The Regulation Of CeRNA Network Cored By MiR-9/miR-181a In The IL-6 /STAT3 Signaling Pathway Promoting EMDSCs Generation And Immunosuppressive Capacity In Breast Cancer

Objective:The purpose of this study was to verify the effects of IL-6/STAT3 pathway on breast cancer-derived eMDSCs generation and immunosuppressive activity,and prove the existence of dysfunctional negative feedback of JAK/STAT/SOCS3 in breast cancer eMDSCs,and clarify the ceRNA network cored by miR-9 and miR-181a regulating JAK/STAT signaling pathway and their key target genes in eMDSCs,and finally confirm the role of miR-9 and miR-181a in promoting IL-6induced eMDSCs infiltration and breast cancer immune escape.Methods:（1）50 peripheral blood samples from healthy donors were collected to isolate CD33⁺cells and induced human breast cancer eMDSCs in vitro.The proportion of eMDSCs was detected by flow cytometry.The protein expression of IL-6R,ADAM10,ADAM17,SOCS1-SOCS3,JAK/STAT pathway in eMDSCs was detected by qRT-PCR and Western Blot.ELISA,CCK8,Annexin V were used to detect the effects of eMDSCs on the cytokines secretion（IL-10 and IFN-γ）,proliferation and apoptosis of T cells.（2）The wild-type and IL-6 knockout(IL-6^low)type 4T1 breast cancer mouse model was constructed.MiRNAs expression in eMDSCs from tumor-bearing mice was detected by miRNA microarray,and miR-9and miR-181a positively correlated with IL-6 but negatively correlated with SOCS3were filtered out.（3）Bone marrow cells of normal BALB/C mice were isolated and transfected with mmu-miR-9 and mmu-miR-181a mimics,mmu-miR-9 and mmu-miR-181a inhibitor and their corresponding NC,respectively.Flow cytometry was used to detect the percentage of CD11b⁺Gr-1^-MHC-II^-F4/80^-cells.The expression of SOCS3,Ptpn3,Ptpn4,Ptpn9,PIAS1,PIAS3,IL-10,and IDO was detected by RT-PCR.Luciferase reporter gene assay verified the relationship between miR-9,miR-181a and SOCS3,PIAS3,and Ptpn4 respectively.Western Blot analyzed SOCS3,PIAS3,Ptpn4,and JAK/STAT pathway related proteins expression.Proliferation and apoptosis of T cells were detected by Brdu and Annexin V methods.The expressions of miR-9 and miR-181a in 4T1 and 4T1^IL-6lowL-6low cells were detected to investigate the source of miRNA.（4）miR-9 and miR-181a agomir and NC transfected eMDSCs were injected into tumors of 4T1 tumor-bearing mice,and the change of tumor was observed,and the tumor growth curve was plotted.The infiltration ratio of eMDSCs and T cell proliferation and apoptosis were detected by flow cytometry.The expression of JAK/STAT signaling pathway was detected.（5）hsa-miR-9 and hsa-miR-181a mimics,hsa-miR-9 and hsa-miR-181a inhibitor,and NC were transfected into human CD33⁺cells,and the same method was used to detect the proportion of eMDSCs,T cell proliferation and apoptosis,SOCS3,PIAS3,Ptpn4,IL-10,IDO,and JAK/STAT pathway proteins expression.Luciferase reporter gene identified the relationship between miR-9,miR-181a and SOCS3,PIAS3.Results:（1）IL-6 increased eMDSCs generation,which was significantly reduced after blocking IL-6.eMDSCs promoted IL-10 but inhibited IFN-γsecretion,and promoted T cell apoptosis but inhibited T cell proliferation,which were reversed after blocking IL-6.（2）The expressions of CD126 and gp130 in eMDSCs increased,but the membrane-bound CD126 decreased and the soluble CD126 increased.JAK1,JAK2,TYK2,STAT1,STAT3 protein in eMDSCs were high phosphorylated and STAT1,STAT3 showed continuous phosphorylation in eMDSCs.SOCS1 increased,while SOCS3 decreased in eMDSCs.And SOCS2 amplification was not detected both in eMDSCs and control group.After blocking IL-6,the activity of JAK/STAT pathway decreased,SOCS1 decreased,and SOCS3 increased.Finally,it was found that adding exogenous ADAM10 increased soluble CD126 induced sustained activation of p-STAT3 and p-STAT1 and decreased SOCS3 in eMDSCs.（3）Methylation results showed none methylation were detected in human and mice SOCS3 promoter regions.MiRNA microarray results showed 23 miRNAs were related to IL-6 in eMDSCs and qRT-PCR confirmed that 11 miRNAs were consistent with the microarray.We also found that miR-9 and miR-181a had negative correlation with SOCS3 in eMDSCs.miR-9 and miR-181a in eMDSCs were derived from tumor cell exosomes,which decreased after inhibiting exosome release.（4）miR-9 and miR-181a in induced mice eMDSCs also increased,which decreased after reducing IL-6.miR-9 and miR-181a increased CD11b⁺Gr-1^-MHC-II^-F4/80^-eMDSCs,but decreased CD11b⁺Gr-1⁺MDSC,increased IDO and IL-10 expression,enhanced the inhibitory effects of eMDSCs on the proliferation and apoptosis of T cells,which were reversed after reducing miR-9 and miR-181a.PCR,WB and luciferase reporter genes showed miR-9 and miR-181a could inhibit SOCS3 and PIAS3 respectively.Furthermore,SOCS3 had a positive relationship with PIAS3.miR-9 and miR-181a synergistically activated JAK/STAT signaling pathway.（5）eMDSCs with increased miR-9 and miR-181a promoted breast tumor growth.miR-9 and miR-181a promoted CD11b⁺Gr-1^-MHC-II^-F4/80^-eMDSCs infiltration in tumor and spleen of tumor-bearing mice and enhanced the effects of eMDSCs on T cells through stimulating JAK/STAT signaling pathway activation.（6）Finally,miR-9 and miR-181a highly expressed in induced human eMDSCs.miR-9 and miR-181a promoted eMDSCs amplification and enhanced the anti-proliferation and pro-apoptosis effects of eMDSCs on T cells.miR-9 and miR-181a activated JAK/STAT signaling pathway through targeting SOCS3 and PIAS3,in which SOCS3had a positive relationship with PIAS3.Conclusion:（1）Tumor-derived IL-6 promoted eMDSCs production and their immunosuppressive effect on T cells by activating the JAK/STAT3 pathway;（2）Continuous IL-6 stimulation induced dysfunctional of SOCS3 negative feedback leading to continuous activation of JAK/STAT3 pathway,which regulated the generation and T cell suppression of eMDSCs;（3）The abnormal JAK/STAT/SOCS3signaling pathway was correlated with the soluble CD126-mediated IL-6trans-signaling pathway;（4）In human and mouse eMDSCs,IL-6-related miR-9 and miR-181a through targeting SOCS3 and PIAS3,which formed ceRNA network synergistically activated JAK/STAT signaling pathway,regulated eMDSCs generation and immune activity,and promoted the immune escape of breast cancer.