LncRNA Snhg6 regulates the differentiation of MDSCs by regulating the ubiquitination of EZH2

Abstract

Myeloid-derived suppressor cells (MDSCs) are derived from bone marrow progenitor cells commonly, which is a heterogeneous cell group composed of immature granulocytes, dendritic cells, macrophages and early undifferentiated bone marrow precursor cells. Its differentiation and immunosuppressive function are regulated by complex network signals, but the specific regulation mechanisms are not yet fully understood. In this study, we found that in mouse of Lewis lung cancer xenograft, long non-coding RNA Snhg6 (lncRNA Snhg6) was highly expressed in tumor-derived MDSCs compared with spleen-derived MDSCs. LncRNA Snhg6 facilitated the differentiation of CD11b⁺ Ly6G^- Ly6C^high monocytic MDSCs (Mo-MDSCs) rather than CD11b⁺ Ly6G⁺ Ly6C^low polymorphonuclear MDSCs (PMN-MDSCs), but did not affect the immunosuppressive function of MDSCs. Notably, lncRNA Snhg6 could inhibit the expression of EZH2 by ubiquitination pathway at protein level rather than mRNA level during the differentiation of mouse bone marrow cells into MDSCs in vitro. EZH2 may be an important factor in the regulation of lncRNA Snhg6 to promote the differentiation of Mo-MDSCs. So what we found may provide new ideas and targets for anti-tumor immunotherapy targeting MDSCs.

Keywords: Differentiation; EZH2; MDSCs; lncRNA Snhg6; ubiquitination.

Fig. 1

LncRNA Snhg6 promotes Mo-MDSCs but not affects the differentiation of PMN-MDSCs. a LncRNA Snhg6 expression in Tu-MDSCs compared with SP-MDSCs measured by qRT-PCR. b, c Typical flow cytometry of CD11b⁺ Gr-1⁺ MDSCs. d–g The percentage and absolute number of CD11b⁺ Gr-1⁺ MDSCs by statistical analyses. h, i Typical flow cytometry and the percentage of CD11b⁺ Ly6G⁺ Ly6C^low PMN-MDSCs and CD11b⁺ Ly6G⁻ Ly6C^high Mo-MDSCs after downregulating the expression of lncRNA Snhg6. j, k Typical flow cytometry and percentage of CD11b⁺ Ly6G⁺ Ly6C^low PMN-MDSCs and CD11b⁺ Ly6G⁻ Ly6C^high Mo-MDSCs after increasing the expression of lncRNA Snhg6. Each expression had three replicates, ns: no significance; * p < 0.05; ** p < 0.01

Fig. 2

LncRNA Snhg6 reduces the stability of EZH2 protein. a The cellular localization of lncRNA Snhg6 was detected by Cy3-labeled lncRNA Snhg6 probe. Cy3-labeled 18S probe was used to indicate plasmid localization and Cy3-labeled U6 probe was used to indicate nuclear localization. DAPI was used to evaluate the cell nucleus. b Subcellular fractionation was isolated of MDSCs, and lncRNA Snhg6 localization was examined by qRT-PCR. 18S and U6 were used as cytoplasmic and nuclear indicators, respectively. c, d qRT-PCR were used to detect the expression of EZH2 at mRNA level. e, f Western blot were used to detect the expression of EZH2 at protein level. g Western blot were used to detect the expression of EZH2 with CHX (40 µg/ml) treated 0 h, 3 h and 6 h after transfecting si-Snhg6. h The statistical graph corresponding to the left. i RIP assays were used to investigate the ubiquitination of EZH2. Each expression had three replicates, ns: no significance; *p < 0.05; **p < 0.01