The Recovered Expression Of WIF1in Gallbladder Could Inhibit Tumor Growth And Promote Tumor Cell Apoptosis, And Can Regulate The Expression Of Proteins Such As EpCAM

BackgroundThe incidence of the gallbladder ranked fifth in the digestive tract malignant tumor, isthe most common malignant tumor of the biliary system. Early diagnosis andtreatment was the only effective mean to cure gallbladder at present, but most patientswas to late to go to a doctor so that was inoperable. The main reasons affecting theprognosis of patients with gallbladder were the rapidity of the gallbladderdevelopment, the easy to occurrence of the invasion of adjacent tissues and metastasis,etc. So, to research the mechanisms of invasion of the gallbladder and the metastasisof gallbladder growth, to find the cure for controlling the invasion and metastasis ofgallbladder in improving the survival of the patients with gallbladder was of greatsignificance.Wnt signaling pathway is a conservative signaling pathway, playing veryimportant roles in diverse physiological processes such as embryonic differentiation,cell proliferation,etc.The pathway normally closed in the normal cells, and its abnormal activation, is oftenassociated with the development of tumor. Previous studies have shown that the WIF-1could inhibit tumor growth by blocking the Wnt pathways as one of the inhibitors ofWnt metabolic pathway in some tumors. But WIF-1expression in the gallbladderand gallbladder for the biological behavior of the influence of the mechanism of itsaction, and there was still no report.This study on the basis of the relevant previous literature reports for the first time todiscuss WIF-1gene in expression and its clinical significance, pathology andprognosis of gallbladder; Research in gallbladder proliferation, invasion andmetastasis process function. Gallbladder in situ nude mice transplantation tumormodel was build. Research the Influence of for WNT WIF-1metabolic pathway to the protein expression. So as to deepen the understanding of the mechanisms ofgallbladder disease and progress, and exploring the gallbladder new therapeutictargets.The first part: WIF-1expression and its significance in clinical pathology andprognosis in the gallbladderObjective: this study was to explore the Relationship of the WIF-1expression andthe clinical pathological characteristics and prognosis of gallbladder.Method: used immunohistochemical to evaluate the expression of the WIF-1proteinin40cases of gallbladder and45cases of chronic cholecystitis.combined with clinicaldata to analyze. used SPSS13.0software package to statistical analysis, count dataused chi-square test, WIF-1expression and clinical pathological features of relationsused chi-square test, P <0.05was statistically significant.Result: WIF-1in the specimens of40cases of gallbladder, only9cases of WIF-1expressed positive,the expression rate was22.5%(9/40), and in50cases ofcholecystitis, the expression of WIF1rate was100%(45/45), the positive ratebetween the two has the remarkable difference (P <0.001). In gallbladder tissue, theWIF-1gene expression and age, gender, tumor size, with or without associated withgallbladder stone, histological type, differentiation degree and clinical pathology datacorrelation was not found (P>0.05), prompted lack of expression may be WIF-1gallbladder occurred early in the process of the event.The second part: The WIF-1gene expression in gallbladder cells and the buildidentification of the overexpression of gallbladder WIF-1cell lineObjective: to test the expression of the WIF–1in cell lines in three gallbladder cellsand to preliminary discuss on the cause of this situation, and then build a gallbladderGBC-SD express WIF-1cell lines, and carried on the test.Method: using RT-PCR, Western Blot, promoter methylation specific PCR to analysis theWIF-1mRNA of the GBC-SD, NOZ and SGC–996cells, the promoter regionmethylation status of the protein WIF-1and WIF-1gene.And through the plasmidtransfection GBC–SD of the gallbladder cell line, used RT-PCR and Weatern-Blotto detect the GBC-SD WIF-1mRNA and protein expression of the gallbladder cellafter transfection.Result:The WIF-1of the GBC-SD, NOZ and SGC–996were missing expression, inthree cells,there were all the promoter methylation status of the WIF-1, this may bethe reason of the missing expression of the WIF-1. after the overexpression plasmidWIF-1functing in gallbladder cell lines GBC-SD, WIF-1mRNA expression wassignificantly raised, WIF1protein expression was restore.The third part: The influence of overexpression WIF1gene to the cellproliferation, invasion, metastasis and apoptosis of the human gallbladdercarcinoma cellObjective: to study the overexpression WIF–1gene on the cell proliferation,invasion, metastasis and apoptosis of the human gallbladder carcinoma cell.Method: the gallbladder GBC-SD cells were divided into three groups: the GBC-SD untreated group, the GBC-SD empty plasmid group (GBC-SD-MOCK) andthe