Role And Mechanism Of Gastric Cancer-Derived MiRNA-107 In Regulating Myeloid-Derived Suppressor Cells

Background and objective Gastric cancer is one of the most common malignant tumors worldwide.The incidence of gastric cancer is ranked fourth and the mortality rate is ranked second in the world.Despite a significant improvement has been made in diagnosis and treatment of gastric cancer,the high morbidity and mortality remain to be major issues to be resolved in the field of gastric cancer research and therapy.Therefore,it is important both practically and theoretically to further study the genesis and development of gastric cancer by molecular biology and to identify new approaches to treat gastric cancer.Immune escape of tumor is inextricably associated with the occurrence and development of gastric cancer.Studies have shown that tumor cells can release a series of immunosuppressive factors and induce the production of tumor-associated immunosuppressive cells,including regulatory T cells,tumor-associated dendritic cells,myeloid-derived suppressor cells,and tumor-associated macrophages.Through these mechanisms,tumor cells can induce immune suppression leading to immune escape of tumor cells.In recent years,increasing evidences suggest that tumor cells have the ability to secrete exosomes,tumor-derived exosomes(TDEs).As a carrier,TDEs can carry a large number of molecular components from host cells,participating in intercellular transportation and signal transduction.TDEs can promote tumor immune escape through inhibiting the activation of immune cells or regulate the expression of cytokines.On account of the important role in tumor immune escape,studying TDEs has been becoming a hot topic in cancer immunology research.Myeloid derived suppressor cells(MDSCs)are pivotal immunosuppressive cells that were discovered recently in the immune system.Studies found that MDSCs were massively proliferated during progressive tumor growth and these overgenerated bone marrow cells can induce immune escape of tumor cells.Recent studies have confirmed that TDEs can be swallowed by bone marrow precursor cells.Proteins and small soluble molecules carried by TDEs can induce the accumulation and proliferation of MDSCs through multiple mechanisms.Although recent findings demonstrated that TDEs carried a large number of micro RNA(miRNA)form host cells,it is currently not clear whether the exogenous miRNAs can be transported effectively to MDSCs.It is far from clear whether these exogenous miRNAs possess the same biological function as the endogenous ones.It is also not clear what is the biological role and mechanisms of involving molecular pathways.In this study,we for the first time have predicted and verified what the functional targets of exogenous mi R107 in MDSCs were.We also systematically studied the roles of gastric cancer cell-derived miRNA-107 in the regulation of MDSC proliferation,differentiation,and immune function and explored the potential molecular mechanisms underlying these regulations.This study includes four parts: Part I: The proliferation and differentiation of MDSCs in the presence of gastric cancer and abnormal changes of immune function;Part II: Prediction and validation of target genes in exogenous miRNA-107 in MDSCs cells;Part III: In vitro studies on the regulatory effect of miRNA-107 derived from gastric cancer on human MDSCs;Part IV: In vivo study on the regulation of mouse MDSCs by miRNA-107 from gastric cancer.Part Ⅰ Abnormal changes of MDSCs in proliferation,differentiation and immune function under gastric cancer condition.Methods 1 Peripheral blood samples were collected with EDTA anticoagulant from 20 gastric cancer patients and 20 age-matched healthy volunteers.In addition,tumor tissues and adjacent tissues were collected from 6 gastric cancer patients and were used to make single cell suspensions.2 HLA-DRneg cells were isolated by magnetic bead method from single cell suspension of gastric cancer and adjacent normal tissues and from peripheral blood samples.3 Immune phenotype was determined in HLA-DRneg cells from MDSCs using BD FCSCanto TM-Ⅱ flow cytometry.ARG1 expression in MDSC subsets were then detected by flow cytometry.4 Relative expressions in both m RNA and protein levels were determined between ARG1 and NOS2 were determined using QRT-PCR and western blot.Results 1 The purity of MDSC isolated by magnetic