| Background: Pancreatic malignancies are one of the most deadly malignancies,and their pathologic types are varied,with ductal adenocarcinoma being the most common.The five-year survival rate of pancreatic ductal adenocarcinoma is extremely low,and more than 80% of pancreatic ductal adenocarcinoma patients have lost the opportunity for surgery at the time of diagnosis due to various reasons,such as distant metastasis of the tumor,or local invasion is too severe.In conclusion,pancreatic ductal adenocarcinoma(PDAC)is a very aggressive malignant tumor,which is very prone to distant metastasis and recurrence.Therefore,the control of distant metastasis of pancreatic cancer has become one of the key directions of oncology researchers.We know that EMT refers to the differentiation of static epithelial cells into interstitial,motor phenotypes observed during early development.EMT plays a decisive role in many pathologic processes,especially in malignant tumor progression and metastasis.Pramlintide is a drug approved by the FDA in 2005 for the adjuvant treatment of type 1 and type 2 diabetes mellitus.The drug belongs to the endogenous isletamyloidpolypeptide(IAPP).A non-polymeric isomer of amylin.IAPP is produced within the beta cells of the pancreatic islets,and insulin is in a fixed ratio(under normal physiological conditions 1:100,the proportion will change under pathological conditions)accompanied by secretion.According to a literature report in nature in 2015,our study found that pancreatic cancer cell lines with IAPP overexpression showed a downregulation of proliferation ability,and through further demonstration,it was concluded that overexpression of IAPP or use of Pramlintide could improve the drug resistance of gemcitabine-resistant pancreatic cancer strains.We know that drug resistance and EMT process are inextricably linked,but there is no report on whether IAPP or Pramlintide can regulate EMT process in pancreatic cancer cells.We know that aerobic glycolysis and NUMB expression may have a positive relationship.NUMB plays an important role in tumorigenesis and development,especially in the initiation of epithelial-mesenchymal transformation(EMT).We know that,given the variable shear relationship between NUMB’s two most common shear isomers,NUMB-PRRS and NUMB-PRRL,perhaps focusing only on the NUMB total protein is not an in-depth exploration of the biological mechanism of its behavior.Therefore,to elucidate the specific relationship between aerobic glycolysis and NUMB-PRRS and NUMB-PRRL,it is very important to explore the relationship between NUMB-PRRS and NUMB-PRRL on EMT of pancreatic cancer.Therefore,this study was intended to observe the regulatory effects of Pramlintide on the EMT process of pancreatic cancer cell lines PANC-1,SW1990 and Miapaca-2,and further explore the molecular mechanism of Pramlintide inhibiting the EMT pathological process of pancreatic cancer cell lines,and demonstrate the important molecular pathway mechanism.In order to provide a new way to prevent and cure the metastasis and invasion of pancreatic cancer.Methods: 1.Human pancreatic duct adenocarcinoma cell lines PANC-1,SW1990 and Miapaca-2 were cultured with Pramlintide.The change trend of glycolysis level in cancer cells was verified by ECAR,and the data were analyzed by wave2.6.Transwell experiment was performed to demonstrate the changes of invasion and migration ability of pancreatic cancer cell lines after the change of glycolysis level.Image J was used to count and process the basic data.Westernblotting test was used to verify the changes in the expression of EMT-related phenotypes and other proteins after the change of glycolysis level of pancreatic cancer cells.Image J was used for gray scale analysis.MTS assay and clonal formation assay were used to verify the changes of cell activity and proliferation ability of pancreatic cancer cell lines after glycolysis modification.The apoptosis of pancreatic cancer cells after the change of glycolysis ability was detected by flow cytometry AV-PI.2.Human pancreatic ductal adenocarcinoma cell lines PANC-1,SW1990 and Miapaca2 were cultured by Pramlintide,and the mRNA expression of NUMB-PRRS in pancreatic cancer cell lines cultured by Pramlintide was detected by qRT-PCR.Westernblotting is used to detect the protein expression changes of pramlintide-cultured pancreatic cancer cell line NUMB-PRRS.After activating glycolysis with ASA-58,qRT-PCR and westernblotting are used to detect the changes of NUMB-PRRS in Pramlintide-cultured pancreatic cancer cell line.The EMT phenotype and invasive ability of pancreatic cancer cell lines were detected by lentiviral overexpression of NUMB PRRS,westernblotting and transwell assay.3.After the constructed sh RNA is used to inhibit the expression of NUMB-PRRS,the target proteins of WNT signaling pathway in cells are detected by Westernblotting,and the activity of WNT signaling pathway is evaluated by the TOP/FOP Flash luciferase reporting system.To demonstrate the negative regulatory effect of NUMB-PRRS on EMT of pancreatic cancer cells through WNT pathway.Results: 1.Cells in PANC-1,SW1990 and Miapaca-2 groups were cultured with 10%RPMI-1640 complete medium containing 10mg/L of Pramlintide as the most suitable concentration.The aerobic glycolysis inhibition effect of Pramlintide on cells in each group was confirmed by ECAR experiment.We set up control group(CON),Pramlintide group(PLT),high-glycemic group(HG),and Pramlintide plus high-glycemic group(PLT+HG)to observe the changes of EMT-related protein expression(E-cadherin,N-cadherin,and Vimentin)in pancreatic cancer 24 h