells,respectively.Results After RNA interference of EGFL7, there was no significant difference on cell proliferation activity among the three groups(P>0.05); the invasion ability of si-PANC-1 cells were decreased significantly(P<0.05).The expression of epithelial biomarker ECadherin was increased and that ofmesenchymal biomarkers N-Cadherin,Vimentin and Fibronectin was decreased(P<0.05).Conclusions EGFL7 may promote the invasion of pancreatic cancer by modulating EMT.