bead method was determined by flow cytometry to be more than 95%.2 The number of HLA-DR-CD33+ARG1+,HLA-DR-CD11b+ARG1+,and HLA-DR-CD14+ARG1+subtype MDSCs in peripheral blood of GC patients′ was significantly higher(P<0.05)than in those of healthy controls quantitatively.Among these subtypes,the amount of HLA-DR-CD33+ARG1+MDSCs was the highest(>95%),which was significantly higher(P<0.05)than other subtypes.HLA-DR-CD33+ARG1+MDSCs subset is significant differences with the other subgroup.Further immunophenotyping indicated that MDSCs with HLA-DR-CD33+ARG1+MDSCs immune phenotype,outcome indicated that HLA-DR-CD33+CD14-CD15-ARG1+MDSCs amounted for ≥90% of the total cell population,which were significantly higher than(P<0.05)other subgroups.3 The number of HLA-DR-CD33+ARG1+,HLA-DR-CD11b+ARG1+,and HLA-DR-CD14+ARG1+MDSCs was significantly higher(P >0.05)in tumor tissues than in adjacent tissues in gastric cancer patients.However,no significant difference was observed among three subgroups(P >0.05).4 The relative m RNA level of ARG1 and NOS2 in peripheral blood-isolated HLA-DRneg cells was 3.01 and 6.4 times higher(P<0.05 in both cases),respectively,in GC patients than in healthy controls.The relative protein level was also 3.89 and 3.49 times higher(P<0.05 in both cases).The relative m RNA level of ARG1 and NOS2 in HLA-DRneg cells isolated from tumor tissues was 1.79 and 4.14 times higher(P<0.05 in both cases)than those from adjacent tissues,respectively,in GC patients.The relative protein level was also 2.2 and 4.63 times higher(P<0.05 in both cases).Part Ⅱ Predict and validate target genes of miRNA-107 in MDSCsMethods 1.Target Scan,Pic Tar and mi Randa were used to predict miRNA-107 target genes.2.Fluorescent reporter plasmid h-DICER1-3’UTR and h-PTEN-3’UTR were constructed and were used together with miRNA-107 mimics to co-transfect 293 T cells.Dual luciferase detection kit were used to determine fluorescence intensity of reporter gene to confirm miRNA-107 target gene.3.miRNA-107 mimics,miRNA-107 inhibitors and unrelated miRNA were synthesized and used to transfect HLA-DRnegcells,individually.These cells were cultured at 37℃ and 5%CO2 for 72 hours.The m RNA and protein levels of DICER1 and PTEN were measured by QRT-PCR and Western blot,respectively to verify the target genes of mi R-107 in transfected HLA-DRneg cells.Results 1.mi R-107 complementary sequence AUGCUGC was identified in the DICER1 and PTEN genes.h-Dicer1 m RNA-3’UTR contained six potential binding sites,and h-PTEN m RNA-3’UTR possess two potential binding sites.2.After 293 T cells were co-transfected with miRNA-107 mimics and hDICER1-3’UTR or h-PTEN-3’UTR,the ratio of two luciferases were significantly lower in wildtype group than in mutation group and control group.3.The m RNA levels of DICER1 and PTEN were not significantly changed in HLA-DRneg cells transfected with miRNA-107 mimics.However,the protein levels of both Dicer1 and PTEN were significantly decreased(P<0.05).Part Ⅲ Study on role of gastric cancer-derived miRNA-107 in regulating human MDSCs in vitroMethods 1.Exosomes were isolated from the culture medium of gastric cancer cell lines,respectively.The morphology of exosomes was observed by JEM-1400 high contrast transmission electron microscope.Western blot was performed to identify antibodies used for labeling exosomes.Exosomes were then determined quantitatively using analysis of BCA protein quantitation method.2.q RT-PCR was employed to determine the relative expression level of miRNA-107 in the exosome isolated from gastric cancer cells.3.Fluorescent microscope was used to examine the transportation of miRNA-107-Cy3 through the exosomes between gastric cancer cells and HLA-DR-CD33+MDSCs in vitro.Then q RT-PCR was used to determine the relative expression level of miRNA-107 and pre-miRNA-107 in HLA-DR-CD33+MDSCs.4.miRNA-107 mimics and unrelated miRNA were synthesized and then used for transfecting HLA-DRneg cells,while untransfected HLA-DRneg cells were used as controls.Three groups of cells were cultured at 37℃ in a humidified 5% CO2/95% air atmosphere incubator for 96 hours.Flow cytometry was applied to assay the proliferation and differentiation of MDSCs and the expression of ARG1 proteins in MDSC subsets.q RT-PCR was then used to examine the expression of ARG1-m RNA in HLA-DRneg cells.5.We first prepared exosomes with enriched or lack of the miRNA-107.The