RNAi-mediated Knockdown Of Notch1 Enhance Immune Responses Against Tumor In Vivo

Background:Malignant melanoma(Malignant melanoma,MM),a kind of cutaneous tumor,is originated from neural crest melanin cells in basilar part.It has a fatal outcome and poor prognosis if it goes into growth spurt.The incidence of malignant melanoma increased rapidly in recent decades in the global,and the annual growth rate reached almost 3-5%.The incidence of melanoma in caucasian populations is higher than other races.It is low in Asian countries including China and Japan,but it has a rapidly rising trend.Malignant melanoma is one of the most highly aggressive tumors in the world.It is usually difficult to be detected in early stage and easy to metastasize distally.The median survival time of advanced melanoma is only 7.5 months.The 2-year survival rate is 15%and the 5-year survival rate is only 5%.Patients with advanced metastatic malignant melanoma need to be treated with systemic treatment,but melanoma cells resists to conventional chemotherapy and radiotherapy.Immunotherapy,which acts through stimulating the body’s immune system to improve the antitumor immunity effect,is a promising strategy to treat melanoma.IL-2 and IFN-a immunotherapy which were approved by the FDA(US Food and Drug Administration,FDA)show notable curative effects on melanoma,but their long-term effects are limited.That is mainly due to the immune suppression induced by tumor.Melanoma cells often create an immunosuppressive microenvironment that can facilitate tumor growth and block antitumor immune responses through producing cytokines,such as TGF-β,VEGF and IL-10.Melanoma cells can also recruit and activate MDSCs(Myeloid-derived suppressor cells,MDSCs)and Treg cells(Regulatory T cell,Tregs)directly,which play a crucial role in tumor-induced immune suppression.Notch signaling regulates cell fates and plays a fundamental role in cell differentiation,proliferation,apoptosis and angiogenesis.The Notch family of receptors is a highly conserved pathway that controls the onset and progression of tumors.Aberrant Notch signaling has been identified in various kinds of tumor,which plays an important role in proliferation,invasion,migration and angiogenesis of tumor.Recent studies indicated that Notch play a dual role as either an oncogene in breast cancer,medulloblastoma,colorectal cancer,pancreatic cancer,melanoma and leukemia,or a tumor suppressor in keratinocyte derived from carcinoma,prostatic epithelium,hepatocellular carcinoma,small cell lung cancer.Moreover,signaling through Notch has been proved to be a critical influence on the differentiation,development and function of immune cells including inherent immune cells and adaptive immune cells.Notch signaling can affect the occurrence of hematopoietic stem cells in the process of embryonic development.Notch signaling may interact with TLR/NF-κB pathway to regulate the biological function of macrophagocyte.Furthermore,Notch signaling has some regulating effects on the development and differentiation of dendritic cell.Notch signaling can regulate intestinal mucosal immunity induced by mastocyte through facilitating the expression of MHC-Ⅱmolecule and OX40L in mastocyte.In addition,Notch signaling is essential for the development and differentiation of T cells and B cells.Notch pathway has something to do with the occurrence of immune-related disorders including virus infection,inflammatory reaction,hypersensitivity and autoimmunity disease.siRNA technology,as immediate area of regulating gene expression in recent years,can induce gene-silencing processes in eukaryotes through the degradation of homologous mRNAs.Compared with the traditional tumor treatment,siRNA technology can specificity silence genes expression simply and efficiently.Moreover,siRNA intratumoral injection has been widely used in the treatment of various kinds of solid tumor and show a positive therapeutic effect.Currently,the phenomenon of siRNA targeting Notchl gene inhibiting tumor cell growth and proliferation has been confirmed in glioma,kidney cancer,prostate cancer,pancreatic cancer,acute T lymphocyte leukemia etc.Aberrant Notch signaling has been identified in malignant melanoma,which plays an important role in proliferation,invasion,migration and angiogenesis of melanoma.Moreover,signaling through Notch has been proved to be a critical influence on the development and function of T cells.But little is known about the effect of aberrant activation of this pathway in melanoma on tumor-induced immunosuppressive microenvironment.In this study,we aimed to value the effects of a knockdown of Notchl upon immune suppression induced by melanoma cells.We used siRNA targeting Notchl gene to transfect melanoma cell line B16F1 and induce RNA interference.After