Anti-melanoma Effect And Mechanism Of PD-1-siRNA Combination Therapy

Objective:Melanoma is a highly malignant tumor.Its occurrence and development is a complex disease with multiple factors and stages.The treatment of a single factor often fails to achieve an ideal tumor inhibition effect.Therefore,according to the mechanism of its occurrence and development,improving and combining the existing treatment methods to find more effective treatment methods is the prospect of melanoma treatment.Currently,the monoclonal antibody against the immune checkpoint,PD-1,has made progress in the treatment of melanoma,but the patient response rate still needs to be improved.Previous studies found that PD-1-si RNA can inhibit the expression of PD-1 to treat melanoma in mice.However,the therapeutic efficiency of PD-1-si RNA still needs to be improved,so PD-1-si RNA combining with other drugs can provide new direction for the treatment of melanoma.Cp G ODN(oligonucleotide rich in guanine and cytosine),as a synthetic deoxygenated oligonucleotide single strand DNA containing Cp G motif,can activate NK cells and B cells of the body,stimulate spleen cells,plasma cell like dendritic cells(p DC),and stimulate the secretion of interferon,showing a strong immune stimulation function,which has obvious clinical application prospects.We combine the Cp G ODN with the si RNA carried by attenuated salmonella that targets PD-1.The si RNA sequence is carried by attenuated salmonella to treat murine melanoma,and explore the potential anti-tumor mechanism.At the same time,considering that the development of new drugs is a very costly process,and there are disadvantages of long time consuming and great risk of failure.In this paper,Buspirone hydrochloride,a clinical anti anxiety drug,was used to explore its new function in anti melanoma and its combined anti melanoma effect with PD-1-si RNA.Methods:1.Colony forming method was used to detect the targeting capacity and enrichment of attenuated Salmonella in tumors and the distribution of attenuated Salmonella in other organs.2.Modeling,grouping and treatment:female C57BL/6mice were selected that were 6 weeks old with a similar body weight.A volume of 0.1ml containing B16 melanoma cells(approximately 1×10~6 cells in total,i.e.,a concentration of 1×10~7 cells/ml)was injected subcutaneously into the root of the right hind leg in each mouse.Approximately 7 days after the injection of tumor cells,a local tumor bulge with a diameter of 6-8 mm was observed.If no tumor bulge was observed,inoculation failure was indicated.On day 7 after inoculation with B16 cells,the C57BL/6 mice were randomly divided into 5 groups,with 5 mice in each group:the PBS group,Scramble group,PD-1-si RNA group,Cp G ODN group and Cp G ODN~+PD-1-si RNA group.The treatment scheme was as follows:each mouse in the PBS group was injected subcutaneously with 100μl PBS inside the tumor;each mouse in the Scramble group was administered 100μl Salmonella carrying si RNA with a random RNA sequence inside the tumor;each mouse in the PD-1-si RNA group was administered 100μl Salmonella carrying si RNA targeting PD-1 inside the tumor;each mouse in the Cp G ODN group was injected subcutaneously with 20μg of Cp G ODN in the inguinal drainage lymph nodes;and each mouse in the Cp G ODN+PD-1-si RNA group was administered 100μl Salmonella carrying si RNA targeting PD-1 inside the tumor and 20μg of Cp G ODN in the inguinal drainage lymph nodes.In addition,Cp G ODN was administered once a day four times in total,and a second injection of Salmonella carrying si RNA was performed one week after the first administration.3.Detection of therapeutic effect of the Cp G-ODN+PD-1-si RNA combination therapy on tumor-bearing mice:the therapeutic effect after administration was judged by analyzing the tumor weight and survival time of different groups.Biochemical indexes were used to detect whether it would have obvious side effects on mice.4.To explore the anti-tumor mechanism of the Cp G-ODN+PD-1-si RNA combination therapy:proteins related to cell cycle(cyclin D1),proliferation(Stat3,p-Stat3),apoptosis(cleared caspase 3),migration(MMP-2)and cellular immunity(CD4,CD8 and PD-1)were detected by Western blotting;TUNEL method was used to detect the apoptosis of tumor cells in each group;CD4,CD8 and PD-1 proteins were detected by immunohistochemistry;The cell proliferation marker protein(Ki67)and macrophage marker protein(CD86 and CD11b)were detected by immunofluorescence;The number of lymphocytes and NK cells in spleen were detected by flow cytometry;NK cell killing test was used to detect the cytosis of NK cells after administration;T cell proliferation test was used to detect the proliferation ability of T cells after administration;The cytokines involved in the antitumor effect of the Cp G-ODN+PD-1-si RNA combination therapy were detected by ELISA.5.CCK8 test,cell scratch test and Western blot were used to detect the effect of oncomelanin hydrochloride on melanoma cells.6.The melanoma bearing mice were divided into PBS group,buspirone hydrochloride group,PD-1-si RNA group and PD-1-si RNA+buspirone hydrochloride group.Each mouse in the PD-1-si RNA group was injected with 4×Attenuated Salmonella carrying PD-1-si RNA of 105 CFU;Intratumoral injection of buspirone hydrochloride 200μG of buspirone hydrochloride;The Cp G-ODN+PD-1-si RNA combination therapy group was given attenuated salmonella and buspirone hydrochloride respectively.Frequency of administration:Buspirone hydrochloride was administered once a day,7 times in total;The attenuated Salmonella sequence carrying PD-1-si RNA was administered twice,respectively on the first day and the seventh day of treatment.The tumor size and the expression of related proteins in tissues of mice in each group were detected.7.Statistical