| Pancreatic cancer is one of the cancer with high mortality,and the incidence is increasing year by year.Due to the difficulty of early diagnosis,most patients have developed to the advanced stage of cancer by the time of diagnosis,and the effect of existing treatment methods is not effective.Therefore,it’s an important work to find a specific biological marker for pancreatic cancer diagnosis and targeted therapy.Tumor protein D52(TPD52)gene family is a class of proto-oncogenes which have been discovered recently.TPD52 is related to the occurrence and development of a variety of solid tumors.However,there are relatively few in-depth investigations of TPD52 in the tumorigenesis and development of tumor,and its specific mechanism in pancreatic cancer is not clear.In this study,the expression of TPD52 in pancreatic cancer tissues and its relationship with clinical biological behavior of pancreatic cancer are firstly studied at the tissue level.Then the effect of TPD52 on the biological behavior of pancreatic cancer cells(such as cell proliferation,migration and invasion)and its mechanism are studied at the cellular level.Finally,the subcutaneous tumor model of nude mice is established to observe the effect of TPD52 on the growth of subcutaneous tumor of nude mice.All of these are to provide a basis for the comprehensive evaluation of pancreatic cancer and molecular targeted therapy.Part one Study on the Relationship between TPD52 Expression andObjective:To investigate the TPD52 expression of pancreatic cancer tissue and its relationship with the clinical biological behavior,and further clarify the clinical value of TPD52 in the diagnosis and treatment of pancreatic cancer.Methods:1. The paraffin-embedded tissues are collected from 115 pancreatic cancer patients who were admitted to Cangzhou Central Hospital between January 2014 and October 2018.2.The protein expression levels of TPD52 in pancreatic cancer tis-sues and corresponding paracancerous tissues are determined by immun-ohistochemistry.3.The relationships between the TPD52 expression in pancreatic cancer tissues and the clinicopathological features are analyzed by Chi-square test or Fisher’s exact probability method.4.The relationships between the TPD52 expression in pancreatic cancer tissues and the patient’s prognosis are analyzed by Kaplan-Meier method and COX regression model.Results:1. TPD52 is mainly expressed in cell cytoplasm and cell membrane.In the pancreatic ductal adenocarcinoma tissues,TPD52 is positively expressed to different degrees,with a positive expression rate of 85.22%(98/115).2.The TPD52 expression levels in pancreatic cancer tissues are not associated with sex,age,tumor site,primary focus of tumor,regional lymph node metastasis,hepatic metastasis and portal vein invasion(P?0.05).3.According to the degree of pathological differentiation,the patients are divided into well differentiation group,moderate differentiation group and poor differentiation group.The positive expression rates of TPD52 are 33.30%,70.50%and 72.20%,respectively.The difference of the TPD52 positive expression rates between the three groups is statistically significant(X~2=9.593,P<0.05).4.The positive expression rates of TPD52 are 74.00%and 50.00%in the tumor nerve infiltration group and the tumor nerve non-infiltration group,respectively.The difference of the TPD52 positive expression rates between the two groups is statistically significant(X~2=6.754,P<0.05).5.According to the pathological TNM stage,it is divided into stage I group,stage II group,stage III group and stage IV group.The positive expression rates of TPD52 are 42.90%,71.00%,75.00%and 80.00%,respectively.The difference of the TPD52 positive expression rates among the four groups is statistically significant(X~2=7.829,P<0.05).6.The m OS is 21 months in the TPD52 positive expression group and 31months in the TPD52 negative expression group.The difference in the survival time between the two groups is statistically significant(X~2=9.986,P<0.05).The regional lymph node metastasis,hepatic metastasis,and the level of TPD52 expression are identified as the independent risk factors affecting OS(P<0.05).The associated risk levels for OS are 2.517,12.467and 2.070,respectively.Summary:1.The TPD52 expression level in pancreatic ductal adenocarcinoma is independent of sex,age,tumor site,primary focus of tumor,regional lymph node metastasis,hepatic metastasis and portal vein invasion.2.The expression level of TPD52 is related to the degree of pathological differentiation,the tumor nerve infiltration and the pathological TNM stage.3.The m OS of the TPD52 positive expression group is lower than that of the TPD52 negative