later.Westernblot results showed that compared with CON group and HG group,the expression of N-cadherin and Vimentin was decreased in PLT group,while the expression of E-cadherin was significantly up-regulated.In the PLT+HG group,the expression of N-cadherin and Vimentin decreased,while the expression of E-cadherin decreased significantly.Changes in invasion and migration of pancreatic cancer under the above treatment conditions.The results of Transwell experiment showed that compared with CON group and HG group,the invasion and migration ability of cells in PLT group was significantly weakened,while the invasion and migration ability of cells in PLT+HG group was increased under the influence of high glucose.MTS assay was performed to verify that Pramlintide inhibited the activity of pancreatic cancer cell lines by inhibiting their aerobic glycolysis ability.The proliferative activity of the four pancreatic cancer cells cultured by pramlintide was lower than that of the control group.Annexin V-FITC/PI flow cytometry double staining apoptosis assay showed that the four pancreatic cancer cells cultured with Pramlintide showed an increased proportion of early apoptotic cells compared with the control group.After re-activation of glycolysis,westernblotting detected EMT related indexes.Westernblot results showed that with the increase of glycolysis ability,N-cadherin and Vimentin were significantly up-regulated,while E-cadherin expression was significantly down-regulated.transwell experiment verified the invasion and metastasis of cells in each group.With the increase of glycolysis ability,the invasion and metastasis ability of cells also increased significantly.2.We used the string database to predict the cell membrane channel that Pramlintide enters the pancreatic cancer cytoplasm to produce tumor inhibition,and the rationality and correctness of this inference were verified by ECAR experiment.The expression of NUMB-PRRS in PANC-1,SW1990 and Miapaca-2 cells cultured with 10%RPMI-1640 with a concentration of 10mg/L of Pramlintide may be detected by qRT-PCR method.The results showed that compared with the control cells,the expression of NUMB-PRRS in the three groups was significantly lower than that in the control group.The expression of NUMB-PRRS in Pramlintide cultured cells was significantly up-regulated.The expression of total NUMB protein and NUMB-PRRS protein in PANC-1,SW1990 and Miapaca-2 cells cultured by10%RPMI-1640 with pramlinpeptide concentration of 10mg/L were detected by westernblotting method.The results showed that compared with the control cells,the expression of NUMB-PRRS in Pramlintide cultured cells was significantly up-regulated,but the total NUMB expression was not significantly changed.After DASA-58 activated glycolysis,qRT-PCR and westernblotting were used to detect the changes of NUMB-PRRS in Pramlintide cultured pancreatic cancer cell lines.After the glycolysis activity of Pramlintide cultured cell lines was activated by DASA-58,qRT-PCR and westernblotting methods were used to detect the mRNA expression of NUMB-PRRS and the expression of total NUMB protein and NUMB-PRRS in PANC-1,SW1990 and Miapaca-2 cells.The results showed that,compared with Pramlintide cultured cells,the expression of NUMB-PRRS in the reactivated glycolytic cells was significantly reversed.3.We predict the possible interaction mechanism between NUMB-PRRS and WNT pathway through string database.The protein levels of WNT signaling pathway target proteins Cyclin-D1,C-myc,Axin-2 and β-catenin in PANC-1,SW1990 and Miapaca-2 cells were detected by Westernblotting.Inhibition of NUMBPRRS expression significantly increases the levels of β-catenin,Cyclin-D1,C-myc,and Axin-2,while overexpression of NUMB-PRRS significantly reduces the levels of these proteins.The WNT signaling pathway activity was evaluated using the TOP/FOP Flash luciferase reporting system.Inhibition of NUMB-PRRS significantly increased fluorescence intensity,while overexpression of NUMB-PRRS significantly decreased fluorescence intensity.WNT signaling pathway activity was modulated using either WNT signaling pathway inhibitor IWR1 or WNT signaling pathway activator WNT-3A.Through experimental observation,it was found that the inhibition of NUMB-PRRS resulted in significant changes in EMT-related phenotypes of pancreatic cancer(E-cadherin down-regulation,N-cadherin and Vimentin up-regulation),while the WNT pathway inhibitor IWR-1 was used to inhibit NUMB-PRRS cell lines.The EMT-related phenotypes of pancreatic cancer significantly reversed.Conclusion: 1.Pramlintide may be a potential drug to control metastasis of pancreatic cancer.Pramlintide has an important effect on the aerobic glycolysis of different pancreatic cancer cell lines.Pramlintide has a pharmacological effect on EMT of pancreatic cancer by improving the level of aerobic glycolysis,providing a new perspective for the treatment of pancreatic cancer.2.Among the different types of shear isomers existing in NUMB,NUMB PRRS may be an important molecule affecting the EMT phenotype and other biological behaviors of pancreatic cancer.The regulation of this molecule may have a profound and beneficial effect on the biological behavior of pancreatic cancer.3.Through a series of changes in Pramlintide→ glycolysis →NUMB-PRRS→WNT→EMT,we believe that the use of Pramlintide to control the level of aerobic glycolysis in pancreatic cancer cells during the occurrence and development of pancreatic cancer may be a potential way to control EMT,inhibit the progression and metastasis of pancreatic cancer. |
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