| Background:Pancreatic cancer is a malignant digestive tract tumor with rapid onset and poor prognosis.It is a serious threat to the life and health of patients.Although the 5 year survival rate of most cancer patients has improved over the past few decades,it is regrettable that the 5 year survival rate of patients with pancreatic cancer is still very low and has not achieved a significant therapeutic effect.Only a few patients with locally resectable pancreatic cancer can be controlled,but only about 5-10%patients have the chances to get operation.Overall,the 5 year survival rate of pancreatic cancer patients is only 8%.Faced with this dilemma,we should constantly strengthen the research of pancreatic cancer,and try every possible way to improve the survival rate of patients with pancreatic cancer.There are many signaling pathways involved in the development of pancreatic cancer,especially in the part of malignant biological behavior of pancreatic cancer,such as K-Ras,JNK,Hedgehog,Wnt/Notvh signaling pathways and so on.As a member of the transducin-like enhancer,TLE1 is an inhibitor of some transcription factors and plays an important role in the growth and development.Besides,it plays an important role in the process of tumor occurrence,invasion and metastasis.Unfortunately,there is no in-depth study of the specific mechanisms of TLE1 in the process of tumor development.Moreover,there were no related papers to explore the relationship between TLE1 and pancreatic cancer,and the relationship between the results of TLEl and the follow-up of the prognosis.Therefore,the exploration of the relationship between TLE1 and pancreatic cancer is of great significance to the study of the specific mechanism of malignant biological behavior of pancreatic cancer cells and the evaluation of the prognostic value.It also explores new targets for the treatment of pancreatic cancer.Objectives:1)To find the effects of TLE1 on the migration,proliferation and cell cycle of pancreatic ductal adenocarcinoma cells;2)To understand the mechanism of TLE1 in the migration,proliferation and cell cycle of pancreatic ductal adenocarcinoma cells.3)To analyse of the value of TLE1 for the prognosis of pancreatic cancer patients.Methonds:1)The transfection reagents were used to transfect TLE1 silencing and overexpressed plasmids into pancreatic duct adenocarcinoma cell lines MiaPaCa-2 and T3M4 to establishment of transient transfection strain;2)Wound-healing test and Transwell test were used to determine the migration ability of cells in each group.CCK8 method was used to observe the proliferation ability of each group.PI method was used to observe cell cycle activity.Western Blot was used to detect the expression of EMT and cell cycle marker protein;3)The RNA of the cell line after transfection was collected and the transcriptional group was sequenced and the sequencing result was analyzed;4)Using immunohistochemical staining to evaluate the prognostic value of TLE1 in patients with pancreatic cancer.Results:1)The wound-healing test showed that in both MiaPaCa-2 and T3M4,the healing velocity of the silenced group was significantly shorter than that in the control group,while in the over expression group,the healing time of the overexpressed group was significantly higher than that of the control group.In the Transwell migration experiment,up regulation of the expression of TLE1 in MiaPaCa-2 and T3M4 inhibited the migration of pancreatic cancer cells,while the silenced expression of TLE1 increased the migration ability of two cells;2)The detection of EMT pathway marker protein showed that in the silenced group,both MiaPaCa-2 and T3M4 cell lines,N-Cadherin and Vimentin were up-regulated,while TLE1 and E-Cadherin were down regulated.In the overexpressed group,the situation is the opposite.3)The results of CCK-8 proliferation test showed that the proliferation rate of the silenced group was far greater than that of the normal control group for the MiaPaCa-2 cell line,and the proliferation rate of the two groups was different from the second day(P<0.05).In the comparison between the over expression group and the control group,the proliferation rate of the two groups was also statistically different(P<0.05),but the proliferation rate of the group was lower than that of the normal control group.For T3M4 cell lines,the cell proliferation rate in both the silenced group and the overexpression group was lower than that of the corresponding MiaPaCa-2 cell line.However,the phenotype of the proliferating phenotype is the same as that of MiaPaCa-2.4)The cell cycle was analyzed by PI method.In MiaPaCa-2 and T3M4,overexpression of TLE1 resulted in cell arrest at G0/G1 stage(MiaPaCa-2:83.12±11.22 vs 88.4± 1.60,P=0.011;T3M4:57.33±3.16 vs 69.48±2.69,P=0.007),while S phase cells decreased(MiaPaCa-2:8.81±0.14 vs 7.01 ±0.78,P=0.018;T3M4:37.01±13.37 vs 21.27=3.90,P=0.007).In the silenced group,the situation is the opposite.Detection of cell cycle marker protein showed that overexpression of TLE1 protein reduced the Gl/S phase marker protein cyclin D1 of MiaPaCa-2 and T3M4 cell lines,and the G2/M period marker protein cyclin B1 and cyclin A.For the two cell lines with knockout,the cyclin D1 cyclin B1 and cyclin A were increased.5)The transcriptional group was sequenced,and the 16 genes involved in the regulation of TLE1 on MiaPaCa-2 and T3M4.6)The immunohistochemical results of pancreatic cancer patients who had gotten operation showed that the expression of TLE1 in pancreatic cancer tissues was higher than that of paracancerous tissues(p<0.001)and has connection with the age(p=0.003),whether drinking(p=0.045),whether the skin sclera yellow dye(p=O.034),whether the degree of differentiation(p=0.005).Conclusions:1)TLE1 inhibits the migration and proliferation of pancreatic cancer cells,and blocks the mitotic cycle of pancreatic cancer cells,showing the function of inhibiting cancer.2)TLE1 can inhibit EMT,which blocks the migration of pancreatic cancer cells by up regulating E-Cadherin.3)TLE1 inhibits the proliferation of pancreatic cancer cells by blocking G1/S phase and G2/M phase.4)There was no correlation analysis in the expression of TLE1 and the overall survival of patients with pancreatic ductal adenocarcinoma.5)The expression of TLE1 protein in pancreatic ductal adenocarcinoma has correlation analysis with age,drinking,sclerotic yellow staining and tumor differentiation.6)Whether the family history of pancreatic cancer,the loss of weight,the degree of differentiation and lymph node metastasis are the risk factors that affect the prognosis,and whether there has the loss of weight,the degree of tumor differentiation,and the lymph node invasion is an independent risk factor that affects the prognosis of the patients with pancreatic cancer. |
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