MiR-20b Inhibits Apoptosis Of MDSCs By Targeting FOXO1

Background:Myeloid derived suppressor cells?MDSCs?are a heterogeneous group of myeloid cells,including myeloid precursor cells and immature myeloid cells.The latter include immature macrophages,granulocytes and dendritic cells.Under the pathological conditions of infection,tumor,inflammation,trauma or autoimmune disease,MDSCs accumulate in the bone marrow,spleen,lymph nodes,blood and peripheral tissues.MDSCs play an immunosuppressive role and need to be recruited from the bone marrow to the periphery by chronic inflammation-related factors?IL-6,GM-CSF,SCF,VEGF,etc.?,and further induced activation by tumor-derived factors?TDFs?.Activated MDSCs inhibits innate and acquired immunity through a variety of pathways.MicroRNAs?miRNAs?are a class of highly conserved,non-coding RNA molecules containing 17-25 nucleotides that regulate the expression of target genes by specifically binding to the base of the 3,-end non-coding region?3,-UTR?of the target genes.More and more studies have shown that miRNAs play an important regulatory role in the proliferation and immune function of MDSCs.MiR-20b is a member of the miRNA 106a-363 gene cluster family,located on the X chromosome of mammals.Currently,most studies on miR-20b focus on its effect on tumor cells.MiR-20b overexpression increases the apoptosis of teratoma cells and promotes their differentiation.In colorectal cancer,upregulated miR-20b inhibits the expression of PTEN,leading to B7-H1 overexpression in colorectal cancer,thereby inhibiting the activation and proliferation of T cells and promoting the immune escape of tumor cells.However,some studies have found that miR-20b can also inhibit the differentiation of Th17 cells,thereby reducing the severity of EAE disease.Recent studies have shown that overexpression of miR-20b can reduce the activity of macrophages and induce their apoptosis.However,it is not clear whether miR-20b can affect the activity and biological function of MDSCs.Objective:To investigate the effect of miR-20b on the activity and biological function of MDSCs and its related mechanisms.Methods:The mouse liver cancer cell line H22 was used to construct the model of liver cancer transplantation in mice.The bone marrow,spleen and local tissues of normal mice and tumor-bearing mice were selected by immunomagnetic bead method,and the expression of miR-20b in MDSCs cells was detected by fluorescence quantitative PCR.H22 tumor cell culture supernatant?TCCM?or IL-33 or IL-6+GM-CSF stimulated MDSCs derived from bone marrow of normal mice for 24h,and fluorescence quantitative PCR detected the expression of miR-20b in MDSCs cells.IL-33 stimulated MDSCs at different time points?0h,6h,12h,24h?,and fluorescence quantitative PCR was used to detect the expressions of miR-20b,ARG1 and MMP2 in MDSCs cells.Immunomagnetic beads were used to select MDSCs from bone marrow of normal mice,spleen and local tumor tissues of tumor-bearing mice,and fluorescence quantitative PCR was used to detect the expressions of miR-20b,ARG1and MMP2 in MDSCs cells.MiR-20b expression was detected by fluorescence quantitative PCR 72h after MDSCs transfection with miR-20b inhibitor lentivirus?lv-sponge?and its control lentivirus?lv-ctrl?.After lv-sponge or lv-ctrl was used to infect the spleen source MDSCs of the tumor-infected mice for 48h,TCCM was added to stimulate it for 24h,and the fluorescence quantitative PCR was used to check the expression of ARG1 and MMP2.MDSCs from spleen of tumor bearing mice were transfected with lv-sponge or lv-ctrl for 72h,and then H22 cells labeled with CFSE fluorescence were separately or mixed with various MDSCs labeled with CFSE and put into the upper chamber of Transwell coated with Matrigel.RPMI-1640complete culture medium was added into the lower chamber.After 8h,the lower chamber cells were collected and counted.MDSCs from spleen of tumor-bearing mice were transfected with lv-sponge or lv-ctrl lentivirus,and CCR2 expression was detected by 72h post-flow cytometry.MDSCs from the spleen of the tumor-bearing mice were put into the upper chamber of the Transwell chamber,CCL2 or PBS were added into the culture medium of the lower chamber for cell migration experiments,and the chamber was removed after 8h,the lower layer