Effects Of Silencing Lymphocytes Cbl-b Gene On The Cytotoxicity Against Murine Prostate Cancer Cells RM-1 In Vitro

Background Prostate cancer is the most common male cancer in the United States and Europe. It accounts for the second most frequent death in male cancer. As the population aging and diet changing in China, the incidence of prostate cancer is increasing year by year. The growth of prostate cancer is hormone-dependent. The majority of advanced prostate cancer treated with androgen deprivation therapy can draw out satisfactory outcomes in the beginning. But after a period of time of androgen deprivation therapy, almost all the prostate cancer will convert to hormone-independent prostate cancer (also called androgen-independent prostate cancer, AIPC). The mechanism of AIPC is not yet clear. Untill now we don't have effective treatment on it. Its prognosis is very poor. Cancer immunotherapy as an important means of modern cancer treatment has been paid more and more attention. In recent years, the areas in the research of prostate cancer immunotherapy mainly include:cytokines, monoclonal antibodies, tumor-associated antigen targeted therapy, dendritic cell immunotherapy and tumor vaccines. Some researches have shown that the tumor-specific lymphocytes can be rapidly induced into the status of immune tolerance in the local microenvironment in the prostate cancer. Therefore they can not attack tumor effectively. A variety of ways have be taken to increase the Immunogenicity of tumor cells and activate the tumor-specific lymphocytes. But they can not reverse the immune escape and immune tolerance status. So none of them manages the prostate cancer effectively. It is noteworth that in recent years studies have shown that the silencing of cbl-b gene in lymphocytes might be able to overcome these obstacles. Cbl-b (Casitas B-cell lineage lymphoma-b) is a member of Cbl family. It is a negative regulating molecule of lymphocytes. It plays a key role in the regulation of peripheral T cells activation and tolerence. cbl-/- CTL can be activated directly in the absence of costimulatory ligands. cbl-/-T cells are more resistant to the inhibition of the inhibitory cytokine TGF-0 and inhibitory lymphocyte Treg. Targeting this molecule may bring a way to overcome tumor induced immune tolerance. Studies have found that ablation of cbl-b gene could effectively reduce tumor incidence in spontaneous lymphoma model and regressed the tumor which had grown up. Untill now there is no report concerning immunotherapy of prostate cancer by silencing cbl-b gene in lymphocytes.Objective The cbl-b gene in lymphocytes was silenced by mouse cbl-b specific siRNA. Then T-cell activation and cytokine expression changes were examed as well as mouse prostate cancer cell RM-1 killing activity in order to understand the changes of anti-tumor immunology in prostate cancer. Thus to provide a theoretical basis for the immunotherapy in prostate cancer and to futher explorer the escape mechanisms of prostate cancer.Methods1. Using an online siRNA design software to design mouse cbl-b gene specific siRNA sequence. Then they were blasted in the genomic database by genome sequence comparison in order to ensure a high degree of specificity. The recombinant expression plasmid containing the green fluorescent protein were constructed and packed with lentiviral vector packing system.293T cells was chosen as the platform for transfection. The inhibition of the Cbl-b expression levels were detected by Western blotting.2. Mononuclear cells were obtained from the spleen of adult healthy C57 mice by gradient centrifugation and then cultured. These lymphocytes were transfected by lentivirus vector containing cbl-b-specific siRNA. They were divided into three groups:control group, blank group and test group. In the control group C57 mouse spleen lymphocytes were isolated and untreated. In the blank group lymohocytes were transfected with blank lentivirus vector. In the test group lymohocytes were ransfected with lentivirus vector carring cbl-b-specific siRNA. Lymphocyte surface molecule CD69 was detected by flow cytometry. IL-2 and INF-y secretion were detected by enzyme-linked immuno-sorbent assay. Lymphocyte proliferation was detected by CCK8 assay.3. Mouse prostate cancer cells RM-1 grew adherently. They were cultured in IMDM culture medium with 10% fetal calf serum and without androgen. RM-1 cells were added with either untreated lymphocytes or lymphocytes transfected cbl-b-specific siRNA lentiviral vector. They were mixed and cultured for 7 days. RM-1 killing activity were detected by CCK8 method. Then TGF-βwas added in the same grouping and tumor-killing activity of lymphocytes were detected again by CCK8 method.Results1. Four different sequence targeting the cbl-b gene were designed. Then the recombinant plasmids containing each of these cbl-b-specific siRNA sequences were successfully constructed. As observed by fluorescence microscopy 90% of the 293T cells showed fluorescence after transfetion, suggesting that the sequence had been transferred into the target cells. As detecting by western bloting, the second cbl-b-specific siRNA cbl-b₂ could knock down the cbl-b gene to the most extend. The Cbl-b protein expression inhibition rate reached 93.4%. So this sequence was the best one and was chose for the following experiments. The virus vector titer was 2E+7 TU/mL as examed by hole dilution method. Its titers reached more than 2.00E+7 TU/ml as as examed by real-time PCR. The two results were comparable and can be used in the following experiments.2. C57 murine spleen mononuclear cells were obtained and cultured. The proliferation of cultured lymphocytes went the strongest in the seventh day. They showed a status of the cluster growth, and were easy to stir into single cells. Lentiviral vector was transfected into lymphocytes successfully and the transfection rate reached about 92%. The inhibition rate of Cbl-b protein in lymphocytes was 89.4%. The surface antigen CD69 of lymphocytes was significantly higher in the group of lymphocytes transfected cbl-b-specific siRNA lentivirus vector as examed by flow cytometry (P<0.01). The cytokines IL-2 and INF-y secretion of lymphocytes were also significantly higher as detected by ELISA (P<0.05).3. The Cell Counting Kit 8 was used in the determination of prostate cancer cells RM-1 killing activity of lymphocytes in each group. In vitro experiments showed that the lymphocytes transfected with cbl-b-specific siRNA had the strongest taget killing activity (while E/T ratio was 40:1, the taget killing activity was 40.98%). It was significantly enhanced compared with the control (P<0.05). After adding TGF-β, RM-1 killing activity of lymphocytes transfected cbl-b-specific siRNA was not significantly inhibited.Conclusion1. In this study we constructed cbl-b-specific siRNA expression plasmid and then packed into lentiviral vector. The second sequence of siRNA cbl-b₂ was chosen. It could effectively suppress lymphocyte cbl-b gene expression.2. The activation of lymphocytes transfected with cbl-b-specific siRNA markedly enhanced. Its cytokines IL-2 and INF-y secretion significantly increased. These suggested that the negative regulation of Cbl-b in peripheral lymphocytes was inhibited. The activity of lymphocytes increased.3. RM-1 killing activity of the lymphocytes transfected with cbl-b-specific siRNA was significantly enhanced. It was resistant to TGF-P inhibition. These showed that when cbl-b gene in lymphocytes was silenced, the lymphocytes would had stronger anti-tumor effect. It could be a promising taget in the treatment of prostate cancer. This may provide a strong basis to overcome the immune tolerance and immune silence in immunotherapy of prostate cancer. It also made a certain experimental and theoretical basis to find a new immunotherapy for AIPC.