Long Noncoding RNA Snhg6 Regulates The Differentiation And Immunosuppression Of MDSCs

Objective:Study on the regulation and molecular mechanism of long noncoding RNA Snhg6（lncRNA Snhg6）on the differentiation of myeloid-derived suppressor cells（MDSCs）,and further explore its effection on the immunosuppressive function of MDSCs,providing new ideas and targets for anti-tumor immunotherapy targeting MDSCs.Methods:（1）Immunomagnetic beads were used to isolate and purify MDSCs derived from spleen,and immunomagnetic beads combined with flow cytometry（FCM）were used to isolate and purify MDSCs derived from tumor tissues of mice with lung cancer xenografts,FCM was used to detect the purity of MDSCs.Analysed of Arraystar LncRNA microarray and finally filtered out lncRNA Snhg6.The expression of lncRNA Snhg6 in MDSCs from different tissues were verified by qRT-PCR.Using tumor cells conditioned medium（TCCM）to simulate tumor microenvironment,qRT-PCR was used to detect the expression of lncRNA Snhg6 after TCCM treatment,FCM was used to detect the influence of TCCM on differentiation of MDSCs.（2）The differentiation system for inducing MDSCs by mouse bone marrow cells was constructed in vitro.The specific siRNA（si-Snhg6）of lncRNA Snhg6 was used to knock down the expression of lncRNA Snhg6 in mouse bone marrow cells and then induced MDSCs in vitro,the percentage and number of MDSCs,PMN-MDSCs and Mo-MDSCs were detected by FCM.（3）Fluorescence in situ hybridization（FISH）,nuclear plasma separation combined with qRT-PCR were used to detect the subcellular localization of lncRNA Snhg6 in MDSCs.（4）Using si-Snhg6 to knock down the expression of lncRNA Snhg6 in mouse bone marrow cells,and then MDSCs were induced in vitro.qRT-PCR and Western blot were used to detect the expression of EZH2 mRNA and protein levels after induction respectively.（5）Reduce the expression of lncRNA Snhg6 by si-Snhg6 during the differentiation of mouse bone marrow cells to induce MDSCs in vitro.After adding the protein synthesis inhibitor Cycloheximide（CHX）,Western blot was used to detect the regulation of lncRNA Snhg6 on the stability of EZH2 protein.Immunoprecipitation（IP）was used to detect the regulation on EZH2 protein ubiquitination by lncRNA Snhg6.（6）Using si-Snhg6 to knock down the expression of lncRNA Snhg6 in tumor-derived MDSCs,immunosuppressive function of MDSCs on CD4⁺T cell proliferation was measured by CFSE labeling method,the activity of arginase（Arg-1）and the content of nitric oxide（NO）were measured by colorimetric assay,moreover,the expression of reactive oxygen species（ROS）was determined by FCM.Results:（1）Compared with spleen-derived MDSCs,lncRNA Snhg6 was highly expressed in tumor-derived MDSCs of tumor-bearing mice（p<0.01）.Compared with the untreated group,the expression of lncRNA Snhg6 and the percentage of MDSCs were significantly increased after TCCM treatment（p<0.05）.（2）Reducing the expression of lncRNA Snhg6 in bone marrow cells,and then inducing MDSCs in vitro,the percentage and number of CD11b⁺Gr-1⁺MDSCs and the percentage of CD11b⁺Ly6G⁺Ly6C^low PMN-MDSCs showed no significant changes（p>0.05）,but the percentage of CD11b⁺Ly6G^-Ly6C^high Mo-MDSCs was significantly decreased（p<0.05）.（3）LncRNA Snhg6 was expressed both in the cytoplasm and the nucleus of MDSCs,and was mainly expressed in the cytoplasm of MDSCs.（4）After knockdown of lncRNA Snhg6 during the induction of MDSCs differentiation in vitro,the mRNA level of EZH2 was not significantly changed（p>0.05）,but its protein level was significantly increased.Further results showed that the stability of EZH2 was significantly increased,while its protein ubiquitination level of EZH2 was significantly decreased after downregulating the expression of lncRNA Snhg6.（5）After inhibiting the expression of lncRNA Snhg6 in tumor-derived MDSCs,the immunosuppressive function of MDSCs on the proliferation of CD4⁺T cells did not change significantly,nor did the immunosuppressive effector molecule Arg-1activity,NO content and the expression of ROS change（p>0.05）.Conclusions:（1）LncRNA Snhg6 is involved in regulating the differentiation of MDSCs,but does not affect the immunosuppressive function of MDSCs.（2）In the process of inducing MDSCs in vitro,lncRNA Snhg6 impacts EZH2protein stability by regulating the level of ubiquitination.