| Pancreatic cancer is present known one of high degree malignant tumors, with amortality/incidence ratio of0.99:1. Among cancers, pancreatic cancer is the fourthleading cause of cancer-related deaths in the United States. Recently, the incidence ofpancreatic cancer in our country is on the rise. It is the eighth malignant tumor morbidity,attributable to about1311deaths in2000in Shanghai. The incidence of pancreatic cancerin Shanghai is10per100,000people and with age-standardized incidence rate of6.0/100,000people. Due to non-specific symptoms, aggressive growth, drug resistanceand early dissemination, pancreatic adenocarcinoma patients are often diagnosed duringthe late stages. Therefore, there is an urgent need to discover novel, early, diagnosticbiomarkers and new therapeutic strategies.MicroRNAs are short (approximately18~25nt), noncoding RNAs that mainlyregulate gene expression at the posttranscriptional level. Partial complementaritybetween the seed region of miRNA and the3’UTR region of the target gene leads totranslation inhibition, whereas perfect complementarity results in target mRNAdegradation. MiRNAs are predicted to control the activity of about30%of allprotein-coding genes in mammals, with their targets forming a complex regulationnetwork, taking part in many biological processes, such as proliferation、apoptosis、differentiation、development and stress response. Altered expressions of miRNAs arereported in various cancers and may associate with cancer pathogenesis, apoptosis, andcell growth, thereby functioning as either tumor suppressors or oncogenes. Therefore, thelatest study confirmed the role of certain treatment while againsting or restoring theeffect of miRNA. So, miRNA may have diagnostic and prognostic value in severalhuman malignancies.Recently, it has been reported that miRNAs play a critical role in human cancersincluding pancreatic cancer. The microRNAs are studied more and more deeply inpancreatic cancer. Not only do we obtained the expression profiles of pancreatic cancerby the microarray assay, getting the number of microRNA in pancreatic cancer, but alsogained the expression of microRNAs that main different in pancreatic cancer by variousmethods, such as miR-21, miR-18a, miR-196a, miR-10b, miR-16, miR-155, miR-181a,miR-181b, miR-200b、c, miR-210are upregulated；miR-34a, miR-101, let-7a、b,miR-217,miR-146, miR-148are downregulated. Previous reports also showed that these differential expression miRNAs play important roles in pancreatic cancer biologybehavior, for instance, proliferation, invasion, migration, apoptosis, drug resistance, andespecially in the research of pancreatic cancer pathogenesis. MicroRNA functionalnetwork of pancreatic cancer is from biology to biomarkers of disease. MicroRNA canbe used for the diagnosis of pancreatic cancer, prognosis, the curative effect of drugs andsurgical resection.miR-26a is21nt in length, located in3p23-21．31，which are fragile chromosomalregions in many cancers．MiR-26a was identified as a cooperating component of afrequently occurring amplicon that also contains CDK4and CENTG1, two oncogenesthat regulate the RB1and PI3kinase/AKT pathways, respectively. The poly (A) tail ofmiR-26a length was maintained by Zcchc11which is a ribonucleotidyltransferase with apreference for uridine. The role of miR-26a was also regulated by myc. It was reportedthat the identified direct acting targets include Ezh2、PTEN、cyclinD2、cyclinE2、GSK-3β,and functionally relevant targets of miR-26a include RB1、 MAP3K2/MEKK2andSMAD1. Through acting on these targets, miR-26a regulated the biological behaviors ofmany tumor cells. But the role of miR-26a in carcinogenesis appears to be a complicatedone, in the sense that both oncogenic and tumor suppressive effects were reported incancers such as glioblastoma and hepatocellular carcinoma, respectively. As reported,miR-26a exists in the microarray profiling of pancreatic cancer, without deeply reseach.Based on the review of miR-26a research and the role in the origin and development oftumor, this research project aim to identify the expression characteristics of miR-26a inpancreatic cancer tissues, and observe the influence on the characteristics of pancreaticcancer cell biology when miR-26a expression level altered. We made some researches toelucidate the characteristics and regulatory mechanisms of miR-26a in pancreatic cancerwhich help understand the mechanisms and remedy of pancreatic cancer．1. To identify the expression characteristics of miR-26a in pancreatic cancer tissuesTo examine the expression of microRNA-26a in pancreatic cancer, we measuredthe expression of miR-26a in15pairs of pancreatic cancer tissues and matched adjacentnormal tissue samples by real-time PCR. The expression of miR-26a was lower in thepancreatic cancer tissues than in adjacent normal tissues(p＜0.05, student’s t-test).Hybridization in situ technique was employed to detect the expression of miR-26a inpancreatic cancer tissues and matched adjacent normal tissue samples. The resultsshowed that the miR-26a was expression in both cytoplasm and nuclear of pancreatic ductal epithelial cells and that the expression of miR-26a in the malignant ductalepithelial cells of pancreatic cancer was lower than in the normal ductal epithelial cells.2. The influence of overexpression of miR-26a on biological characteristics of pancreaticcancer cells.The recombinant vector with pri-miR-26a sequence (pTM) and blank control vector(pT) were transfected into the pancreatic cancer cell SW-1990and Capan-2respectively.Real-time PCR was used to investigate the transfection efficiency, and the results showthat the transcription of miR-26a increased significantly in two tumor cell lines(3.34-fold in SW-1990and6.16-fold in CAPAN-2) compared with the control vector (P <0.05).The CCK-8assay and flow cytometric analysis were carried out to detect thechange of proliferation and cell cycle distribution; the western blot assay was used todetect the change of target protein cyclinE2.The results showed that the cell growth wasreduced and G1-phase was arrested, and the cyclinE2was down regulated.3．MiR-26a suppressed tumor growth of pancreatic cancer cells in nude mice．The results showed that the volumes of tumors induced by miR-26a transfectedcells(pTM-transfected SW-1990cells) were significantly lower than those by thescramble-transfected controls(pT-transfected SW-1990cells).That’s to say,miR-26a canreduce the growth of pancreatic cancer cells in vivo.Moreover, immunohistochemistry was also performed to detect the expression ofcyclinE2in randomly selected tumors derived from miR-26a (pTM) and scramble (pT)transfected SW-1990cells. The miR-26a-overexpressing tumors expressed lower levelsof cyclin E2than the controls. Similarly, immunohistochemical analysis was used tomeasure the protein levels of PCNA in the tumor tissues, showing decreases of PCNA inmiR-26a transfected cell tissues. PCNA is molecular evidence of proliferation.Conclusions:1. The real-time PCR and in-situ hybridization results showed that the expression ofmiR-26a was downregulated in pancreatic cancer tissues．2. The real-time PCR and Fluorescence microscope observations demonstrated thatpancreatic cancer cells were stably transfected with the vectors of pri-miR-26a sequence(pTM) and negative control (pT) vectors respectively successfully.3. Overexpression of miR-26a in vitro caused the pancreatic cancer cell growthsuppression and G1-phase arrest.4. Overexpression of miR-26a may inhibit the tumorgenesis of pancreatic cancer cell in vivo.5. Then we measured the cyclinE2protein of SW-1990and Canpan-2cells transfectedwith miR-26a by western blotting and found that miR-26a positively down-regulated theexpression of cyclinE2in the pancreatic cancer... |
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