co-culturing B16F1 cells transfected by siNotch1 with splenic CD3+T lymphocyte,the effects of siNotch1 on inhibiting lymphocyte proliferation were assessed by CCK8(Cell Counting Kit 8).The mRNA expression and supernatant secretion of immunosuppressive cytokines TGF-β,VEGF and IL-10 were measured by RT-PCR and ELISA.We further tested the immune effects of siNotchl intratumoral injection after using mouse cell line B16F1 to establish mice tumor models.The ratios of CD3+CD8+cytotoxic T cells and CD4+CD25+CD127-Treg cells in tumor were examined by flow cytometry.The concentration-of IFN-y and immunosuppressive cytokines TGF-β in tumor homogenate supernatant were determined by ELISA,and immunohistochemistry was used to detect the number of CD11b+MDSCs in tumor.Objective1.This study was to evaluate the effects of Notch]siRNA on tumor-derived immunosuppressive cytokines and CD3+T lymphocyte proliferation.2.To value the immune effects against tumor of siNotchl intratumoral injection in vivo.Materials and methods1.Materials1.1 Cell and animalMurine malignant melanoma cell line B16F1 of low metastatic,was purchased from China Center for Type Culture Collection.Female C57BL/6 mice,6-8weeks old and weighing approximately 18-22g,were purchased from Laboratory Animal Center of Southern Medical University.Animal care and treatment were in accordance with institutional guidelines.1.2 Experimental reagentsFetal bovine serum,High-glucose DMEM culture medium,LipofectamineTM 2000,Notchl siRNA Oligo and negative control,Trizol,PrimeScript(?)RT reagent Kit,SYBR(?)Premix Ex TaqTM,Notchl primers,Goat anti-mouse Notchl polyclonal antibody,RIPA protein lysate,SDS-PAGE gel configuration Kit,Cell counting kit8,DMSO,Rabbit anti-mouse CDllb monoclonal antibody,CD3,CD8,CD4,CD25,CD117 or matched isotypy control antibodies,TGF-β1、IL-10、VEGF-A IFN-y ELISA Kits.Horseradish peroxidase(HRP)labeled secondary antibody anti-Goat or anti-rabbit IgG kit,Ficoll lymphocyte separation medium.DAB chromogenic agent,AEC chromogenic agent,Hematoxylin,Anhydrous ethanol,95%Alcohol,1%Hydrochloric acid alcohol etc.1.3 Experimental instrumentCarbon dioxide incubator,Clean bench,Optical microscopy,Fluorescence microscope,FACSCablibur flow cytometer,High-speed refrigerated centrifugal machine,Bench centrifuge machine,Electrophoresis,LightCycler 480 Realtime PCR System,Cell strainer,Paraffin slicing machine,-80℃ refrigerator,Culture dishes,Straw,Pipettes,EP tube,Freezing tube,Slides,Coverslips etc.2.Experimental procedure2.1 Transfer murine malignant melanoma cell lines B16F1 with siRNA targeting Notch1The siRNA Oligo targeting Notchl and negative control were designed through website and synthesised by Genephanna,The plasmids were transfected into B16F1 cells using LipofectamineTM2000 transfection reagent according to the manufacturers’instructions.Three groups were established in the experiment:A:siNotchl group(B16F1 cells transfected with siRNA targeting Notch1);B:siNC group(B16F1 cells transfected with scrambled negative control siRNA);C:mock group(Mock transfection with only lipofectamineTM2000 in B16F1 cells).Transfection efficiency was analyzed by both RT-PCR and western blot.2.2 To evaluate the effects of Notchl siRNA on tumor-derived immunosuppressive cytokines.To figure out the impact of Notchl siRNA on tumor-derived immunosuppressive cytokines in melanoma cells,the mRNA expressions and the supernatant secretions of immunosuppressive cytokines TGF-β,VEGF and IL-10 in melanoma cells were measured by RT-PCR and ELISA assay.2.3 To evaluate the effects of Notch1 knockdown in B16F1 cells on inhibiting CD3+T lymphocyte proliferation.Splenic lymphocytes were isolated from splenic cell suspension using density gradient centrifugation.CD3+T lymphocytes were purified using microbead isolation kits followed by magnetic-activated cell sorting according to the manufacturer’s instructions.siRNA transfection was performed after B16F1 cells was adhered to the bottom of the culture plate for 18h.CD3+T lymphocytes were then added to the B16F1 cells in a 1:1 ratio so that B16F1 cells and lymphocytes were cultured in the same well.The plates were then incubated for 66 hours and subjected to the CCK-8 assay.The inhibition rate of lymphocytes proliferation was calculated according to the equation.2.4 Animal melanoma model and siNotch1 intratumoral injection in vivo.For the tumor challenge experiment,24 mice were injected subcutaneously with 1×106 B16F1,when the tumors were palpable(tumors volume reached around 90-110 mm3),tumor-bearing mice were randomly assigned into three groups of 8 mice each.Mice then received intratumoral injections of either 25ug siNotch1,25ug siNC orPBS,All treatments were performed twice a week,for a total of eight injections.Mice were observed carefully,and tumor