GBC-SD overexpression group (GBC-SD-WIF-1). Used the CCK method todetect the cell proliferation ability of the three groups; Transwell Chambersmovement attacking tested cell migration and invasion ability; Flow cytometryinstrument detected the ability to induce apoptosis of the WIF-1; the change of theMMPs of the Cell supernatant was measured by gelatin zymography.Result: The overexpression WIF-1gene could inhibit the cell proliferation, migration,and invasion of the GBC-SD, and can promote the apoptosis of the GBC-SD (P <0.01). The empty plasmid group and untreated group between cell proliferation andapoptosis of invasion and metastasis ability has no obvious difference (P>0.05).Overexpression WIF1could inhibit the secretion of MMP-2and MMP-9(P <0.01),the ability of the GBC-SD-MOCK and GBC-SD group secreting MMP2,9were no obvious difference (P>0.05).The fourth part:The influence on the overexpression WIF1gene to humangallbladder cells in nude mice subcutaneously transplanted tumor invasion andmetastasisObjective: to study The influence of overexpression WIF-1gene on gallbladder cellsin nude mice subcutaneously transplanted tumor growth and invasion.Method: the gallbladder GBC-SD cells were divided into three groups: the GBC-SD untreated group, the GBC-SD empty plasmid group (GBC-SD-MOCK) andthe GBC-SD overexpression group (GBC-SD-WIF-1),15nude mice wererandomly divided into3groups, each group5only. Collected three groups of cells inlogarithmic phase, and injected100mu l,1x106cells/mouse cells suspension intothe left anterior axillary subcutaneous nude mice in the experiments, mice wereeuthanized at observation after five weeks, measuring weighing volume, collectedspecimen made pathological section.Result:1, successfully build gallbladder subcutaneous cells GBC-SD nude micetransplantation tumor model;2, The tumor rate of3groups of nude mice was100%, overexpression WIF-1set ofnude mice tumor growth and into tumors had accumulated weight were significantlyless than the rest of the other two groups (P <0.001); The empty plasmid group anduntreated negative tumor without obvious difference (P>0.05); showed thatoverexpression WIF-1inhibit the growth of human gallbladder cells in nude mice invivo.The fifth part:The regulation of the overexpression WIF-1for the downstreamprotein and the influence of the micro structure of the cellObjective: to study the overexpression of WIF-1for the influence of the GBC-SD protein expression in the cell, to analysis the WNT mechanism of action of metabolicpathway of the WIF-1and to observe the GBC-SD cell ultrastructure change of theoverexpression WIF1proteinMethod: used qualitative Raybiotech protein chip to check the GBC-SD part changesin protein expression of the overexpression WIF1protein.Observed the ultrastructuralchange by the scanning electron microscopy.Result: WIF-1could lead to DKK-1, and other protein expression change, theseproteins were involved in many important physiological and pathological processes inthe cell, prompted WIF-1might affect the GBC-SD qualitative process betweenepithelial cells; the Scanning electron microscopy (sem) results suggested the GBC-SD-WIF-1lysosomes in the cell secretion was significantly increased, the GBC-SD cells and the GBC-SD-MOCK, you did not see the change.Conclusion:1, The majority of gallbladder tissues were lack of the WIF–1expression, there werenot significant correlation between the WIF-1expression and the tumor size, degreeof differentiation and TNM staging, suggested that the deactivation of the WIF-1might be the early event in the process of occurence and development of thegallbladder.2,The expression were missing of the WIF-1mRNA in the GBC-SD, NOZ-996andSGC-996of the gallbladder cell line, WIF1protein wasnot expressed, this might berelated to its promoter region methylation.Build overexpression WIF-1plasmid to acton human gallbladder GBC-SD cell line, the WIF-1mRNA expression level, WIF1protein expression was restored after it.3, Overexpression WIF-1gene could inhibit the ability of the proliferation,migration and invasion of the GBC-SD cell, and could improve the apoptosis of theGBC-SD,, and at the same time MMP2,9protein expressed in overexpression WIF-1the GBC-SD cells decreased, also proved the inhibitory effect of WIF-1to theGBC-SD.4, The GBC gallbladder cells-SD could be induced by subcutaneous to inoculate the nude mice to produce the transplantation tumor; Overexpression WIF-1gene couldinhibit the growth of human gallbladder cells in nude mice in vivo.5, Overexpression WIF-1could lead to GBC-SD part intracellular protein expressionchange, these proteins involved in cell proliferation, differentiation, and otherphysiological and pathological process, and might be associated with qualitativeprocess between epithelial tumor cells. Scanning electron microscopy (sem) resultsshowed that the lysosomes in the cell significantly increased, which may be related toits protein secretion change.