two types of exosomes were added to the HLA-DRneg cells,which were cultured at 37℃ and 5%CO2 for another 96 hours.Flow cytometry was used to determine the rates of MDSCs’ proliferation and differentiation as well as the expression level of ARG1 proteins in MDSC subsets.q RT-PCR was then used to detect the expression of ARG1 m RNA in HLA-DRneg cells.6.Wild h-DICER1-3’UTR-WT plasmid and mutant h-Dicer1-3’UTR-MUT plasmid were constructed and were then used to transfect two groups of HLA-DRneg cells.These cells were then co-cultured with miRNA-107-enriched or-deficient exosome,respectively for 96 hours at 37℃ and 5% CO2.The relative level of Dicer1 proteins were determined by western blot and the proliferation and differentiation of MDSCs was measured using flow cytometry assays.7.In order to further confirm whether suppression of the DICER1 gene can result in enhanced proliferating and differentiating of MDSCs,h-Dicer1-and scramble-si RNAs were synthesized and then used to transfect HLA-DRneg cells,which were cultured at 37℃ and 5%CO2 for an additional 96 hours.The relative level of Dicer1 proteins were determined by western blot and the proliferation and differentiation of MDSCs was measured using flow cytometry assays.8.First,wild h-PTEN-3’UTR-WT plasmid and mutant h-PTEN-3’UTR-MUT plasmid were constructed and were then used to transfect two groups of HLA-DRneg cells.Each group of these cells were then co-cultured with miRNA-107-enriched and-deficient exosomes,respectively,at 37℃ and 5%CO2 for an additional 96 hours.The relative expression level of PTEN was measured by western blot experiments and the expression of ARG1 m RNA and PI3K/AKT/m TOR/NF-κBm RNA were determined by q RT-PCR in HLA-DRneg cells.Meanwhile,the expression level of ARG1 and PI3K/AKT/m TOR/NF-κB/P-AKT/p-m TOR/p-NF-κB p65 were also determined by western blot.The expression of ARG1 proteins in MDSC subsets was determined using flow cytometry after HLA-DRneg cells were labelled by fluorescent antibody.9.h-PTEN-si RNA and scramble si RNA were constructed and used to transfect two groups of HLA-DRneg cells,which were cultured at 37℃ and 5%CO2 for an additional 96 hours.The expression of ARG1 m RNA and PI3K/AKT/m TOR/NF-κB m RNA were measured by q RT-PCR in HLA-DRneg cells.The level of ARG1 and PI3K/AKT/m TOR/NF-κB/P-AKT/p-m TOR/p-NF-κB p65 proteins were measured by western blot.Flow cytometry was used to assay the expression of ARG1 in MDSCs subsets.Results 1.We successfully separated and extracted exosomes from cell culture medium.The diameter of exosome ranged from 30 to 100 nm determined by using an electron microscope.Protein concentration was maintained at 5-10μg/μl.The expression of surface protein marker CD9 and CD63 were also detected by western blot.2.Relative level of total mi R-107 in cell lines MGC-803,BGC-823,and SGC-7901 were 2-3 times more than in 293 T cell.The level of exosome-derived mi R-107 from MGC-803,BGC-823,SGC-7901 were 3-5 times more than from 293 T cells.3.Cy3 red fluorescence was observed by fluorescence microscope in HLA-DRneg cells.The level of mi R-107 had a significantly increased(P<0.01)in HLA-DR-CD33+MDSCs.However,pre-miRNA-107 level had no significant changes(P>0.05).These results suggested that high level of mi R-107 were likely came from the exogenous carriers,namely exosomes.4.Our results showed that the number of HLA-DR-CD11b+ and HLA-DRCD33+subset MDSCs was significantly increased than that of the control cells after HLA-DRneg cells were transfected with miRNA-107 mimics.The number of HLA-DR-CD11b+ and HLA-DR-CD33+ subset MDSCs was significantly decreased than that of the control cells after HLA-DRneg cells transfection with miRNA-107 inhibitor.These experiments suggested exogenous mi R-107 could enhance the proliferation of HLA-DR-CD11b+ and HLA-DR-CD33+ MDSCs subset cells.5.After co-cultured with miRNA-107-enriched exosome,the ratios of HLA-DR-CD11b+ and HLA-DR-CD33+ subset MDSCs were significantly elevated in culture(P<0.05).However,the ratios of these two subsets were significantly reduced in the presence of miRNA-107-deficient exosomes(P<0.05).6.After co-cultured with HLA-DRneg cells transfected with h-Dicer1-3’UTR-WT plasmids,EXO-miRNA-107 enr cells showed significantly reduced relative level of DICER1 proteins than in other three groups(P<0.05).Flow cytometry analysis found that a significant enhancement in the ratio