LncRNA RNCR3 promotes Chop expression by sponging miR-185-5p during MDSC differentiation

Abstract

Myeloid-derived suppressor cells (MDSCs) play a critical role in regulating immune responses in cancer and other pathological conditions. Mechanism(s) regulating MDSC differentiation and function is not completely clear, especially epigenetic regulation. In this study, we found that MDSCs express retinal non-coding RNA3 (RNCR3), and the expression in MDSCs is upregulated by inflammatory and tumor associated factors. RNCR3 may function as a competing endogenous RNA (ceRNA) to promote Chop expression by sponging miR-185-5p during MDSC differentiation. RNCR3 knockdown suppressed differentiation and function of MDSCs in vitro and in vivo. Quantitative RT-PCR showed that RNCR3 was negatively regulated by miR-185-5p in MDSCs. MiR-185-5p affected the expansion of MDSCs and reversed the effect of RNCR3 on MDSC differentiation and function through directly targeting Chop. Thus, our results suggest a RNCR3/miR-185-5p/Chop autologously strengthening network to promote MDSC differentiation and suppressive function in response to extracellular inflammatory and tumor-associated signals.

Keywords: Chop; RNCR3; epigenetic modification; miR-185-5p; myeloid-derived suppressor cells.

Figure 1. RNCR3 is upregulated in tumor microenvironment

(A) Flow cytometric analyses (left) and RNCR3 qRT-PCR (right) of CD11b⁺Gr1⁺ cells in the spleen of tumor-free mice and B16 tumor-bearing mice. The MDSCs were sorted from the splenocytes of tumor-bearing mice and tumor-free mice. Iso.ctrl., antibody isotypic control. (B) Flow cytometric analyses (left) and RNCR3 qRT-PCR (right) of CD11b⁺Gr1⁺ cells in the tumor (tMDSC), spleen (sMDSC) and bone marrow (bMDSC) of B16 tumor-bearing mice. The MDSCs were sorted from tumor tissues, spleen and bone marrow (BM) of B16 tumor-bearing mice. (C) Flow cytometric analyses (left) and RNCR3 qRT-PCR (right) of CD11b⁺Gr1⁺MDSCs induced by GM-CSF or GM-CSF plus IL-6. Fresh CD11b⁺Gr1⁺ BMCs (BMC) were used as control. (D) QRT-PCR of RNCR3 in CD11b⁺Gr1⁺ MDSCs isolated from the BM of WT or IL-6^-/- tumor free and tumor-bearing mice (Left), and in CD11b⁺Gr1⁺MDSCs isolated from the BM of B16 tumor-bearing WT or IL-6^-/- mice (right). The data are representative of at least three separate experiments. ^*P < 0.05, ^**P < 0.01, ^***p<0.005; Ns, no significance.

Figure 2. RNCR3 knockdown interrupts MDSC differentiation in vitro

(A) QRT-PCR of lncRNA RNCR3 in MDSCs transfected with SiNC (siRNA control), SiRNCR3-1(RNCR3 siRNA 1) or SiRNCR3-2 (RNCR3 siRNA 2). (B) Flow cytometric analyses of MDSCs transfected with SiNC or SiRNCR3 (RNCR3 siRNA 2). (C) Statistical analyses of MDSCs transfected with SiNC or SiRNCR3 (RNCR3 siRNA 2). (D) QRT-PCR of RNCR3 in MDSCs transduced with control shRNA/lentiviruses (Lv-shNC) or RNCR3 shRNA/Lentiviruses (Lv-shRNCR3) (left), and fluorescence microscopy of lncRNA shRNA/lentiviruses transduced mouse MDSC (right). Blue (DAPI), nuclei; Green(GFP), lentivirus shRNA lentivirus. Rule bar =100μM. (E) Flow cytometric analyses of MDSCs transduced with control shRNA/lentiviruses (Lv-shNC) or RNCR3 shRNA/Lentiviruses (Lv-shRNCR3). (F) Statistical analyses of MDSCs transduced with control shRNA/lentiviruses (Lv-shNC) or RNCR3 shRNA/Lentiviruses (Lv-shRNCR3). The data are representative of at least three separate experiments. ^*P < 0.05, ^**P < 0.01, ^***p<0.005; Ns, no significance.

Figure 3. RNCR3 knockdown impairs immunosuppressive function of MDSCs in vitro

(A and B) Suppressive capacity of RNCR3 siRNA (SiRNCR3) transfected (A) or RNCR3 shRNA/Lentivirus (Lv-shRNCR3) transduced (B) MDSCs. Activity of T cells was measured by their capacity to produce IFN-γ upon OVA-MHCI specific peptide stimulation. (C and D) QRT-PCR and immunoblotting of Arg-1 and iNOS in RNCR3 siRNA (SiRNCR3) transfected (C) or RNCR3 shRNA/Lentivirus (Lv-shRNCR3) transduced (D) MDSCs. (E and G) NO production in RNCR3 siRNA (SiRNCR3) transfected (E) or RNCR3 shRNA/Lentivirus (Lv-shRNCR3) transduced (G) MDSCs. (F and H) Flow cytometric analyses of ROS in RNCR3 siRNA (SiRNCR3) transfected (F) or RNCR3 shRNA/Lentivirus (Lv-shRNCR3) transduced (H) MDSCs. SiNC, siRNA control; Lv-shNC, control lentiviruses. The data are representative of at least three separate experiments. ^*P < 0.05, ^**P < 0.01, ^***p<0.005; Ns, no significance.

