| Introduction:Pancreatic ductal adenocarcinoma (PDAC) is characterized by high malignancy, rapid progress, difficult diagnosis, poor treatment and poor prognosis, which is a serious threat to human health. PADC is located in the cause of cancer death fourth,5 year survival rate is only 3%～5%, and the median survival is not more than six month. How to effectively intervene in the occurrence and treatment of PDAC patients, improve the survival rate has become a major pressing problem at present. So it is very important to find an effective therapeutic target for PDAC. More and more studies have confirmed that, in addition to the PDAC gene mutations, the miRNA expression imbalance will also affect the development of PDAC. MicroRNA (miRNA) is found in recent years, a wide range of animals and plants in the body of non encoding RNA, about 18-26 base. binding to the 3’UTR of target gene, and play a negative regulatory role in gene transcription and translation. It was confirmed that miRNA was involved in all the processes of life, including the early embryonic development, cell proliferation, differentiation and apoptosis, cell immune regulation, etc.. In recent years, miRNA also plays a role as tumor suppressor gene and oncogenes, miRNA expression in different types of tumor tissues, miRNA and tumor occurrence, development, diagnosis, treatment and prognosis are closely related. At present, the study found that the expression of many miRNA in pancreatic cancer is abnornal, including miR-122, miR-13a, miR-21, miR-195, miR-221, miR-301, miR-101 and so on, and they play an important role in the development of pancreatic cancer. The abnormal expression of miRNA can affect the development of PDAC by effectting cell proliferation, apoptosis, migration and invasion. However, the molecular mechanism of miRNA involved in the occurrence and development of PDAC needs to be further studied. The foundig of abnormal expression of miRNAs in PDAC and the study of their function and mechanism, and the new specific marker molecules of PDAC, will provide a new theoretical basis for the diagnosis, treatment and prognosis of PDAC.Objective:The purpose of this study was to analyze the expression levels of miR-21 in pancreatic ductal adenocarcinoma, and to analyze the relationship between miR-21 and its potential target gene of PDAC and the relationship between miR-21 and the clinical pathological characteristics of pancreatic ductal adenocarcinoma, and then to analyze the biological function of miR-21 in pancreatic duct adenocarcinoma cells, analyze its target gene, explore the possible molecular mechanism in PDAC.Methods:The expression of Bcl-2, c-Myc and PTEN mRNA and protein was was detected by QRT-PCR and western blot. The relationship betweeen miR-21, PTEN, bcl-2, c-Myc and PDAC was statistical analysized. The effect of miR-21 on pancreatic ductal adenocarcinoma cells was studied by in vitro cell transfection of pre-miR-21 and miR-21 inhibitor, and detectived with MTT, colony formation, flow cytometry, scratch test, Transwell chamber experiment. The target gene of miR-21 was studied with dual luciferase reporter gene, over expression experiments, western blot method. and the changes of cell cycle, apoptosis, invasion related molecules was detectived in cells in transfected with miR-21 and PTEN, further explored the molecular mechanism of miR-21 in pancreatic ductal adenocarcinoma cell.Results:1. The expression of miR-21 was detected in 38 pancreatic ductal adenocarcinoma tissues and their adjacent tissues,3 pancreatic cancer cell lines and 1 normal pancreatic ductal epithelial cells.the results showed that the expression level of miR-21 in pancreatic ductal adenocarcinoma tissues and pancreatic ductal adenocarcinoma cells was significantly increased, the difference was significant.2. The correlation between miR-21 and clinical pathological features of pancreatic ductal adenocarcinoma was analyzed, and the result showed that the expression level of miR-21 wasn’t correlated with the age, gender, tumor location and tumor size, but was closely related to tumor grade, TNM stage and lymph node metastasis, suggested MiR-21 can be used as a marker for the prognosis of patients with pancreatic ductal adenocarcinoma.3. The expression of Bcl-2, c-Myc and PTEN in pancreatic ductal adenocarcinoma tissues was studied with QRT-PCR and Western-blot, the result showed that the expression of c-Myc, Bcl-2 in cancer tissues was significantly high, but the expression of PTEN in cancer tissues was significantly low, the difference was statistically significant. Correlation analysis of clinical and pathological characteristics of pancreatic ductal adenocarcinoma revealed that PTEN, Bcl-2 and c-Myc were not associated with age, gender, tumor location and other pathological factors, but were closely related to the pathological grade, clinical stage and lymph node metastasis. suggested that the expression of PTEN, Bcl-2, c-Myc could reflect the biological behavior of pancreatic cancer, and they could be used as a marker for the diagnosis of pancreatic ductal adenocarcinoma.4. The expression of MiR-21 was negatively correlated with the expression of PTEN, but was positively correlated with c-Myc, was no significant correlation with Bcl-2, suggested that PTEN may be a target gene directly regulated by miR-21.5. In vitro study showed that miR-21 could promote the proliferation of pancreatic ductal adenocarcinoma cells by MTT and clone formation assay. Flow cytometry revealed that miR-21 could inhbit the apoptosis of PDAC cells, and induce the transition from G to S phase. The scratch test showed that the high expression of miR-21 promoted the migration of cancer cells. Transwell test indicated that miR-21 could significantly enhance the invasive ability of cancer cells.6. Cell phenotype change and cell regulatory factor assay, hinted that PTEN, Bcl-2 and c-Myc would be the candidate target genes of miR-21, blot Western analysis showed that the expression of PTEN, Bcl-2 and c-Myc proteins in pancreatic ductal adenocarcinoma cells was significantly affected by miR-21.7. Luciferase reporter vectors of PTEN 3’-UTR wild-type and mutant, were transfected into cells with pre-miR-21, the results showed that the wild-type transfection group was significantly decreased, the mutant transfection group had no obvious change, which indicated that the miR-21 could be directly target PTEN 3’-UTR and down regulated PTEN expression.8. The expression of pAKT, pFOXO3a, Cdk2, CyclinD1, P21 protein in PTEN/AKT signaling pathway was significantly up-regulated, and the expression of Bax and MMP2 was down regulated by miR-21. It is indicated that miR-21 is involved in the regulation of cell apoptosis, cycle regulation, invasion, metastasis and so on.Conclusions:1. The expression of miR-21 in pancreatic ductal adenocarcinoma was significantly up-regulated, suggesting that high expression of miR-21 was closely related to the development of pancreatic ductal adenocarcinoma. The expression of miR-21 in pancreatic ductal adenocarcinoma is closely related to the pathological parameters of pancreatic cancer, and it is suggested that miR-21 can be used as a potential predictive index for the diagnosis of pancreatic ductal adenocarcinoma and its prognosis.2. The expression of Bcl-2 and c-Myc in pancreatic ductal adenocarcinoma tissues were significantly up-regulated, and the expression of PTEN in pancreatic ductal adenocarcinoma tissues was significantly decreased, and the binding was correlated with the progression of pancreatic cancer, suggested that PTEN, c-Myc and Bcl-2 could be used in the regulation of pancreatic ductal adenocarcinoma, which could be used as potential molecular targets for the diagnosis of pancreatic cancer.3. MiR-21 promotes the proliferation, clone formation, invasion and migration of pancreatic ductal adenocarcinoma cells, and inhibit cell apoptosis.4. PTEN was identified as a direct target gene for miR-21. Bcl-2, c-Myc was the indirect function target of miR-21.5. MiR-21 was involved in cell apoptosis, cycle regulation, invasion and metastasis by regulating the expression of CyclinDl, Bcl-2, c-Myc, MMP2. |
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