The Mechanism And The Effect Of Interaction Of CypA And HIF On The Ability Of Invasion And Metastasis Of Pancreatic Cancer

Object iveHIF-1is closely related with the biological behavior of pancreatic cancer. Early study results suggest that there might be a relationship between the expression of HIF-1a and CypA. This study aimed to explore the molecular regulation mechanisms, as well as HIF-1and CypA influented on biological behavior of pancreatic carcinoma and study on its mechanism; Explicited CsA and2ME on tumor growth and the effects of drug toxicity; Validated CypA and HIF expression in the histopathology of pancreatic cancer and Influence on the pathologic outcomes.MethodsPart I:The regulation relationship of HIF-1a to CypA was verified.Applied Si-RNA-a HIF-1interference pancreatic cancer cell lines BXPC-3to inhibit HIF-1α expression, and applied PC-DNA3.0-HIF-1α to MIA-paca-2cells to over express HIF-la, application of real time PCR, western blot authentication to detect the expression of CypA and HIF-1mRNA and protein; using Chromatin immune-precipitation techniques to determine the combination of HIF-la and CypA promoter, further clarify the regulation mode between HIF-1α and CypA.Part II:The influence of CypA on pancreatic carcinoma cell lines’biological behaviors and mechanism.Application Si-RNA-HIF-1a inhibited BXPC-3cell lines HIF-la and CypA, to MIA-paca-2cells with PC-DNA3.0-HIF-1a overexpressed HIF-1a, and by giving2ME and CsA to inhibit HIF-la and CypA expression, flow cytometry was used to detect the changes of cell cycle and apoptosis. MTT assay was used to detect cell proliferation ability. PCR, Western blot, immunofluorescence methods were used to detect cellular HIF-1alpha, CypA, NFKB, ERK1/2, p-ERK1/2, P53expression. Transwell cell assay was used to detect cell migration.Part III:Validation HIF-la and CypA signaling pathway and the influence of tumor growth in vivo.16BALB/c nude mice were randomly divided into four groups, the control group:3mice, fed saline20ml;2ME (2-methoxyestradiol,2ME) group:4mice, with2ME 50mg/ml/kg and20ml saline; CsA group:4mice, with CsA (Cyclosporin A, CsA)20mg/ml/kg and20ml saline; group2ME+CsA:5mice,25mg/ml with2ME/kg and CsA1Omg/ml/kg and20ml saline. Every Tuesday, Thursday morning for two weeks, an interval of one week of a cycle, row a total of3cycles. Mice were sacrificed after the end of the administration. Harvested the tumors, liver and kidney tissue, measuring tumor size and weight; immunohistochemistry detected the expression of HIF, CypA, NFkB, ERK1/2, P53; HE stained with the liver and kidney tissues to evaluate the toxicity.Part IV:The correlation of HIF-la and CypA in pancreatic carcinoma and its effect on prognosis of patientsSelected specimens of79cases with pancreatic cancer in Tian jin cancer hospital. Immunohistochemical staining was used to detect the expression of HIF-1α and CypA. Patients were followed-up and calculated the survival rate by Kaplan-Merier method.Resul tsPart I:The relationship of HIF-1a and CypACHIP:There was3HRE in the CypA promoter, HIF-1α combined of the second HRE located at-77bp of CypA promoter.Western blot results showed:MIA-paca-2cells couldn’t express HIF-la and highly express in BXPC-3cells, CypA protein in BXPC-3cells expressed more than that of MIA-paca-2cells. MIA-paca-2cells in hypoxic condition training (1%O2), the expression of HIF-la and CypA at1h and3h were upward compared with the Control group(HIF-1α:2.062and4.739times; CypA:2.058and20.833times, respectively). Interference of HIF-la to BXPC-3cells:expression of HIF-1a protein reduced, expression of CypA protein reduced too (0.559and0.187, respectively). Expression of HIF-la in MIA paca-2cells, expression of CypA also increased (3.22and1.88, respectively).PCR results showed:After the Si-RNA interference of HIF-la to BXPC-3cells, expression of HIF-1a mRNA decreased, and expression of CypA mRNA also decreased. Over expression of HIF-1a to MIA-paca-2cells by pc-DNA3.0-HIF-1a, expression of HIF-la and CypA mRNA were increased. MIA-paca-2cells cultured under hypoxia (oxygen concentration1%), the expression of HIF-1mRNA and of CypA mRNA on1h and3h:the expression was significantly increased (p<0.05). Part Ⅱ:Effect of CypA expression on pancreatic carcinoma cell line biological behaviors and mechanismScratch test results showed:Detected the scratch space at Oh,6h,12h and24h of BXPC-3cell after transfection by siRNA-HIF-la and siRNA-CypA. After transfection, the width in the transfer groups were smaller than that of control group (p<0.05).2ME and CsA to the BXPC-3cells at Oh,6h, cell migration in every group there was no statistically significant differences (p>0.05). CsA group and2ME+CsA group of cell migration were faster than the control group (p<0.05) at12h;2ME+CsA group of cell migration were significantly lower than single drug group and control group at24h (p<0.05).MTT results showed:After interference of CypA and HIF-la to BXPC-3cells, cell proliferation was the same in the interference group and control group (p>0.05). The proliferation of two interference group ability decreased significantly at48h-72h, especially the CypA interference group. After giving inhibitors of HIF-la and CypA to BXPC-3cell, single2ME and CsA might