expression group.The independent risk factors affecting the OS of pancreatic cancer patients are the regional lymph node metastasis,hepatic metastasis,and the level of TPD52 expression.4.TPD52 may be a specific biological marker to determine the clinical biological behavior and prognosis of patient with pancreatic cancer.Part two Effect of Knockdown TPD52 on Biological Behavior of Pancreatic Cancer CellsObjective:To investigate the effect of knockdown TPD52 on the biological behavior of pancreatic cancer cells.Methods:1.The sh RNA plasmid against TPD52 gene is successfully built and then transfected pancreatic cancer cells.The stable transfection cells are selected.The cells are divided into parental group,NC group and sh RNA group.2. RT-PCR and Western blot are used to observe the effect of TPD52-sh RNA transfection on the levels of TPD52 m RNA and protein in pancreatic cancer cells.3.CCK-8 assay and flow cytometry are used to observe the effect of TPD52-sh RNA transfection on cell proliferation and cell cycle.4.Hoechst staining,flow cytometry and western blot are used to observe the effect of TPD52-sh RNA transfection on cell apoptosis and the expression levels of apoptosis-related proteins.5.Cell scratch assay is used to observe the effect of TPD52-sh RNA transfection on cell migration in vitro.6.Transwell Chambers model is used to analyze the effect of TPD52-sh RNA transfection on cell invasion in vitro.Results:1.The results of RT-PCR show that the level of TPD52m RNA in the transfection group is significantly lower than that in the parental group and the NC group(P<0.001).Western blot results show that the expression level of TPD52 protein in the transfection group is significantly lower than that in the parental group and the NC group(P<0.001).2. The results of cell proliferation detected by CCK-8 assay show that the OD value of the transfection group is significantly lower than that of the NC group at 24h,48h and 72h(P<0.05).The results of cell cycle detected by flow cytometry show that the G0/G1 phase cells in the transfection group are significantly increased compared with the NC group(P<0.05),and the S phase cells in the transfection group are significantly decreased compared with the NC group(P<0.01).3.The results of cell apoptosis detected by Hoechst staining show that the apoptotic positive cells in the transfection group increase significantly compared with the NC group(P<0.001).The results of cell apoptosis detected by flow cytometry show that the transfection group has more apoptotic cells than the NC group(P<0.001).The results of western blot show that the expression levels of cleaved caspase-3 and Bax proteins are significantly up-regulated(P<0.001),while the expressions of Bcl-2 protein is significantly down-regulated(P<0.001),compared with the NC group.4.The results of cell migration detected by cell scratch assay show that the cell migration ability of the transfection group is weaker than that of the control group,and the 24h migration rate is significantly reduced(P<0.05).5.The results of cell invasion detected by Transwell Chambers model show that the transfection group has significantly lower invasion capacity than the control group,and the number of transmembrane cells significantly decrease(P<0.05).Summary:1.Knockdown of TPD52 can significantly inhibit the proliferation of pancreatic cancer cells,lead to cell cycle arrest in the G0/G1 phase,and promote cell apoptosis.2.Knockdown of TPD52 can inhibit the migration and invasion of pancreatic cancer cells.3.Knockdown of TPD52 can regulate the biological behavior of pancreatic cancer cells.Part three Study on the Mechanism of TPD52 Regulating the Biological Behavior of Pancreatic Cancer CellsObjective:To investigate the mechanism of inhibiting the expression of TPD52 gene on regulating the biological behavior of pancreatic cancer cells by treating cells with Akt activator.Methods:1.The cells are divided into NC+DMSO group,sh TPD52+DMSO group and sh TPD52+SC79 group.2.CCK-8 assay and Hoechst staining are used to detect the effect of Akt activator on TPD52-sh RNA transfection to inhibit pancreatic cancer cell proliferation and promote cell apoptosis.3. Cell scratch assay and Transwell Chambers model are used to detect the effect of Akt activator on TPD52-sh RNA transfection to inhibit the migration and invasion of pancreatic cancer cells.4.Western blot is used to observe the effect of TPD52-sh RNA transfection on the levels of Akt and p-Akt protein in pancreatic cancer cells treated with Akt activator.Results:1.The results of cell proliferation detected by CCK-8 assay show that the OD value of the sh TPD52+DMSO group is significantly lower