of the experimental subsets of HLA-DR-CD11b+MDSCs(P<0.05)and HLA-DR-CD33+MDSCs(P<0.05)than other groups.7.The protein level of Dicer1 in HLA-DRneg cells were obviously suppressed by h-DICER1-si RNA(P<0.01).Flow cytometry analysis indicated that the population ratios of experimental subsets of HLA-DR-CD11b+MDSCs(P<0.05)and HLA-DRCD33+MDSCs(P<0.05)were significantly increased than other three groups.8.Compared with other three groups,HLA-DRneg cells transfected with h-PTEN-3’UTR-WT plasmid expressed significantly lower level of PTEN proteins(P<0.01)and higher level of ARG1 m RNAs(P<0.01).ARG1 protein level was significantly increased(P<0.01)in the subsets of HLA-DR-CD11b+MDSCs and HLA-DR-CD33+MDSCs.A significant increase was found in both PI3K/Akt/m TOR /NF-κB m RNA level(P<0.05)and PI3K/AKT/m TOR/NF-κB/P-AKT/p-m TOR/p65-NF-κB protein level(P<0.05).9.PTEN protein level in HLA-DRneg cells was significantly suppressed by knocking down PTEN gene expression(P<0.01).ARG1 m RNA relative expression has a significant increase(P<0.01).ARG1 protein level was significantly increased(P<0.01)in the subsets of HLA-DR-CD11b+MDSCs and HLA-DR-CD33+MDSCs.A significant increase was found in PI3K/Akt/m TOR/NF-κB m RNA level(P<0.01)and in PI3K/Akt/m TOR/NF-κB protein level(P<0.05).Part Ⅳ Role of gastric cancer-derived miRNA-107 in regulating mice MDSCs in vivoMethods 1.Exosomes were extracted from the medium of cultured SGC-7901 cells transfected with miRNA-107 mimics-Cy3.Extracted exosomes were administrated into mice through the tail vein injection.Tail venous blood was collected 72 hours after injection.CD11b+Gr1+/highMDSCs in the blood samples were then collected by magnetic bead method.Fluorescent signals of miRNA-107 mimics-Cy3 was observed under a fluorescence microscope. 2.Exosomes secreted from SGC-7901 cells,extracted from the serum of gastric cancer patients,and secreted from SGC-7901 cells transfected with miRNA-107 inhibitors were administrated into mice through the tail vein injection.The mice were injected 7 times,once every another day,including 20μg total proteins(by protein quantification)per injection.Tail venous blood samples were collected 72 hours after injection.3.Nucleated cells in mouse venous blood were labeled with fluorescent antibody against Gr-1-FITC(mouse)and CD11b-PE(human and mouse).The amount of CD11b+Gr1+/highMDSCs and CD11b+Gr1-/lowMDSCs in the mice tail venous blood were then counted by flow cytometry.4.CD11b+Gr1-/lowMDSCs were isolated from the Peripheral blood in mice by magnetic bead separation method.The relative m RNA and protein levels of ARG1/ NOS2/DICER1/PTEN were detected by q RT-PCR and western blot,respectively.Results 1.After seven injections of miRNA-107-mimics-Cy3-enriched exosomes,we observed the red fluorescent signals of cy3 by fluorescence microscope in mouse CD11b+Gr1-/lowMDSCs.2.Compared to the control,seven injections of exosomes from different sources significantly enhanced the amplification of CD11b+Gr1+/high cells in the peripheral blood of mice at variable degrees.The percent of CD11b+Gr1+/high cells was 63.5%(P<0.05)in the mice injected with exosomes derived from GC patients,46.6 %(P<0.05)from SGC-7901 cells,36.6 %(P >0.05)from 293 T cells,and 31.2% in the control group.3.Compared the control group using exosomes lacking miRNA-107,both the m RNA and protein levels of ARG1 and NOS2 in CD11b+Gr1+/ high cells in peripheral blood of mice were significantly higher(P<0.05)in the groups using GC cell-and patient-derived exosomes,both the m RNA and protein levels of DICER1 and PTEN were significantly lower(P<0.05).Conclusions 1.Under the condition of gastric cancer,the proliferation and accumulation of HLA-DR-CD33+ARG1+MDSCs,HLA-DR-CD11b+ARG1+MDSCs and HLA-DRCD14+ARG1+MDSCs are enhanced in human peripheral blood and gastric cancer tissue,with a significantly enhanced immune suppressive function.2.Gastric cancer-secreted miRNA-107 can effectively be transported to MDSCs via exosomes,and can post-transcriptionally regulate DICER1 and PTEN expression respectively by targeting their 3’UTRs in MDSCs,and can induce the proliferation and differentiation of HLA-DR-CD33+MDSCs and HLA-DR-CD11b+ MDSCs,and can enhance immune suppressive function,in MDSCs.3.Gastric cancer-derived miRNA-107 can induce the proliferation and amplification of CD11b+Gr1+/high MDSCs and enhance immune suppressive function in mice.