The Biological Effect Study Of Hepsin On Human Prostate Cancer Cell's Migration And Proliferation

Objective: Prostate cancer 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 have been rising gradually in the past decades. The etiology of prostate cancer was shown to be relevant with race and environmental factors, and it results from multiple genetic, molecular abnormality, and its dynamic progression. Since the bone metastasis of prostate cancer is a poor marker of prognosis, studies on the molecular mechanism of local invasion and distant metastasis have become a hot topic in the tumor biology field.The proteolytic activities of membrane-anchored proteins are believed to play a central role in the cell surface activation events. Proteolysis of extracellular matrix (ECM) and cell surface proteins can mediate cell adhesion via integrin binding in cell fusion and cell signaling via interactions of their cytoplasmic domains. The cleavage of ECM components plays an important role in cancer invasion and metastasis.Hepsin mRNA has been identified as a highly overexpressed gene in prostate cancer recently. It is a cell surface expressed chymotrypsin-like serine protease and a member of the family of type â…¡ transmembrane serine protease (TTSP). Currently, the physiological function of hepsin is elusive, and the biological effect of Hepsin on human prostate cancer cell's migration and proliferation is still unclear. Thus, investigation emphasing the effect of hepsin on prostate cancer's progression becomes a necessity. This study is focused on the biological effect of hepsin on theprostate cancer cell's migration and proliferation. Hepsin may become an ideal new prognostic biomarker in prostate cancer as well as an ideal target for prostate cancer therapy.This study was divided into four parts:PART I : Clone the human origin hepsin gene and construct the expression plasmidObjective: Clone the full length cDNA of Hepsin gene and construct the expression vector. Methods: Use RT-PCR to clone the full length cDNA of Hepsin gene of the LNCap Cells and put it into expression plasmid pIRES2-EGFP by gene recombination method. Results: Full length cDNA of Hepsin was cloned and constructed into the expression plasmid of pIRES2-EGFP successfully. Conclusion: This work provides the base for the processing study on the function of Hepsin gene.PART II: Clone the pZeoU6-hepsin shRNAi and construct the expression plasmidObjective: Construct and identify the expression plasmid pZeoU6-Hepsin shRNAi. Methods: Use PCR-based strategy to clone short hairpin sequences in order to construct eukaryotic expression vector pcDNA3. shRNA sequences were converted into a single ~72nt primer sequence onto which were added the human U6 promoter. The vectors were applied to the relative analyses of enzyme cutting and sequencing identification. Results: The expression plasmid pZeoU6-Hepsin shRNAi was constructed successfully. Conclusions: This work could make a base for further study on the effect of hepsin by RNAi.PART HI: Construct and identify PC3 cells stable expressing hepsin gene and establish 22RV1 cells stable silencing hepsin geneObjective: Construct and identify PC3 cells stable expressing hepsin gene and establish 22RV1 cells stable silencing hepsin gene. Methods: Eukaryotic expression vector pIRES2-EGFP expressing hepsin gene was successfully transfected by Lipofectin into PC3 cells and hairpin-shaped siRNAs vector was transfected into 22RV1 cells, which resulted in stable hepsin-expressed PC3 cell lines and hepsin-depressed 22RV1 cell lines after selection with G418, respectively. Empty vector was also transfected as a control. Furthermore, RT-PCR was used for relative identification. Results: The PC3 cells stable expressing hepsin gene and 22RV1 cells stable silencing hepsin gene were estblished. Conclusions: This work could make the basis for further study on the biological effect of Hepsin on prostate cancer cell's invasion and prolification.PART IV : The biological effect study of Hepsin on human prostate cancer cell's migration and proliferationObjective: Study the malignant phenotype of different Hepsin-expression level's PC3 cells and 22RV1 cells. Methods: Wound healing and matrigel coated transwell assay were used to study the migration of PC3 cells and 22RV1;MTT, BrdU and cell counted methods were used to study the proliferation of PC3 cells and 22RV1 cells. Furthermore, the gene expression related ECM was investigated by RT-PCR. Results: Cell proliferation activity analysis showed that overexpression of Hepsin in PC3 cells and down-regulation of Hepsin by RNAi in 22RV1 have no significant promotion to the growth of PC3 cells and 22RV1, respectively.Overexpression of Hepsin in PC3 cells promotes cell migration and the cell's migation ability was improved by about 160%;Down-regulation of Hepsin in 22RV1 cells inhibits cell invasion and the cell's migration ability was inhibited by about 45%, besides down-regulated MMP9 gene expression in 22RV1 cells. Conclusions: Hepsin is one kind of cell surface serine protease that has no impact on cell proliferation;hepsin promotes cell invasion in prostate cancer.Conclusion:From the present study, it has been demonstrated that overexpression of hepsin gene in prostate cancer cells plays a role in prostate cancer development and progression. Hepsin has no impact on cell proliferation, but it promotes' cell migration. In addition, there may be a cross-talk between hepsin and MMP9. Further investigation will emphasize the biological mechanism of hepsin, which may provide insight into the pathways involved in prostate cancer progression. Hepsin can be a future clinical biomarker as well as a target for drug development that may prevent tumor metastasis.