| BackgroundNowadays, pancreatic ductal adenocarcinoma is one of the most malignant and fataltumors, and the incidence increases year by year. In addition, the5-year survival rate isabout4%. Although the diagnosis and treatment of pancreatic ductal adenocarcinoma haveobviously enhanced, the efficacy of therapy is not still satisfactory. Meanwhile, the5-yearsurvival rate after surgery is below15%. Because of pancreatic ductal adenocarcinomalacking of visibly early clinical characteristic, when to be diagnosed most patients arestayed in middle-and late-stage cancer, and only about20%of patients can acceptpancreaticoduodenectomy. Thus the development of new diagnostic and treatmentstrategies is extremely urgent.Recently, it has been confirmed that epigenetic changes (e.g. microRNAs) are crucialto malignant tumor development. MiRNAs are short noncoding RNAs (approximately21–23nt in length) that can regulate gene expression by base pairing with the3.0untranslated region (UTR) of their target mRNAs, which triggers either translationrepression or RNA degradation. MiRNAs maybe regulate upon30%human genes. Risingresearch reveals that lots of miRNAs are directly concerned with the pathogenesis ofvarious cancers. MiRNAs expression manners have the disease specificity, and contributeto the classification of certain cancer histotypes.B cell-specific moloney murine leukemia virus insertion site1(Bmi-1) is part ofpolycombgroup (PcG) family. During embryonic development, Bmi-1can maintain theself-renewal of hematopoietic system and proliferation of neural stem cells. A lot of studieshave confirmed that Bmi-1is over-expressed in many malignant tumors, such asendometrial cancer, ovarian cancer, breast cancer, prostate cancer, and non-small cell lungcancer. In pancreatic ductal adenocarcinoma, Bmi-1is also significant over-expression and is closely involved in the histological grade and clinical stage. In addition, some reportsshow that the over-expression level of Bmi-1could reduce the survival time of patients andpromote lymph node metastasis in patients with endometrial and ovarian cancers. So,Bmi-1may play a vital oncogenic role in pancreatic ductal adenocarcinoma. Meanwhile,the related evidence has been demonstrated that Bmi-1can regulate the epithelial tomesenchymal transition (EMT), which is a key step in the promotion of tumor metastasis.The reduction or deletion of E-cadherin is the symbal of EMT and this can closely beassociated with invasive behavior. The regulation of Bmi-1is complex, it has been revealedthat the expression of Bmi-1is regulated in many malignant tumors by different miRNAs(e.g., miR-15a, miR-16, miR-128, miR-194, miR-302, and miR-200c). Therefore, weassume that the expression of Bmi-1mediated by miRNAs maybe help to the developmentof pancreatic ductal adenocarcinoma, but this speculation has no reports.To objectively and comprehensively investigate the significance of Bmi-1regulated bymiRNAs in pancreatic ductal adenocarcinoma, in combination with the recent relatedstudies, we put forward the following hypothesis that miRNAs inhibits cell proliferationand EMT in pancreatic ductal adenocarcinoma by down-regulating Bmi-1expressionObjectiveTo confirm that miRNAs can inhibit cell proliferation and epithelial-mesenchymaltransition in pancreatic ductal adenocarcinoma by the down-regulation of Bmi-1expression.Methods and Results1. To evaluate the alterations of miRNAs expression in pancreatic ductaladenocarcinoma tissue and normal pancreas tissue, normal pancreas tissue (n=20) andpancreatic tumor tissue (n=43). Firstly, all the fresh-frozen pancreatic ductaladenocarcinoma samples and twenty normal pancreas tissues were obtained from theInstitute of Hepatopancreatobiliary Surgery, Southwest Hospital, Third Military MedicalUniversity. Subsequently, miRNAs expression analysis was performed by using real-timePCR in pancreatic ductal adenocarcinoma samples and normal pancreas tissues. The resultsof miRNAs expression analysis were calculated with the△△CT method based on thehouse keeping microRNA, miR-16, normalized to the average value in control group, andplotted as relative quantifications (RQs). Our results showed that only miR-15a was obviously reduced in pancreatic ductaladenocarcinoma tissues compared with normal pancreas tissues. At the same time, ourresults revealed that the expression level of miR-16had no distinction in any of thesespecimens.2. To evaluate the alterations of Bmi-1expression in pancreatic ductal adenocarcinomatissue and normal pancreas tissue and the relationship between Bmi-1and miR-15a.Immunohistochemistry (IHC) was accomplished to detect Bmi-1expression level inpancreatic ductal adenocarcinoma and normal pancreas tissues. Using cell transfection andwestern blot was identified the relationship between Bmi-1and miR-15a.Our study showed that Bmi-1was significantly high expression in pancreatic ductaladenocarcinoma tissue compared with normal pancreas tissue and the negative control.After transfecting miR-15a into the pancreatic cancer cell line PANC-1, Bmi-1expressionlevel was obviously decreased compared with blank control by western blot analysis. What’more, we also found a close connection between Bmi-1and some clinicopathologicalfeatures and survival time.3. To evaluate the influence of alterations of Bmi-1/miR-15a expression on pancreaticductal adenocarcinoma cell proliferation and EMT. We investigated the role of miR-15a insuppressing the proliferation of pancreatic ductal adenocarcinoma cell and regulating EMTby miRNA transfection, plasmid transfection, western immunoblot analysis and cellproliferation assay.Our results showed that Bmi-1expression was enhanced in cells which beentransfected with Bmi-1gene plasmid but not with blank control plasmid, and after miR-15awas transfected into PANC-1, the proliferation of pancreatic ductal adenocarcinoma cellwas significantly decreased when compared with blank control. Moreover, E-cadherinexpression level was distinctly increased in pancreatic ductal adenocarcinoma cell aftermiR-15a transfection while it was decreased in the blank control. However, after the Bmi-1gene plasmid was transfected into PANC-1cells pre-transfected with miR-15a, E-cadherinwas decreased compared with control cells transfected with empty plasmid.Conclusions:1. The expression of miR-15a is significantly reduced in pancreatic ductaladenocarcinoma as compared to normal pancreas samples.2. Bmi-1is over-expressed and directly regulated by miR-15a in pancreatic ductal adenocarcinoma. In addition, there is an important relationship between the Bmi-1expression level and clinicopathological features patient as well as the survival time aftersurgery.3. miR-15a can inhibit the proliferation of pancreatic ductal adenocarcinoma cells andthe development of EMT via down-regulation of Bmi-1. |
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