Screening Of Mucinous Cystic Neoplasms Of Pancreas Related MicroRNAs And Establishment Of The Cell Lines Of Mucinous Cystic Neoplasms Of Pancreas

Background：Mucinous cystic meoplasms of pancreas (MCNs) are benign lesions of cysticneoplasms of pancreas with underlying malignancy. Once detected, it should beresected at early stage for favorable prognoses and low recurrence rates. Whenprogressing, some people may loss surgery opportunities and the neoplasms may notbe sensitive to chemotherapy drugs or radiotherapy treatment. So it is significant forpatients of MCNs if making precise diagnosis. But the pathogenesis has not beenelucidated until now, and no biomarkers with better sensitivity and specificity can beused to confirm histological type of neoplastic tissues. microRNAs (miRNAs), asendogenous small non-protein-coding RNAs,19-25nt, can regulate biologicalfunctions abroadly though targeting down stream mRNA3’UTR regions. Differentlyexpressioned miRNAs have been reported in hepatocellular Carcinoma, gastic cancer,colon cancer, esophageal carcinoma and so on.There are also aberrant expressions ofmiRNAs playing important roles in pancreatic diseases such as acute pancreatitis(AP) or chronic pancreatits (CP), intraductal papillary mucinous neoplasia (IPMN),pancreatic intraepithelial neoplasia (PanIN) and pancreatic cancer. Accordingly, inthis study we focus on differently expressions of miRNAs and their putative targetgenes in MCNs to explore the mechanisms of miRNAs, which can also provideevidence for the pathegenesis of neoplasms and seeking biomarkers at early stage fordiagnosis.Objective:Screen aberrant expressions of miRNAs, predict putative target genes andestablish the cell lines of mucinous cystic neoplasms of pancreas initially.Methods：1. The tissue samples of patients who underwent surgery were obtained at ourChanghai Hospital from May to December in2011. Total miRNAs of these pairedsamples (neoplastic tissues and the corresponding adjacent normal tissues) wereextracted after histological diagnoses were verified, and qualified miRNAs were usedfor microarray screening.2. Verify significantly differentially expressed miRNAs screened by the microarrays in MCNs with Reverse Transcription-Polymerase Chain Reaction(RT-PCR), using Taqman miRNA probes.3. Identify the putative genes with target prediction engines miRGEN (includingTargetscan, picTAR, miRanda) and analyse their relative functions for furtherstudies.4. Methods of explant culture, enzymatic digestion or xenograft models in nudemice were used in primary culture. Preliminary identification of the epithelialcomponent origins of the cell lines were confirmed by the features of morphologyand the immunophenotype.Results：1. After confirming quality,3paired samples were chosen for miRNAmicroarray ananlysis and there were4significantly differentially expressed miRNAsscreened from tissue. miR-192was3.31-fold up-regulated, while miR-197,-224,-375were down-regulated, respectively0.43,0.34,0.36.2. Compared with corresponding adjacent normal tiusses by RT-PCR, miR-192was2.14-fold up-regulated, while miR-197,-224,-375were down-regulated(respectively0.47,0.26,0.60) in3paired samples, which were consistent with thescreening results of the microarrays.3. There are thousands of target genes of4miRNAs respectively by usingbioinformatics methods. miR-224and its candidate target genes Jagged1, L1CAM,SMAD4were ultimately selected for functional studies after integrating informationof4miRNAs from Kegg signal pathways, GO annotations and so on. It alsoindicates that the negative correlations between the expression levels of miR-224andtheir target genes.4. The epithelial-like cells and fibroblast-like cells were adherent andco-cultured after dispersed with collagenase V. The epithelial origin was confirmedby a flattened polygonal morphology and the cytoplasmic positive expressions ofbroad-spectrum cytokeratins in MCNs. Conclusions：1. The aberrant expressions of miR-192, miR-197, miR-224and miR-375maycorrelated with the pathogenesis of mucinous cystic neoplasms of pancreas.2. Jagged1, L1CAM, SMAD4may be the candidate target genes of miR-224.3. Collagenase V enzyme digestion method can be used for primary culture ofMCNs.