Figure 4. RNCR3 knockdown affects differentiation and immunosuppressive function of MDSCs in vivo

(A-F) Tumor growth (A, D), tumor size (B, E) and tumor weight (C, F) in C57/BL6 mice bearing B16 tumors (N=6), which were injected with CD45.1⁺MDSCs transfected with SiRNCR3 (RNCR3 siRNA) (A, B, C) or transduced by shRNA (Lv-shRNCR3) (D, E, F). SiNC, siRNA control; Lv-shNC, control lentivirus; CTRL, without MDSC injection. (G) Gated strategies for tumor immune cells. (H and I) Flow cytometric and statistical analyses of MDSCs (H) and their subpopulations (I) in the spleen and tumor from mice injected with CD45.1⁺ MDSCs transfected with SiNC or SiRNCR3. (J) Flow cytometric and statistical analyses of CD4⁺ or CD8⁺ cells in tumor from mice injected with or without CD45.1⁺MDSCs transfected with SiNC or SiRNCR3. The data are from three separate experiments. ^*P < 0.05, ^**P < 0.01, ^***p<0.005; Ns, no significance.

Figure 5. MiR-185-5p regulates differentiation and function of MDSCs

(A) QRT-PCR of miR-185-5p in MDSCs transfected with miR-185-5p mimics or MiR-NC (mimics control) in the presence of GM-CSF and IL-6. (B and C) Flow cytometric (B) and statistical analyses (C) of the MDSCs transfected with miR-185-5p mimics or NC. (D and E) QRT-PCR (D) and Western blot (E) of iNOS and Arg-1 in MDSCs transfected with miR-185-5p mimics control (NC) or miR-185-5p mimics. (F) QRT-PCR of miR-185-5p in the MDSCs transfected with miR-185-5p inhibitor or inhibitor control (NC). (G and H) Flow cytometric (G) and statistical analyses (H) of MDSCs transfected with miR-185-5p inhibitor or inhibitor control (NC). (I and J) QRT-PCR (I) and Western blot (J) of iNOS and Arg-1 in MDSCs transfected with miR-185-5p inhibitor control (NC) or miR-185-5p inhibitor. (K and L) Flow cytometric (K) and statistical analyses (L) of MDSCs transfected with RNCR3 siRNA and miR-185-5p inhibitor (siRNCR3+miR-185-5p inhibitor) or inhibitor control (siRNCR3+inhibitor-NC). NC, siRNA and inhibitor control. The data are representative of at least three separate experiments. ^*P < 0.05, ^**P < 0.01, ^***p<0.005; Ns, no significance.

Figure 6. MiR-185-5p directly targets Chop

(A) Predicted paring of miR-185-5p to the 3’-UTR of Chop and the mutant site (Red) of Chop 3’-UTR in pSiCHECK^™-2 luciferase reporter vector. (B) Dual-luciferase reporter assay of 293T cells co-transfected with Chop 3’-UTR-WT or Chop 3’-UTR-Mut and miR-185-5p mimics or mimics control (NC). (C) Western blot of Chop in MDSCs transfected with mimics control (miR-NC) or miR-185-5p mimics. Untransfected MDSCs as control. (D) Western blot of Chop in MDSCs transfected with inhibitor control (inhibitor-NC) or miR-185-5P inhibitor. Untransfected MDSCs as control. ^*P < 0.05, ^**P < 0.01, ^***p<0.005; Ns, no significance.

Figure 7. Chop knockdown exerts the same effect to miR-185-5p mimics during MDSC differentiation

(A) QRT-PCR of Chop in MDSCs transfected with siNC (control siRNA), siChop-1(Chop siRNA 1) or siChop-2 (Chop siRNA 2). (B) Flow cytometric and statistical analyses in CD11b⁺Gr1⁺MDSCs transfected with siNC or siChop. (C) Flow cytometric and statistical analyses of MDSC subpopulations in MDSCs transfected with siNC and siChop. (D) QRT-PCR of Arg1 and iNOS in MDSCs transfected with siNC and siChop. The data are representative of at least three separate experiments. ^*P < 0.05, ^**P < 0.01, ^***p<0.005; Ns, no significance.

Figure 8. Regulation of RNCR3 in MDSCs is through upregulating Chop by sponging miR-185-5p

(A) QRT-PCR (left) and Western blot (right) of Chop in MDSCs transfected with siRNA control (SiNC) and RNCR3 siRNA (SiRNCR3). (B) qRT-PCR of Chop in MDSCs transfected with RNCR3 siRNA and miR-185-5p inhibitor (siRNCR3+miR-185-5p) or miR-185-5p inhibitor control (siRNRC3+NC). siNC, siRNA control. (C, D, E) QRT-PCR of RNCR3, miR-185-5p and Chop in the MDSCs isolated from the tumor tissues (tMDSCs), spleen (sMDSCs) and BM (bMDSCs) of mice bearing B16 tumor. (F, G, H) QRT-PCR of RNCR3, miR-185-5p and Chop in CD11b⁺Gr1⁺BMCs (BMC), GM-CSF induced MDSCs (GM-CSF) and GM-CSF plus IL-6 mediated MDSCs (GM-CSF+IL-6). Western blot of Chop (lower in H). (I) RNA-FISH in the isolated MDSCs from the spleens of mice bearing B16 tumor. Green, RNCR3; Blue, nuclei. Rule bar = 20 μM. The data are representative of at least three separate experiments. ^*P < 0.05, ^**P < 0.01, ^***p<0.005; Ns, no significance.