than that of the NC+DMSO and sh TPD52+SC79 groups at 24h,48h and 72h(P<0.05).2.The results of cell apoptosis detected by Hoechst staining show that the apoptotic cells in the sh TPD52+DMSO group increase significantly compared with the NC+DMSO group,and the apoptotic cells in the sh TPD52+SC79 group decrease significantly compared with that in sh TPD52+DMSO group(P<0.001).3.The results of cell migration detected by cell scratch assay show that the cell migration ability of the sh TPD52+DMSO group is weaker than that of the NC+DMSO group.Compared with the sh TPD52+DMSO group,the cell migration ability of the sh TPD52+SC79 group is enhanced,and the 24h migration rate increase(P<0.05).4. The results of cell invasion detected by Transwell Chambers model show that compared with the sh TPD52+DMSO group,the cell invasion ability of the sh TPD52+SC79 group significantly increase,and the number of transmembrane cells increase(P<0.05).5. The results of western blot show that compared with the sh TPD52+DMSO group,the level of p-Akt in the sh TPD52+SC79 group is significantly increased(P<0.01).Summary:1.Knockdown of TPD52 can significantly inhibit the proliferation and promote the apoptosis of pancreatic cancer cells,and the addition of Akt activator can reverse its effect.2.Knockdown of TPD52 can significantly inhibit the migration and invasion ability of pancreatic cancer cells,and the addition of Akt activator can reverse its inhibitory effect.3.Knockdown of TPD52 can reduce the level of p-Akt protein in pancreatic cancer cells,and the Akt activator can restore it.4.TPD52 may affect the biological behavior of pancreatic cancer cells through the Akt signaling pathway.Part four Effect of Knockdown TPD52 on Subcutaneous Transplanted Tumor in Nude MiceObjective:To study the effect of knockdown TPD52 on the proliferation of subcutaneous transplanted tumor in nude mice.Methods:1.12 nude mice are randomly divided into two groups.The NC group is those inoculated with As PC-1-NC cells.The sh TPD52 group is those inoculated with As PC-1-sh TPD52 cells.2.A subcutaneous transplanted tumor model of pancreatic cancer cells(As PC-1)in nude mice is established to observe the growth of the transplanted tumor.3.The tissue structure of transplanted tumors is analyzed by HE staining.4.Western blot is used to analyze the levels of TPD52,Akt and p-Akt in subcutaneous transplanted tumor cells of nude mice.Results:1.The volume of the subcutaneous transplanted tumor in the transfection group is significantly smaller than that of the NC group,and the difference is significant at 7 days(P<0.001).The average weight of the subcutaneous transplanted tumor in the NC group is higher than that in the transfection group(P<0.0001).2.In the transfection group,there is necrosis in the subcutaneous tumor tissue,the cell density is decreased,and there is a large amount of fibrous connective tissue hyperplasia.In the NC group,the subcutaneous tumor tissue is rich in cells,the mitosis is more common,and there are abundant blood vessels in the tumor tissue.3.The results of western blot show that compared with the NC group,the level of TPD52 and p-Akt in the transfection group is significantly decreased(P<0.001).Summary:1.Knockdown of TPD52 in pancreatic cancer cells can significantly inhibit the growth of subcutaneous transplanted tumor.2.Knockdown of TPD52 in pancreatic cancer cells can reduce the density of cells and induce hyperplasia of fibrous connective tissue in the subcutaneous transplanted tumor.3.Knockdown of TPD52 in pancreatic cancer cells can reduce the expression level of TPD52 protein in the subcutaneous transplanted tumor,which may reduce the proliferation of pancreatic cancer cells and inhibit tumor growth through the Akt signaling pathway in vivo.Conclusion:1.The TPD52 expression levels in pancreatic cancer tissues are related to the degree of pathological differentiation,the tumor nerve infiltration and the pathological TNM stage.The TPD52 expression level is the independent risk factors affecting the OS of pancreatic cancer patients.2.Knockdown of TPD52 can significantly inhibit the proliferation,migration and invasion of pancreatic cancer cells,and promote cell apoptosis.The Akt activator can reverse the effect.3.Knockdown of TPD52 in pancreatic cancer cells can significantly inhibit the growth of subcutaneous transplanted tumor.4.TPD52 may affect the biological behavior of pancreatic cancer through the Akt signaling pathway.TPD52 may become a specific biological marker to determine the clinical biological behavior of pancreatic cancer and a new target for gene therapy of pancreatic cancer. |
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