The Study Of Roles And Mechanism Of Malignant Activity Of MUC4 Splice Variant-MUC4/Y

Part ?Expression of Mucin 4 is associated with progression and prognosis of pancreatic ductal adenocarcinoma PurposeTo study the association between the expression of membrane mucin MUC4(human)and the progression as well as prognosis of pancreatic ductal adenocarcinoma,then to provide evidence for the early diagnosis and prognosis of PDAC.MethodsWe used the techniques of quantitative real-time PCR combined microdissection to precisely extract MUC4 mRNA and analysis it's expression in 116 microdissected foci in tissue specimens paired of cancerous lesion and para-cancerous samples from 57 PDAC patients,studied the difference MUC4 expression in NP,PANIN-LS,PANIN-HS and PDAC,analysed the correlations of MUC4 expression with clinicopathological variables and survival of PDAC patients,and identified prognostic factors with Cox regression model.ResultsThe mRNA expression of MUC4 increased from normal to precancerous lesions to pancreatic cancer.Multivariate Cox regression analysis showed that high-level MUC4 expression(P = 0.008)and tumor node-metastasis staging(P = 0.038)were significant independent risk factors for predicting the prognosis of 57 patients.ConclusionsMUC4 expression is related closely to the progression and prognosis of pancreatic ductal adenocarcinoma,can be served as an independent poor factor to predict the prognosis of PD AC patients.Microdissection-based can contribute to localize and screen precisely the target cells or tissue in specimens to avoid the problem of cellular heterogeneity by neighboring cells.Combined with microdissection,expression of target genes in specimens can be precisely detected.Part IIThe study on roles of malignant activity of MUC4/Y and NIDO-AMOP-vWD domain areas in pancreatic cancer cell linesPurposeTo study the roles of malignant activity of MUC4 splice variant-MUC4/Y and NIDO-AMOP-vWD domain areas in pancreatic cancer cell lines.Methods1.The Lentiviral expression vector of MUC4/Y ? MUC4/Y-QNAV genes(MUC4/Y-QNAV is abbreviation of NIDO-AMOP-vWD deletion gene,MUC4/Y-@NIDO-AMOP-vWD?)were constructed and identified;2.The PANC-1 cell line of pancreatic carcinoma which not expresses MUC4 was infected by the virus particles with target genes,and stable transfected cell lines were then selected in a medium containing puromycin.By using Real-time PCR,western blot and immunofluorescent techniques,expression and locating of target gene was detected at mRNA and protein level.This's proved that stable transfected cell lines of pancreatic carcinoma which over-express MUC4/Y and MUC4/Y-QNAV genes are successfully constructed,named respectively PANC-1-MUC4/Y and PANC-1-MUC4/Y-QNAV.3.Using wild-type PANC-1,stable transfected cell line PANC-1-EV(Empty Vector)as respectively the blank group and the negative group,stable transfected cell line PANC-1-MUC4/Y and PANC-1-MUC4/Y-QNAV as respectively the experimental group,the experimental research was made on their in vitro function to confirm the influence of over-expressed target gene on malignant biological behavior of pancreatic carcinoma:the ability of cell proliferating,anti-apoptosis,invasion and migration,adhesion with matrix protein was detected by CCK8,FACS(Annexin-V/7-AAD fluorescent staining),Transwell assay and adhesion assay respectively.4.Cell lines were re-infected by Lentivirus containing GFP/Luciferase tag and stable transfected cell line over-expressing target gene containing Luciferase,which named PANC-1-EV-Luc?PANC-1-MUC4/Y-Luc and PANC-1-MUC4/Y-QNAV-Luc respectively were successfully constructed by FACS screening.5.On the baisis of the model of cell lines in subcutaneously tumor-bearing nude mice,the influence of MUC4/Y and MUC4/Y-QNAV genes on malignant biological behavior of pancreatic cancer in vivo was explored with monitoring general condition of animals,combined with in vivo optical imaging technologies,gross laboratory observation and pathological examination to compare proliferation and spontaneous metastatic ability of pancreatic cancer cell in vivo.Results1.The construction and