Prompted The Anti-tumor Effect Of SiRNA-PD-1 By Inhibiting Angiogenesis On Mice Melanoma

BackgroundMelanoma is one of the most common cutaneous malignant tumors in clinical practice.With the development of current immunology research,people begin to focus on tumor immunotherapy.Among them,the representative immune checkpoint is the PD-1 molecule.Blocking the binding of its ligand PD-L1 molecule can effectively improve the body’s anti-tumor immunity and play an anti-tumor role.At the earlier stage,we constructed the siRNA-PD-1 sequence,and it had been verified that this sequence can effectively inhibit the expression of PD-1 in tumor tissues and play an anti-tumor role.However,compared with the effective treatment mice,the number of blood vessels in tumor tissues of ineffective mice was significantly increased.Other studies have also suggested that angiogenesis restricts the therapeutic effect of blocking PD-1.Therefore,this project will improve the therapeutic effect of siRNA-PD-1 in melanoma-bearing mice by inhibiting angiogenesis.ObjectiveThe objective of the study is to explore the therapeutic effect of siRNA-PD-1 on melanoma-bearing mice by inhibiting angiogenesis and to preliminarily explore its mechanism.Methods1.Cell experimentLaying a six-well plate of EL4 cells for 14-16 hours.Four p Endostatin-siRNA-PD-1plasmids were transfected into EL4 cells and set up two wells as control groups and they were cultured continuously for 24h.After extracting the cell proteins,the expression of PD-1 was detected by western blot in transfection cells.2.Animal experimentThe experimental mice were female C57BL/6 with the age of 6-8 weeks and weight of 18-22g.The mice were subcutaneouly injected with B16 cells on the right hind legs for making models.The mice were randomly divided into PBS group,Scramble group,siRNA-PD-1 group,p Endostatin group,and p Endostatin-siRNA-PD-1 group.After one week of modeling,the mice were treated once a week and twice in total.During the period,the changes of tumor size and tumor volume in melanoma-bearing mice were observed.The mice were killed 7 days later after the last time treatment and isolated the tumor tissues.The expressions of PD-1,VEGF,CD34,and MMP2 in tumor tissues were detected by western blot after the tissue proteins were extracted from mice.Immunofluorescence assay was used to detect the infiltration of T lymphocytes in tumor tissue sections and apoptosis assay was used to detect the cell apoptosis.Flow cytometry was used to detect the proportion of CD4~+,CD8~+T cells,and NK1.1 cells in the spleens of mice.Results1.The expressions of VEGF and CD34 in melanoma tissues are significantly higherthan that in para-cancer tissues.2.Co-expression plasmid p Endostatin-siRNA-PD-1 significantly inhibited theexpressions of PD-1 in EL4 cells.3.The co-expression plasmid p Endostatin-siRNA-PD-1 carried by attenuatedsalmonella significantly inhibited the tumor growth and prolonged the survival ofmelanoma-bearing mice.4.The co-expression plasmid p Endostatin-siRNA-PD-1 carried by attenuatedsalmonella significantly increased the cell apoptosis in melanoma tumor tissues.5.The co-expression plasmid p Endostatin-siRNA-PD-1 carried by attenuatedsalmonella significantly increased the necrosis of tumor cells in melanoma tumortissues.6.The co-expression plasmid p Endostatin-siRNA-PD-1 carried by attenuatedsalmonella significantly inhibited the expressions of VEGF and PD-1 molecules inmelanoma tumor tissues.7.The co-expression plasmid p Endostatin-siRNA-PD-1 carried by attenuatedsalmonella increased the infiltration of CD4~+and CD8~+T cells in melanoma-bearingmice tumor tissues.8.The co-expression plasmid p Endostatin-siRNA-PD-1 carried by attenuatedsalmonella increased the ratio of CD4~+and CD8~+T cells and NK cells in the spleensof melanoma-bearing mice.ConclusionThe co-expression plasmid p Endostatin-siRNA-PD-1 delivered by attenuated salmonella can effectively inhibit the tumor growth of melanoma-bearing mice and improve the survival rate of the mice and enhance their anti-tumor immune response.It can increase the anti-tumor effect of siRNA-PD-1 by inhibiting angiogenesis,which provides a new method and related theoretical basis for the clinical treatment of melanoma.Certainly,we still need to make further research on its underlying mechanisms.