| Objective:Prostate adenocarcinoma is the most common primary malignant tumor among male population in the western world. As the age indication increases and the dietary ingredient changes in China, the incidence and death rate of prostate cancer raises gradually in the past decades. The etiology of prastate cancer has been showjyelevant to race and environmental factors, and results from multiple genetic and molecular abnormality and their dynamic progression. The bone metastasis of prostate adenocarcinoma is the poor marker of prognosis; therefore the study on the molecular mechanism of the local invasion and distant metastasis has become the hot topic in the tumor biology field.The adhesive molecules are essential for the tumor's invasive growth and metastasis. As the an adhesive molecule, CD44 involves the cell-cell and cell-matrix interactions, and play an important role in the regulation of cell's proliferation, suvival, differentiation and motility. In addition, CD44 as the co-receptor interacts with numbers of molecules including excellular matrix components, growth factors, matrix metelloproteinases, etc. to regulate tumor cells proliferation and metastasis. In many malignant tumors, CD44 has shed by MT1-MMP extracellularly, and causes the release of the soluble CD44, which facilitate the tumor cells motility. As the result from above processing, CD44AE (lacking the CD44 extracellular domain) is cleaved and the intracellular tail of CD44 is, in turn, liberated from the cell membrane has been reported lately. Presenilins, highly conservative transmembrane protein family, are responsible for this r-secretase proteinase process. However, the subcellular location and the potentia1 function of the released CD44ICD (CD44 intracellular domain) remain uncertain. Therefore, in this study we used molecular biology approach toinvestigate the cleavage and liberation of the CD44ICD and its subcellular location. Furthermore, we observed the proliferative and motile effect of CD44ICD on prostate adenocarcinoma cells. Method:1. Obsever the proliferative and motile effect of presenilins inhibitors on prostate adenocarcinoma cell line PC-3;2. Over expressing CD44FL, CD44 AE and CD44ICD in PC-3 cells, and study their phenotype in the aspects of proliferation and migration;3. Invesitgate the subcellular location of CD44ICD via cell fractionation and confocal microscopy;4. Explore the possible signaling transduction mediated by CD44ICD through performing luciferase reporter genes assay on the commom transcriptional factors;5. Using RNA interference and anti-sense RNA techniques to knock down the CD44 expression in PC-3 cells, and investigate the reverse of prostate adenocarcinoma's malignant phenotype;6. To study the migration of PC-3 cells, we used zymogram assay, wound healing and Matrigel coated transwell assay etc;7. To study the proliferation and apopotisis of PC-3 cells, we used MTT and Tunel assay etc.Results:1. CD44 and presenilin 1/2 expressed in prostate adenocarcinoma PC-3 cells;2. Presenilins inhibitors DAPT and DupE, but not JLK2, down-regulated MMP9 expression in PC-3 cells, and inhibit the motility of PC-3 cells;3. DAPT and DupE, but not JLK2, treated PC-3 cells' proliferate rates started to decrease from day3; DAPT and DupE, rather than JLK2, induce the apoptosis in PC-3 cells;4. The motility of the PC-3 cells over-expressing CD44 AE and CD44ICD significantly higher than the PC-3 cells over-expressing CD44FL; the proliferation rate of the PC-3 cells over-expressing CD44FL, CD44 AE and CD44ICD is higher as well, and transfection did not induce apoptosis;5. DAPT and DupE, rather than JLK2, blocked the liberation of CD44ICD from CD44 AE;6. PC-3 cells transfected with CD44ICD resuced the MMP9 expression under the treatment of DAPT;7. CD44ICD translocated into nuclear after releasing from CD44 AE, and might regulate the downstream signaling trusduction via NF-kB pathway;8. RNAi as well as Anti-sense RNA techniques knocked down b...
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