analysis:the numeric data are expressed in x±SD.Spss19.0 statistical software(SPSS,Inc.,Chicago,IL,USA)was used for analysis.One way analysis of variance(ANOVA)was used to test the differences between groups,and Kaplan Meier method was used to analyze the survival rate.P<0.05 was considered as a statistically significant difference.Results:1.After 48 hours of injection of attenuated Salmonella,attenuated Salmonella was mainly distributed in tumor tissues,which showed that the targeting capacity of attenuated Salmonella was good.2.After different groups of tumor bearing mice were treated correspondingly,the tumor weight of tumor bearing mice in the Cp G-ODN+PD-1-si RNA combination treatment group was the smallest and the survival time of tumor bearing mice in the Cp G-ODN+PD-1-si RNA combination treatment group was the longest.3.There was no significant difference in biochemical indexes between different groups of mice after their corresponding administration,which showed that the Cp G-ODN+PD-1-si RNA combination therapy did not lead to obvious side effects in mice.4.Western blotting showed that compared with the control group,the protein expression levels of p-Stat3,cyclin D1 and MMP2 in the Cp G-ODN+PD-1-si RNA combination treatment group decreased,and the expression level of cleaved caspase 3 increased.This indicates that the antitumor mechanism of the Cp G-ODN+PD-1-si RNA combination therapy is related to cell proliferation(Stat3),cell cycle(cyclin D1),cell migration(MMP-2)and apoptosis(cleaved Caspase 3).5.By immunofluorescence method,it was found that the expression level of cell proliferation related protein(Ki67)in the Cp G-ODN+PD-1-si RNA combination treatment group was significantly lower than that in the control group.By TUNEL method,it was found that the number of apoptotic cells carrying green fluorescence label in the Cp G-ODN+PD-1-si RNA combination treatment group was significantly higher than that in the control group.This further proves that the Cp G-ODN+PD-1-si RNA combination therapy can inhibit tumor proliferation and promote tumor apoptosis.6.By immunohistochemical method,it was found that the expression levels of CD4 and CD8 in tumor tissues in the Cp G-ODN+PD-1-si RNA combination treatment group were significantly higher than those in the control group,while the expression level of PD-1 in tumor tissues in the Cp G-ODN+PD-1-si RNA combination treatment group was significantly lower than that in the control group.The same trend of the protein expression level was also found by Western blotting method.This indicated that the anti-tumor mechanism of the Cp G-ODN+PD-1-si RNA combination therapy is related to the increase of CD4~+and CD8~+T cell infiltration in tumor tissues and the decrease of PD-1 expression on the surface of tumor cells.7.By flow cytometry,it was found that the number of CD4~+,CD8~+T cells and NK cells in the spleens of the Cp G-ODN+PD-1-si RNA combination treatment group increased significantly,compared with the control group.This indicated that the anti-tumor mechanism of the Cp G-ODN+PD-1-si RNA combination therapy is related to the increase of CD4~+,CD8~+T and NK cells infiltration in the spleens.8.Through the immune cell function test,it was found that compared with the control group,the T cell proliferation ability and NK cell killing ability of the Cp G-ODN+PD-1-si RNA combination treatment group were higher than those of the control group,which showed that the anti-tumor mechanism of the Cp G-ODN+PD-1-si RNA combination therapy was not only related to the increase of the number of T cells and NK cells,but also related to the improvement of the function of T cells and NK cells.9.By ELISA,it was found that compared with the control group,TNF-αand IL-6 in the serum of the Cp G-ODN+PD-1-si RNA combination treatment group increased significantly.This indicated that TNF-αand IL-6 played a role in the anti-tumor mechanism of the Cp G-ODN+PD-1-si RNA combination therapy.10.By immunofluorescence,it was found that the protein expression levels of CD11b and CD86 in the Cp G-ODN+PD-1-si RNA combination treatment group were significantly higher than those in the control group.This suggested that the anti-tumor mechanism of the Cp G-ODN+PD-1-si RNA combination therapy was related to the increase of macrophage infiltration.11.Buspirone hydrochloride showed a certain killing effect on melanoma cells.12.Buspirone hydrochloride significantly inhibited the migration of melanoma cells.13.Buspirone hydrochloride significantly inhibited the expression of p-Stat3,MMP2 and cyclin D1 proteins in melanoma cells.14.Buspirone hydrochloride combined with attenuated Salmonella carrying PD-1-si RNA significantly inhibited the growth of melanoma in mice and the expression of p-Stat3,MMP2,cyclin D1 proteins in tumor tissues.Conclusion:1.Compared with the model group and the single treatment group,the combined application of Cp G ODN and PD-1-sirna can more effectively inhibit the growth of melanoma and increase the antitumor immune response of tumor bearing mice.2.The antitumor mechanism of combined therapy may be due to the precise localization and killing of attenuated Salmonella tumor,the blocking of PD-1 pathway,resulting in the release of T cell killing function inhibition and Cp G ODN mediated immune enhancement,so as to play a more powerful role in inhibiting tumor cell proliferation and promoting tumor cell apoptosis.3.The Cp G-ODN+PD-1-si RNA combination therapy can not only enhance the immune response in the tumor microenvironment,but also improve the overall immune level of mice.4.Buspirone hydrochloride can inhibit the proliferation and migration of melanoma cells,showing a certain anti melanoma potential.The combination of PD-1-si RNA and PD-1-si RNA showed a synergistic effect on tumor growth.