| Pancreatic ductal adenocarcinoma(PDAC)is the most common type of pancreatic cancer(PC)and it ranks the fourth to fifth leading cause of cancer-related death in western countries with an average overall 5-year survival of less than 5% and a median survival period of less than 6 months.The aggressiveness of PDAC partly due to the lack of specific symptoms and early detection and chemotherapy resistance.In the past two decades,a lot of time,manpower and resources have been devoted to the researches of improving the treatment and prognosis of patients with PDAC,however,the outcome is still disappointed.At present,surgical treatment and postoperative chemotherapy is still the most effective standard treatment of pancreatic cancer,therefore,it is essential to explore and understand the mechanism of the invasion,metastasis,and recurrence of pancreatic cancer to predict the prognosis of PDAC patients and improve the survival rate of PDAC patient.A series of complex,multifactorial processes such as tumor invasion,metastasis and drug resistance are involved in the development of tumors.In recent years,some scholars have proposed a new concept of cancer stem cell like cell(CSLC),or cancer stem cell(CSC).Emerging evidence supporting the existence of tumor initiating cells or CSCs within tumors provided the biological significance to CSCs in cell growth,migration,invasion,metastasis,drug resistance,the capacity of self-renewal and the potential to regenerate into all types of differentiated cells,which are associated with poor clinical outcome in patients diagnosed with PDAC.Additionally,recent studies revealed that epithelial-mesenchymal transition(EMT)phenotype cells share biological characteristics with CSLCs/CSCs and micro RNA-30 family(miR-30s)is involved in EMT process and working as a powerful regulator of EMT and has been proved to act as tumor suppressors in many kinds of malignant tumors such as breast cancer,non-small cell lung cancer,renal carcinoma and gastrointestinal cancer.Moreover,Snail,which has a perfect match to the seed region of miR-30 s has been proved to be a direct repressor of E-cadherin.In our study,PCSCs of human PDAC cell line PANC-1 were processed for CD24,CD44 and Ep CAM multi-color staining,and sorted out on a BD FACS Aria II machine.RT-qPCR was performed to value the expression of miR-30 family.To find the role of miR-30 in the EMT process of PCSCs,a series of in vitro and in vivo experiments were performed using miR-30 b mimic/agomir transfection.Luciferase reporter assay was performed to verify whether miR-30 b can directly target Snail m RNA in PCSCs.Finally,we discussed the possible mechanisms of miR-30 b in tumor suppression,to provide a new therapeutic target for improving the prognosis of PDAC patients.Part ? The Acquisition and Biological Characteristics of Pancreatic Cancer Stem CellsObjective: To obtain pancreatic cancer stem cells and explore the biological difference between PCSCs and normal PDAC cells and analyze the effects of PCSCs on the development of PDAC.Methods: PCSCs of PDAC cell line PANC-1 were processed for CD24,CD44 and Ep CAM multi-color staining,and sorted out by a flow cytometry.We identified PCSCs after microscopic morphology observation and evaluating the expression of Nanog and Oct4 in PCSCs and PANC-1 cells using RT-qPCR and Western Blot analysis.We investigated the expression levels of EMT markers in PCSCs and PANC-1 cells using RT-qPCR and Western Blot analysis.We investigated the expression levels of miR-30 s in PCSCs and PANC-1 cells using RT-qPCR.Result: In PANC-1 cells,6.02%-9.81% of cells were double-positive(CD24+,CD44+),and 4.52%-8.09% of cells were triple-positive(CD24+,CD44+,Ep CAM+).RT-qPCR and Western Blot analysis showed the expression levels of Nanog and Oct4 in PCSCs were significantly higher than PANC-1 cells.We found PCSCs formed typical cell spheroid under a microscope.RT-qPCR and Western Blot analysis showed the expression of E-cadherin in PCSCs was significantly lower whereas the expression of N-cadherin,vimentin and Snail was remarkablly higher than in PANC-1 cells.RT-qPCR analysis showed the expression levels of miR-30 b,c,d were significantly downregulated in PCSCs,while the expression levels of miR-30 a,e were similar with PANC-1 cells.Conclusion: PCSCs were successfully sorted out;PCSCs were EMT-phenotype PC cells;miR-30 b,miR-30 c,miR-30 d were down-regulated in PCSCs.Part ? The Impact on PCSCs Biological Characteristics by Overexpressing Micro RNA-30 b in VitroObjective: To investigate and discuss the function of miR-30 b in EMT process of PCSCs in vitro.Methods: miR-30 b mimic and NC-mimic were transfected into PCSCs and the transfection efficiency was valued using RT-qPCR.RT-qPCR and Western Blot analysis were performed to investigate the change of the expression of EMT markers in PCSCs after overexpressing miR-30 b.Transwell migration and invasion assay was performed to observe the alteration of the abilities of migration and invasion of PCSCs after overexpressing miR-30 b.Result: RT-qPCR analysis showed miR-30 b mimic efficiently elevated the expression of miR-30 b in PCSCs.RT-qPCR and Western Blot analysis showed the expression of E-cadherin in PCSCs was significantly up-regulated whereas the expression of N-cadherin,and Snail was remarkablly reduced,however,the expression of vimentin showed no significant changes after transfection.Transwell migration and invasion assay showed the abilities of migration and invasion of PCSCs were meaningfully decreased after overexpressing miR-30 b.Conclusion: Overexpression of miR-30 b partly reversed the EMT process and reduced the abilities of migration and invasion of PCSCs.Part ? The Impact on PCSCs Tumorigenic Ability by Overexpressing Micro RNA-30 b in VivoObjective: To determine the impact on PCSCs tumorigenic ability by up-regulating miR-30 b expression in vivo.Methods: miR-30 b agomir and NC agomir were transfected into PCSCs and the transfection efficiency and stability were valued using RT-qPCR.The human pancreatic cancer mode was established with PCSCs in athymic nude mouse and the gross tumor volumes and weights were measured to verify the tumorigenic ability changes of PCSCs with forced miR-30 b expression.Result: RT-qPCR analysis showed miR-30 b agomir efficiently and stably elevated the expression of miR-30 b in PCSCs.In vivo expriment,the volumes of tumor masses of the miR-30 b agomir transfection group were significantly smaller compared with NC agomir transfection group and blank control group.The weights of tumor masses of the miR-30 b agomir transfection group also showed remarkably reduction.Conclusion: Overexpression of miR-30 b reduced the tumorigenic ability of PCSCs in vivo.Part ? The Mechanism of Micro RNA-30 b in Regulating theEpithelial-Mesenchymal Transition of PCSCsObjective: To explore and discuss the potential mechanism of the regulation of miR-30 b in the EMT process of PCSCs.Methods: Luciferase reporter assay was performed to verify whether miR-30 b can inhibit the expression of Snail by directly targeting it.Snail si RNA and NC si RNA were transfected into PCSCs,and the transfection efficiency was valued using RT-qPCR.Western Blot analysis were performed to investigate the change of the expression of E-cadherin in PCSCs after Snail knockdown.Result: Luciferase reporter assay showed that,compared with NC mimic,miR-30 b mimic significantly decreased the luciferase activity of Snail WT-3'UTR.RT-qPCR analysis showed Snail si RNA efficiently reduced the expression of Snail in PCSCs.Western Blot analysis showed the expression of E-cadherin was significantly increased in PCSCs transfected with Snail si RNA,compared with NC si RNA transfected and non-treated PCSCs.Conclusion: miR-30 b negatively controlled the expression of Snail by directly targeting it in PCSCs,releasing its repression to E-cadherin,partly reversing the EMT process of PCSCs. |
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