cells were collected by centrifugation and counted under the microscope.MiRanda's algorithm predicted the existence of miR-20b binding sites in the FOXO1 3'-UTR region.MDSCs from bone marrow of normal and tumor-bearing mice were isolated,and the mRNA and protein expression levels of MDSCs FOXO1 were detected by fluorescence quantitative PCR and western blot,respectively.A dual luciferase reporter gene recombinant plasmid vector containing FOXO1 3'-UTR was constructed.Hela cells were then co-transfected by the recombinant plasmid and miR-20b mimics/scramble to detect the luciferase activity of the reporter gene.MDSCs apoptosis rate was detected by flow cytometry 48h and 72h after lv-sponge and lv-ctrl lentivirus transfected with MDSCs from spleen of tumor-bearing mice.FOXO1 siRNA interfered FOXO1 in MDSCs for 24h,and then lv-sponge or lv-ctrl lentivirus were transfected for 72h.Flow cytometry was used to detect the apoptosis rate of MDSCs.Results:Compared with normal mice,miR-20b expression of MDSCs cells in bone marrow,spleen and local tissues of tumor bearing mice was significantly increased?p<0.05?.After TCCM stimulated the bone marrow-derived MDSCs of normal mice with dilution of 1/2,1/5 and 1/10 for 24h,the relative expression of miR-20b was up-regulated compared with that of the control group?p<0.01?.Compared with the control group,there was no significant change in the relative expression of miR-20b in the IL-6 and GM-CSF combined group?p>0.05?.The relative expression of miR-20b was upregulated by IL-33?p<0.01?.Compared with the TCCM stimulation group alone,the relative expression of miR-20b was down-regulated in the IL-33antibody blocking group?p<0.05?.Relative mRNA expressions of miR-20b,ARG1,and MMP2 were up-regulated at 6h,12h,and 24h after IL-33 stimulation at 0h?p<0.01?.Compared with lv-ctrl lentivirus infection group,the relative expression of miR-20b in lv-sponge lentivirus infection group was down-regulated?p<0.05?.After MDSCs transfection with lv-sponge and lv-ctrl lentivirus for 72h,relative mRNA expressions of ARG1 and MMP2 were significantly decreased compared with lv-ctrl group?p<0.01?.Compared with the MDSCs group transfected with H22+lv-sponge,the number of H22 cells invading into the lower chamber in the MDSCs group was significantly reduced?p<0.01?.There was no significant difference in MDSCs surface CCR2 expression between lv-ctrl lentivirus transfection group and lv-sponge lentivirus transfection group?p>0.05?.Compared with PBS group,the numbers of MDSCs migration in CCL2 group was significantly increased?p<0.01?.However,compared with lv-ctrl lentivirus group,MDSCs transfected with lv-sponge lentivirus significantly reduced the number of chemotactic migration of CCL2 compared with lv-ctrl lentivirus group?p<0.01?.miR-20b binding sites in the 3,-UTR region of FOXO1 can be predicted by miRanda algorithm.The expression level of FOXO1protein in MDSCs in tumor-bearing mice was significantly lower than that of MDSCs in normal mice?p<0.01?,and there was no significant change in FOXO1 mRNA level?p>0.05?.Agarose gel electrophoresis and sequencing method proved the establishment of a pmiR-RB-Report^TM-FOXO1 3,-UTR dual luciferase reporter vector.The luciferase activity of the FOXO1 recombinant plasmid+miR-20b mimics co-transfected roup was significantly lower than that of the null plasmid+miR-20b mimics group?P<0.05?.Compared with lv-ctrl roup,the expression of MDSCs FOXO1 protein was up-regulated in lv-sponge roup?p<0.05?.Compared with control group and lv-sponge group,the proportion of MDSCs apoptotic cells in the48h group was not significantly changed?p>0.05?.Compared with the ctrl and lv–ctrl infection group,lv-sponge infection group of MDSCs annexin?proportion of positive cells increased significantly in 72h?p<0.01?;Compared with lv-sponge infection group,the apoptosis rate of lv-sponge+FOXO1 siRNA group was significantly decreased?p<0.01?.Conclusion:The expression of miR-20b in bone marrow,spleen and tumor tissue of tumor bearing mice is increased.Tumor origin factors?especially IL-33?induce the up-regulation of miR-20b expression in MDSCs.MiR-20b increases the migration of MDSCs and up-regulates its ability to promote the invasion of H22 cell.MiR-20b inhibits apoptosis of MDSCs by targeting FOXO1.