| Background:Pancreatic cancer is one of the most common gastrointestinal malignant tumors,which has become the fifth cause of cancer death in male in western countries,thefourth in female.In our country,the incidence and mortality rates of pancreaticcancer are separately ranked the 7th and 6th among all of the cancers.Due to its lowrate of early diagnosis,half of the patients have developed systemic metastasis.Overall,pancreatic cancer carries an unfavorable prognosis with short survival rateand poor quality life due to lacking of effective treatment,like Low resection rate,the high malignant degree of Pathological tissues.The 5-year overall survival rate isonly 4%and the median survival time is 4-5 months.Therefore,to clarify themolecular biology mechanisms under the genesis and development of pancreaticcancer,has important clinical application value to the early diagnosis and treatmentof pancreatic cancer.Tumor angiogenesis is closely related to tumor growth and metastasis.When thetumor is growing with a size larger than 2mm3,its growth is angiogenesis-dependent.However,the blood could take the tumor cells into the circulation system,which falloff from the original site,thus promotes the metastasis,when it is supplying thenutrition to the situ tumor.The study found that a variety of signaling molecules,receptors and signaling pathways involved in tumor invasion,metastasis and newblood vessels formation,including the VEGF,MMPs and ANG-2 which areimportant known activity factors.Due to the effective treatment of pancreatic canceris very scarce,the studies on the blood vessels formation and metastasis may haveimportant clinical value,even become a new target for the treatment of pancreaticcancer.High molecular weight makes it difficult to over-expresse in eukaryotic cells,whichrestrict the deep study of its biological function in pancreatic cancer.However,MUC4 had its own particularity in many aspects,like bio-genesis,genetic structure,molecular evolution and biological behaviors:first,it is consists of several domainswhich have different bio-genesis,in addition,these structural domains areconservative,and have the relative independence between each other.second;second,two dozens of MUC4 transcripts are found until now,and they are present inpancreatic intraepithelial neoplasia and PC but not in the normal pancreas or inchronic pancreatitis.MUC4/Y is one of the several transcripts of MUC4,lacks fragment from exon 2(encoding for the tandem repeat domain),compared with MUC4.Its molecularweight decreases obviously,which makes the overexpression in cell lines be possibleby the lenti-virus system.This research plans to study the function and possiblemechanism of MUC4/Y in pancreatic cancer.On this basis,using gene deletionmutation technology,further in-depth study of MUC4/Y-AMOP domain.We wish that our research could provide some certain theoretical basis inin-depth look at the function of MUC4 in the development and progression ofpancreatic cancer.In addition,our research could provide a new solution toinvestigate the high molecular weight protein.Objective:Constructed the lenti-virus system of MUC4/Y-deletion AMOP(MUC4/Y-AMOP)through gene deletion mutation technique.Confirmed thesubcellular localisation of MUC4/Y-AMOP.On this basis,to investigate relevantmolecular mechanisms of MUC4/Y in tumor angiogenesis and metastasis inpancreatic cancer,and the role of AMOP domain played in.More,to provide the theoretical and technical bases for the research of MUC4.Methods:1?Constructed the over-expression lenti-virus system of MUC4/Y andMUC4/Y-AMOP.Constructed the over-expressed cell lines,through affecting thepancreatic cancer cell lines.2?Real time PCR,western blot,cell immunofluorescence were used to confirm thesub-cellular localisation and the up-expression of MUC4/Y and MUC4/Y-AMOP inpancreatic cancer cell lines.3?CCK8,trans-well assay,tube formation were used to study the function ofAMOP in the tumor angiogenesis and metastasis.4?To further verified the effect of AMOP domain in tumor angiogenesis ofpancreatic cancer through the nude mice matrigel plug assay in vivo.5?To observe the effect of different group cells(EV,MUC4/Y,MUC4/Y-AMOP)in the survival of the nude mice through the orthotopic injection model.6?To compare the effects of different group cells(EV,MUC4/Y,MUC4/Y-AMOP)in the metastasis of the nude mice through the orthotopic injection model.7?Real time PCR,western blot,cell immunofluorescence and ELISA kit were usedto explore the mechanism of the AMOP participating in the malignant biologicalprocess of pancreatic cancer.Results:1?We successfully constructed the over-expressed cell lines of MUC4/Y andMUC4/Y-AMOP.Real-time PCR and western blotting assays were used to confirmthe expression of MUC4/Y or the MUC4/Y without the AMOP domainMUC4/Y-AMOP?at m RNA and protein levels.Cell immunofluorescence assayshowed that the MUC4/Y and the truncated protein(MUC4/Y-AMOP?)couldanchor on the cytomembrane and cytoplasm.It clearly pointed out that the deletion of the AMOP domain would not change the localization of the MUC4/Y protein.2?The results showed that the conditioned medium collected from PC cellsover-expressing MUC4/Y could significantly promote HUVEC tube formation,migration,invasion and proliferation.However,this phenomenon disappeared whenthe AMOP domain was deleted.3?It could also decrease the ability of migration and invasion of PC cells,whendeleted the AMOP domain.4?The matrigel plug assay showed that MUC4/Y could enhance the ability of tumorangiogenesis.There was no obvious change in MUC4/Y-AMOP?group,comparedwith control group.5?The Kaplan-Meier survival curves showed that mice injected withPANC-MUC4/Y cells had lower survival rates than those injected with PANC-EV orPANC-MUC4/Y-AMOP?cells.It reveal that the AMOP domain may play a criticalrole in the malignant biological behaviors of MUC4/Y promoted in pancreaticcancer.6?We confirmed the metastatic effects of the MUC4/Y-AMOP domain in vivo usingorthotopic injection of infected cells into nude mice.The results showed that theincidence rate of metastasis was higher in MUC4/Y group than the other two groups.This stated that the AMOP domain was very important in metastasis,MUC4/Y promoted.7?The expression of NOTCH3 was dramatically increased.In addition,we alsofound that the NOTCH3 signaling was activated,resulted in the increased expressionof its downstream target genes.This effected disappeared when the AMOP domainwas deleted.It demonstrated that the AMOP domain has an important role in tumorbiological process.Conclusion:1?The deletion of the AMOP domain would not change the sub-cellular localizationof the MUC4/Y protein.2?The AMOP domain has an important role in tumor angiogenesis and metastasis.The possible mechanism maybe that it could affect the NOTCH3 signaling andregulate the expression of its downstream target genes.3?Taken together,the method we used,could be a new method to investigate thespecific domains of other high molecular weight membrane proteins.This mightprovide a new train thought in molecular biology study. |
PDF Full Text Request