lead to a decline in cell proliferation. When combined the two drugs, the inhibition of cell proliferation increased (p<0.05). There was a synergy effect between the two drugs. Their collaborative index was0.58.Transwell results showed:Interference of HIF-1α and CypA, the cell number through the membrane was not statistically significant differences at12h,24h. And wearing membrane cells were significantly less than the control group at48h and72h (p<0.05). After the Crystal Violet stain, OD values were detected. The interference groups of HIF-1α and CypA:OD values were decreased compared with control group (p<0.05); after giving2ME and CsA, cell invasion were significantly less than the control group at12h and48h (p<0.05). In2ME+CsA group the wearing cells number was the fewest (p<0.01).Flowcytometry detection results showed:The apoptosis in CsA group increased compared with control group (p<0.05). Apoptosis increased in2ME+CsA group compared with control group and the single drug group (p<0.05). Transfection by Si-RNA-HIF-1α and CypA, apoptosis of cells increased; but individual interference of CypA, HIF-1α expression cells apoptosis were no statistically significant difference (p>0.05). After suppression of HIF-1α and CypA to BXPC-3, cell cycles were not changed (F=0.599, p>0.05).PCR results showed:The interference of HIF-1α decreased the expression of CypA mRNA and ERK, NFkB mRNA expression decreased (p<0.05); Interference CypA group:the expression of HIF-1α mRNA were increased (p>0.05), while expression of ERK, NFkB, P53mRNA had no significant change (p>0.05). Given2ME and CsA to BXPC-3cells:2ME group the expression of NFkB, P53mRNA were statistically different (p<0.05); the expression of ERK, NFkB mRNA decreased in CsA group than control group (p>0.05);2ME+CsA group, the expression of CypA, HIF-1α mRNA decreased, P53gene expression increased significantly compared with the control group4.2times(p<0.01). MMPs expression had no obvious change in2ME group compared with control group; the expression of MMPs mRNA decreased in group CsA than control group (p>0.05); MMP3and MMP9decreased in2ME+CsA group (p<0.05).Western blot results showed:Interference of CypA and HIF-1α, the expression of P53increased. ERK1/2, NFkB protein trended decrease in interference group and decreased in dosage groups than control group (p<0.05). The expression of HIF-1α and CypA protein decline0.21and0.10times compared with control group, respectively. The expression of P53protein increased, and expression of NFkB, p-ERK1/2, ERK1/2protein decreased in dosage groups than control group (p<0.05). Immunofluorescence results showed: expression of HIF-1α protein in control group and CsA group expressed similar. The expression of HIF-1α protein was significantly decreased expression in2ME group and2ME+CsA group. The expression of CypA protein decreased in each group compared with the control group. The2ME+CsA group was most obvious. The expression of P53protein gradually increased in2ME, CsA and2ME+CsA groups. But expression of NFkB, ERK1/2protein decreased in group2ME+CsA.Part Ⅲ:Effects of HIF-1α→CypA signaling pathway to the tumor growth in vivo The tumor size was significantly decreased in3administration group, especially in 2ME+CsA group. Tumor size reduced to more than half than the single drug groups. Imrnunohistochemical staining results:the expression of P53protein decrease significantly in2ME+CsA group; the expression of ERK1/2and NFkB in the control group was more than that of the3other groups (>70%).HE staining of the liver and renal tissue results showed: Liver and kidney of four dying mice were:the livers were apparent enlarged and soft texture, and the color was light brown. Enlarged kidneys were clear and soft. The sacrificed mice liver tissue were without any obvious lesion under the microscope in2ME and CsA groups; kidney hemorrhage was not significant;2ME+CsA group, three mice had only mild fatty degeneration of the liver and slight bleeding of kidneys.Part IV: HIF-la and CypA correlation between expression in pancreatic carcinoma tissue and its effect on prognosis of patientsThe expression of HIF-la, CypA was relative in the tissues of pancreatic cancer. The expression of CypA was relative with the survival time of the patients. Patients lived9months in the positive of CypA and19months in the negative of CypA. The expression of HIF-la was not effect in the survival time. patients lived12months in both of the expression of HIF-la.Conclusion1HIF-la combined of the second HRE located at-77bp of CypA promoter to regulate the expression of CypA.2The hypertension expression of HIF-1α and CypA can increase the capacity of cell proliferation, infiltration and metastasis.2ME and CsA through up-regulation the expression of P53induce apoptosis of pancreatic cancer cells and through inhibiting the expression of ERK1/2/NFkB/MMP2,3,9to decrease the proliferation, invasion and metastasis ability of pancreatic cancer cell.3CsA and2ME can inhibit tumor hyperplasia, and union application effect was more obvious, but the combination can lead to fatal liver and kidney function damage.4HIF-1α and CypA expression are correlated in the tumor of pancreatic cancer. High expression of CypA in patients can cause poor prognosis.