Lentivirus-mediated Stable Interference Of YAP1 In Prostate Cancer Cell Lines And Effects

Objective: We package and prepare lentivirus by constructing a recombinant lentiviral vector interferring with YAP1. By virtue of high efficiency of lentiviral transfection, we establish a stable prostate cancer cell line with knock-down of YAP1.After that, we define roles of YAP1 in prostate cancer via detecting the growth of prostate cancer cell line and time needed for tumor formation in nude mice,providing tools for further research in genetic pathway related to YAP1.Methods: We detect several prostate cell lines for Yap1 protein expression by western blotting and determine one with highest expression level for research.Then we use plasmids ps PAX2, p MD2.G and the lentiviral vector which has been screened most effectively tranfecting 293 T cells to package lentivirus and collect the supernatant which contains lentivirus to infect C4-2 cell line. We screen further by puromycin and obtain a cell line with stable knock down of Yap1 expression. What’s more,we detect the knock-down efficiency by RT-PCR and western blotting.After that,MTT are used to detect cell viability and aptosis. Next we inoculate control and stable knock down cell line subcutaneously in nude mice,and then compare these two groups for their ability to form tumors.Results: 1.We detected RWPE-1,Lncap,PC3,C4-2 for Yap1 expression by western blotting, Among which C4-2 expressed the most.Then we transfected YAP1-sh RNA into C4-2 cells and found that the second sequence has higher gene silencing effect.2.After concentrating lentivirus with ultracentrifugation,we measure the titers of lentivirus by q PCR to ensure it meeting demand.72 hours after infecting C4-2 cells with concentrated lentivirus,we screened tranfected C4-2 cells by puromycin whch has been explored for the optimal concentration range for two weeks.And e GFP expression was close to 90 percent of all the tranfected C4-2 cells.By western blotting and RT-PCR, we detected nearly 85 percent konck-down efficiency.3.Next,we find deletion of Yap1 gene decreased cell viability and incresed the aptosis detected by MTT.4.Furthermore, after comparing two groups of nude mice,we found cells lacking Yap1 gene formed tumors much smaller than control group.Besides, fluorescence intensity was much weaker than the other one.Conclusions: In this experiment,we successfully construct a prostate cancer cell line with stable knock down of Yap1 expression by lentiviral vector technology,which slows down cell proliferation ability obviously.In vivo animal experiments shows that cells lack of Yap1 gene form tumors much slower than control group.It is of great significance to further study mechanisms of YAP1 in development,recurrence and metastasis of prostate cancer,and maybe it could be one of potential therapeutic targets.