The Effects Of Raf Kinase Inhibitory Protein Expression In Pancreatic Cancer And The Mechanisms Contributing Migration And Invasion

Background:Pancreatic cancer is a highly malignant and aggressive digestive tract tumor,the main treatment for which is early resection.However,the prognosis of pancreatic cancer is poor due to the lack of effective method for early diagnosis presently.There are numerous studies of the molecular mechanism of pancreatic cancer,and Raf kinase inhibitory protein(RKIP)is one of the most important molecules.RKIP is a member of the phosphatidylethanolamine-binding protein(PEBP)family.Human RKIP genes,which is transcripted by four exons and mainly in the cytoplasm and the plasma membrane.RKIP gene is involved in regulation of many signal transduction pathways including Ras/Raf-1/MAPK(ERK),G protein and NF-?B signal pathways and plays an important role in the process such as cell growth,proliferation,differentiation and apoptosis.RKIP may play a part in tumor suppression,and the expression of RKIP has been shown to be associated with the occurrence,invasion,migration and metastasis of many tumors such as prostate cancer,breast cancer,colorectal cancer,and melanoma,which may make RKIP the new target for tumor diagnosis,prognosis evaluation and targeting therapy.Early metastasis to regional lymph nodes or hematogenous spread to distant organs of pancreatic cancer could influence the therapeutic effects and maybe one of the most important reasons for its high rate of mortality.The metastasis of pancreatic cancer is a complicated process which is closely related to the interaction and influence of the body,tissue and tumor cells.To explore the mechanism of the invasion and metastasis of pancreatic cancer is an important direction of research.On the basis of the preliminary studies,we collect tissue samples which were diagnosed histologically with pancreatic cancer,use polymerase chain reaction(PCR)and immunohistochemical staining to determine difference of the expression level of RKIP in tumors and normal adjacent tissues,and analyze the correlation of such difference with the prognosis,and we use the gene transfection technology to build pancreatic cancer cell lines with RKIP stable expressed for the research of the effect of RKIP in cell proliferation,migration,invasion and apoptosis.Part one: The expression of RKIP in pancreatic cancer and its correlation with prognosisMethods: Pancreatic cancer specimens as well as the clinicopathological datas were collected from patients diagnosed histologically,the RNA of tumors and normal adjacent tissues was obtained.Real-time PCR was performed to detect the difference of the RKIP expression.As well we use immunohistochemical staining to detect the protein expression level of RKIP,according to which divide patients into 2 groups(high-RKIP,low-RKIP),compare the follow-up datas to identify the correlation of RKIP expression levels with the clinicopathological features and prognosis.Results: RT-PCR and immunohistochemical staining results showed that mRNA levels of RKIP were down-regulated in areas of pancreatic cancer compared with normal tissues(P<0.01),the low-RKIP group include a significantly larger tumor size(P<0.001),a higher TNM tumor stage(P=0.006)and a greater depth of tumor invasion(P=0.003)when compared with the high-RKIP group.Compared with low-RKIP group,patients with high RKIP expression level had longer overall survival time(P<0.001).Conclusion: The expression of RKIP is inhibited in areas of pancreatic cancer,which is related to the tumor growth,invasion and pathologic TNM staging,the survival of RKIP high expression group is obviously better than the low expression group.These conclusions above show that RKIP may be associated with the invasion and metastasis of pancreatic cancer,and there is certain prognostic value.Part two: The effect of RKIP on the invasion and metastasis of pancreatic cancer cells and its mechanism.Methods: The coding regions of RKIP complementary DNA(cDNA)were cloned into the pc DNA3.1 expression vector.The c DNA was then fully sequenced to ensure the accuracy of RKIP c DNA sequence.SW1990 and As PC-1 cells were transfected with pcDNA3.1-RKIP and pcDNA3.1,respectively.Cell viability was measured using the Cell Counting Kit-8(CCK-8),Flow cytometry was used for detect cell cycle analysis,and cell migration and invasion were detected by Transwell assays.The RKIP-transfected of vector-transfected pancreatic cells were injected into the mice,and injected mice were examined for measuring tumor appearance.Results: The result of CCK-8 showed that RKIP over-expression inhibited the proliferation of pancreatic cells.The results of flow cytometry showed that when compared with the vector groups,there was an increase in apoptosis of pancreatic cells that were transfected with RKIP expression plasmid.Cultured cell migration and invasion were reduced in cells that overexpressed RKIP when compared with control cells.Tumor cells inoculation experiments in vivo showed that the growth of RKIP-overexpressed xenografts was inhibited when compared with those formed by vector cells.Conclusion: Down-regulation of RKIP expression may inhibit the development,invasion and migration of tumors,the mechanism may be related to suppression of cell growth,proliferation,invasion and promotion of apoptosis.