xenografted in mice were measured and recorded twice a week.Tumor volume was calculated using the formula:(length×width2)/2.To verify the gene inhibition effect of siNotchl intratumoral injection on tumor tissues,immunohistochemistry and western blot were used to determine the expression of Notch1 and its downstream heyl protein2.5 Anti-cancer immune effect of siNotchl intratumoral injection in vivo To value the anti-cancer immune effect of siNotchl intratumoral injection in vivo.The ratios of CD3+CD8+cytotoxic T cells and CD4+CD25+CD127’ Treg cells in tumor were examined by flow cytometry.The concentration of IFN-y and immunosuppressive cytokines TGF-P in tumor homogenate supernatant were determined by ELISA,and immunohistochemistry was used to detect the number of CD11b+MDSCs in tumor.3.Statistical analysisData were analyzes using SPSS 13.0.All date were expressed as means±SD(x± s).The results of RT-PCR and western blot,ELISA assay,inhibition rate on lymphocyte proliferation,animal tumor volume,flow cytometry analysis and immunohistochemical were assessed using one-way ANOVA.Differences were considered statistically significant when P<0.05.Results1.24 hours after transfection,the gene knockdown effect of siRNA was measured by real-time PCR.siRNA-Notchl transfection resulted in significant inhibition of Notchl mRNA expression compared to that of siNC group(P<0.01)and mock group(P<0.01).In addition,the result of real-time PCR was confirmed by western blot 72h after transfection.The expression of Notchl and its downstream heyl protein were observed downregulated in siNotchl group compared with siNC and mock group(P<0.01).2.To figure out the impact of Notchl siRNA on tumor-derived immunosuppressive cytokines in melanoma cells,the mRNA expressions and the supernatant secretion of immunosuppressive cytokines TGF-β,VEGF and IL-10 in melanoma cells were measured by RT-PCR and ELISA assay.siNotchl transfected cells showed significant reduction in the mRNA expression and supernatant secretion of TGF-β compared with the siNC(P<0.05)and control mock group cells(P<0.05),but there is no significant differences in the mRNA expression and supernatant secretion of IL-10 and VEGF(P>0.05)3.We used lymphocyte proliferation assay to evaluate the effects of Notch1 knockdown in B16F1 cells on inhibiting CD3+T lymphocyte proliferation.We observed that the proliferation inhibition rate of T cells co-cultured with siNotchl transfected cells was significantly lower than that of lymphocytes co-cultured with siNC transfected cells and B16F1 cells.Notch1 knockdown in B16F1 cells reduced the T cells proliferation by 36%in comparison with the proliferation of the T cells alone(100%).In contrast,The proliferation inhibition rates of siNC and mock group cells were 65%and 68%(P<0.05).Moreover,siNotchl group showed more lymphocyte colony formation than siNC and mock group under the microscope,suggesting that Notch1 knockdown has the potential to reduce the inhibition of melanoma cells on lymphocyte proliferation.4.In vivo antitumor effect of siNotchl:On the day 30 results show that tumor growth was remarkably suppressed in those mice treated with siNotch1.(P<0.05),while treatment with siNC and PBS did not result in any suppressive effects on tumor growth.As shown in flow cytometry analysis,siNotchl treatment has significantly increased the number of CD8 and CD3 double positive cells in tumors compared with siNC(P<0.05)and PBS controls(P<0.05).The flow cytometry analysis also showed that the percentages of CD25+CD127"Tregs among CD4+lymphocytes was lower in siNotchl treatment group than that in siNC(P<0.05)and PBS controls(P<0.05).The immunohistochemical study revealed that tumors from siNotch1-treated mice were rarely infiltrated with CDllb+MDSCs,compared with siNC(P<0.05)and PBS treated mice(P<0.05).In addition,IFN-y secretion has significantly increased in siNotchl treatment group compared with siNC(P<0.05)and PBS treatment groups(P<0.05).The immunosuppressive cytokines ELISA showed a significant decrease in TGF-β secretion in mice tumor homogenate supernatant treated with siNotch1 compared with siNC(P<0.05)and PBS treatment groups(P<0.05).Conclusions1.RNAi-mediated knockdown of Notch1 in melanoma cells can lead to a dramatical reduction of TGF-β secretion,which in return reduces the inhibition of melanoma cells on lymphocyte proliferation in vitro.2.siNotchl intratumoral injection successfully inhibited Notch1 expression and downregulated tumor-derived TGF-β in vivo,which promoted more CD8+cytotoxic T lymphocytes infiltrating and higher IFN-y release in tumor tissue.It can also reduce the ratio of CD4+CD25+CD127-Treg cells and CDllb+MDSCs in tumor microenvironment,and all these contribute to enhancing anticancer immune responses and suppressing tumor growth.