Cbl-b Gene Silence Affects Killing Activity Of Primary Lymphocyte Immune-induced By TRP-2 Vaccine To Melanoma Cell Line B16F10

Malignant melanoma is the most aggressive and deadly form of skin cancer and its therapy remains a huge challenge. Immunotherapy is a novel and relatively safer strategy for the treatment of cancer, and several forms of immunotherapy to melanoma are already in clinical trials but show limited success. E3 ubiquitin ligase Cbl-b is a key molecule in regulating T cells activation and immune tolerance, and a central checkpoint for limiting immune responses. The objective of present study is to investigate the effect of Cbl-b gene silence on the immune activity of primary lymphocytes of mice against B16F10 melanoma cells in vitro.Part Ⅰ:Construction and Evaluation of Interference Vector Targating Cbl-b GeneObjective:To construct the eukaryotic expression plasmid vectors of short hairpin RNA (shRNA) specific to Cbl-b gene and evaluate their interfering effects on Cbl-b gene.Method:Four pairs of shRNA targeting mouse Cbl-b gene were designed and synthesized, and then inserted into the plasmid of PGPU6/GFP/Neo to construct expression vectors. After checking by DNA sequencing, the shRNA expression vector was co-transfected into 293T cells with Cbl-b eukarytic expresson plasmid. The knockdown efficiency of four shRNA vectors on Cbl-b expression were compared at both mRNA and protein level by the methods of q-PCR and Western blotting after 48 hours transfection.Results:Sanger sequencing showed that four pairs of shRNA oligonucleotide sequences were successfully inserted into the PGPU6/GFP/Neo expression vector of. Through cell transfection, it was found that all the four shRNA vectores could downregulate Cbl-b expession, while the NO.1 shRNA expression vector has the highest interference effect on Cbl-b expression at both mRNA and protein level (p< 0.05).Conclusions:The most effective eukaryotic shRNA plasmid expression vectors specific to Cbl-b gene were successfully constructed and selected, which can be applied to further study about the role of Cbl-b in the malignant melanoma immunotherapy.Part II:Selecting the transfection method of RNA interference to primary lymphocytes of mouseObjective:To explore the effective method of transfecting RNA interference sequence of Cbl-b to primary mouse lymphocytes.Methods:The primary lymphocytes were isolated from spleen and lymph node of mouse and cultured at the optimized conditions, then transfected by four methods: siRNA+lipofectamin2000, lentiviral vector+polybrane, lentiviral vector+EnvirusTM-LV and siRNA+EntransterTM-R. The transfection efficiency was evaluated through fluorescent microscope and flow cytometry.Results:The siRNA+EntransterTM-R method was the most efficient way to transfct the Cbl-b specific siRNA seuqence to mouse primary lymphocytes., After 24 h transfection with the concentration of siRNA 100 nmol/L (2μ1) and 50 nmol/L (1μ1), the transfection efficiency were 43.1% and 22.8%, respectively.Conclution:The siRNA+EntransterTM-R method was an efficient way to transfect the Cbl-b specific siRNA seuqence to mouse primary lymphocytes.Part III:Effects ofCbl-b gene scilence on the immune activitity of pepfide vaccine induced lymphocyte against B16F10 melanoma cells in vitroObjective:To investigate the effect of Cbl-b scilenced by specific siRNA on the immune activity of the mouse lymphocytes.Methods:C57BL/6 mice were immune induced by injecting SVY peptide vaccine.,Twenty days later, lymphocytes from the mouse spleen and lymph nodes were cultured and stimulated bySVY peptide and IL-2 in vitro. Then the lymphocytes were interfered with the Cbl-b specific siRNA. The silencing effect of Cbl-b gene were detect at both mRNA and protein level by RT-PCR and western blot. We also tested the proliferation of the lymphocytes and their secretion level of INF-yand TNF-awith CCK-8 kit and ELISA methods respectively after 48 h transfection. Furthmore, the cytotoxicity of lymphocytes to the tumor cells was decteted by CCK8 kit.Results:Cbl-b specific siRNA could silence the Cbl-b gene of lymphocytes effectively. After 48 h transfection, the spectrophotometric values detected by CCK-8 kit in transfection group, nagtive control group were 1.246±0.063 and 0.906±0.039 respectively, which were significantly different between each other (p<0.05). the secretion level of INF-γ and TNF-a were95.80±17.85 and 62.23±4.409 in transfection group, which were significantly higher than those of nagtive control group 49.92±9.89 and 32.29±2.099, respectively. The cytotoxicity of lymphocyte to the tumor cells in transfection group was much higher than that of negative control group.Conclutions:The immune activity and cytotoxicity of mouse lymphocytes to B16F10 melanoma cells could be markedly enhanced by silencing Cbl-b gene with specific siRNA in vitro.