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Effects of Silencing of E2F3 Expression on Cell Dividing Cycle and Apoptosis of Prostate Cancer 3 Cell Line

Author: WangMi
Tutor: WuChangLi
School: Tianjin Medical University
Course: Surgery
Keywords: protate cancer AAV Virus RNA interferenee E2F3 gene Flow cytometry
CLC: R737.25
Type: Master's thesis
Year: 2011
Downloads: 69
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Abstract


Protaste cancer is the most common urological malignant tumor, more common in western countries and regions.The incidence of our country has been lower,but recent years have rising trend. At present the main treatments of early prostate cancer (≤T2 N0 M0) are watchful waiting, neoadjuvant endocrine therapy and adjuvant endocrine therapy,radical surgery, internal radiation or external radiation therapy; locally advanced Prostate cancer (T3 N0 M0, C period) were not considered appropriate to accept radical prostate surgery; advanced prostate cancer (≥T3,≥Nl orM1,≥D1 period) were treated with combined androgen blockade, continuous and intermittent endocrine therapy endocrine therapy.Although endocrine therapy and radiation can prevent and delay recurrence and progression of prostate cancaer.But after endocrine therapy,many prostate cancers change into hormone refractory prostate cancer. We need new therapies. With the development of molecular biology, oncology, immunology,It is hope that gene therapy has become a human thoroughly cure for cancer. Currently effectively targets and the selection of treatments are clinical barriers for gene therapy application, and RNA interference (RNA), RNAi devices for gene therapy found brings new ideas of targets.RNA interference (RNAi) is a kind of plants and animals, exist widely induced by double-stranded RNA, homology of mRNA specificity silent process, including small signal RNA generated infancy and target the effects of mRNA degradation stage. The phenomenon of RNA interference refers to endogenous and exogenous double-stranded RNA and cell mRNA homologous sequence combination and degradation phenomenon, is a sequence specific transcription gene film, its role to knockout, resulting in lack of corresponding gene phenotype. Compared with conventional technology, advantages of RNAi are less money,short cycle, easy operation.It is a fast and effective method to identificate gene, a new research to study gene.It is useful to be used to signal transduction pathways and cell growth differentiation process. Currently RNAi technology has been widelhy applied to cancer research.Out of control of regulation is closely related with development of tumor Initially E2F is found as E2 promoter of activator. E2F family plays an important role in cell cycle regulation. The abnormal expression of E2F family promotes cell proliferation and malignant transformation. Prostate cancer,breast cancer and other tumors have abnormal E2F expression. In this article we want to know if E2F3 play the key role in cell cycle regulation, if E2F3 can be a new target in protate cancer gene therapy and if knocking-down E2F3 expression by using RNAi can inhibit the proliferation of cancer cells in vitro.Recently Adeno-associated virus vector is researched extensively in gene therapy.because of its broad host、low immunogenicity、high safety and stability,AAVs is considered as one of new generation virus vectors. AAV vector has been used for treating tumor in different animal models, the results show that it is a potential vector.Objective:In this article we have developed Potent RNAi AAVs correponding to E2F3 gene transfected the AAVs into human PC3 cell, assessed the cell cycle and cell apoptosis in protate cancer 3 cells. Preliminnary we want know that if it is feasible for a target gene.Methods:Transfection of AAV virus are provided by Dr hu in this experiment. In this study,we used the optical microscopy to watch the following aspects to know cell growth:cell size, particles within the cytoplasm,nuclear chromatin,cell gap,debis and so on;different doses of pAAV-siRNA-E2F3 were transfected into PC3 cells to determine the optimal transfection of virus titer and time; the transfection efficiency can be acquired by observing green fluorescence of cells. FCM was used to assessed the cell cycle and cell apoptosisb of protate cancer 3 cells before and after transfection. Data from experimental data using statistical software SPSS 16 for statistical processing.Results:Non-treated cells growed in good condition, empty virus cells also slightly affected, while the recombinant AAV group significantly reduced the number of PC3 cells and irregular cell was shrunken, particles increased, the cell gap increased, a large number of cell debris, and the small number of cells floating in the culture medium; recombinant AAV MOI after transfection to determine the optimum value of 1*105v.p.cell, optimal transfection time was 1h; we can watch the green fluorescence after 30-60 minutes transfection, greenfluorescence observed in the expression of transfected into 72h, about 60% of the prostate cancer PC3 cells found greenfluorescence; Detection of cell cycle by flow cytometry showed that:untreated cell group G1 and S phase period (30.20±9.08)%,(58.30±3.22)%; emp-ty virusgroup of PC3 cells in G1 phaseand S phase were (32.07±3.21)%, (57.64±3.58)%; the rAAV group were (79.59±5.01)%, (13.08±0.58)%, untreated cell grou-p and the empty virus group compared no significant diffrence(P>0.05);untreated ce-lls group andr AAV group were significantly (P<0.01); empty virus group phase and rAAV group were significantly (P<0.01); FITC-PI Detection of cell apoptosis by flow cytometry showed that:an empty virus group apoptosis rate was (2.77±1.80)%; rAAVgroupapoptosis ratio was(28.22±1.88)%; the difference was significant (P<0.01).Conclusion:PC3 cells can be transfected efficiently by rAAV. PC3 cells can be blocked in G1 phase,rAAVcan block cell cycle progression significantly and induced apoptosis.Silencing E2F3 may be developed into a potential tool for protate gene therapy.It provides a theoretical basis for follow-up experiments.

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CLC: > Medicine, health > Oncology > Genitourinary tumors > Male genitalia tumors > Prostate cancer
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