identification of stable transfected cell line over-expressing target gene of pancreatic cancer:the significant up-regulation of MUC4/Y gene in stable transfected cell line of PANC-1-MUC4/Y was detected by Real-time PCR,1262 times of the control Empty Vector cell line PANC-1-EV,and the significant up-regulation of MUC4/Y-QNAV gene in stable transfected cell line of PANC-1-MUC4/Y-QNAV increased to 1472 times of that in control Empty Vector cell line PANC-1-EV.Protein of target gene of pancreatic cancer was expressed and located in cytoplasm and membrane,which was the same as that of wild type MUC4 protein.2.The function tests in vitro showed that:MUC4/Y gene correlates positively with the proliferation ability of pancreatic cancer cell in undernourished condition,late anti-apoptotic ability as well as that of promoting carcinoma metastasis.The deletion of 3 adjacent structures attenuates the late anti-apoptotic ability as well as that of promoting carcinoma metastasis of MUC4/Y gene.The over-expression of MUC4 increases the adhesion ability between pancreatic cancer cell and matrix protein such as Collage ??Fibronectin and Laminin.NIDO-AMOP-vWD domain could be a key locus conferring the interaction among MUC4/Y protein and matrix protein such as Collage ??Fibronectin and Laminin.3.Experiments in vivo of the model of subcutaneously tumor-bearing nude mice prove that MUC4/Y has the function of promoting growth and metastasis of carcinoma.The deletion of 3 adjacent structures attenuates the ability of promoting growth,oncogenesis and metastasis of MUC4/Y.Conclusions1.MUC4/Y gene can enhance the malignant function of pancreatic cancer such as proliferation,anti-apoptotosis,adhesion,tumorigenesis in vivo and metastasis.2.NIDO-AMOP-vWD domain is associated with malignant function of pancreatic cancer,the function of MUC4/Y splice variant can not get away from the contribution of the 3 conservative and special domains.Part ?Studies on Transcriptome and Digital gene expression of MUC4/Y of Pancreatic Cancer and Prediction and Verification of the signaling mediated by MUC4/Y on invasion and metastasisPurposeTo construct the transcriptome library of pancreatic cancer cell lines PANC-1-EV and PANC-1-MUC4/Y and offer new clues for experimental and clinical study on pancreatic cancer with bioinformatics analysis.Methods1.The transcriptome library of pancreatic cancer cell lines PANC-1-EV and PANC-1-MUC4/Y is constructed,sequenced and assembled by RNA-high-throughput sequencing approach with bioinformatics analysis.2.Baesd on the results of DGE,the prediction and verification of the invasion and metastasis signaling mediated by MUC4/Y is performed by Real-time PCR,Western blot and the signal transduction pathways blockage experiments.3.To elucidate whether Elk-1,the key transcription factor that cross-links to ERK,AKT and JNK,was involved in the MUC4/Y regulated MMP7 gene transcription,luciferase activity assays were performed.Results1.The transcriptome library of pancreatic cancer cell lines PANC-1-EV and PANC-1-MUC4/Y was successfully constructed.A total of 22748 genes were identified,in which 147 transcripts were differentially expressed:128 of them were up-regulated while 19 of them were down-regulated,involved in 111 signaling pathways.2.The invasion and metastasis signaling of pancreatic cancer mediated by MUC4/Y,(MUC4/Y-ErbB2-FGFR3-Ras-Raf-MEK-MAPK/ERK1/2,MUC4/Y-ErbB2-PI3K-AKT-mTOR,MUC4/Y-ErbB2-Src-FAK-ERK1/2,MUC4/Y-ErbB2-Src-FAK-JNK-cJUN)were predicted and verified.3.MAPK/ERK,AKT and JNK signal transduction-mediated ELK-1 activation was involved in MUC4/Y regulated MMP7 gene transcriptionConclusionsThe differential expression of organism genes in biological processes is studied at global transcriptional level with digital gene expression profiling,thus the tumor-related genes were quickly and efficiently screened and determined by elucidating the biological processes as well as the etiopathogenesis of tumor,so it would be a meaningful approach to uncover critical clues to molecular tumorigenesis and a valuable measure for clinical diagnosis and individual treatment of tumor.