Bladder-cancer-derived exosomal circRNA_0013936 promotes suppressive immunity by up-regulating fatty acid transporter protein 2 and down-regulating receptor-interacting protein kinase 3 in PMN-MDSCs

Abstract

Background: Polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) is one of the causes of tumor immune tolerance and failure of cancer immunotherapy. Here, we found that bladder cancer (BCa)-derived exosomal circRNA_0013936 could enhance the immunosuppressive activity of PMN-MDSCs by regulating the expression of fatty acid transporter protein 2 (FATP2) and receptor-interacting protein kinase 3 (RIPK3). However, the underlying mechanism remains largely unknown.

Methods: BCa-derived exosomes was isolated and used for a series of experiments. RNA sequencing was used to identify the differentially expressed circRNAs. Western blotting, immunohistochemistry, immunofluorescence, qRT-PCR, ELISA and Flow cytometry were performed to reveal the potential mechanism of circRNA_0013936 promoting the immunosuppressive activity of PMN-MDSC.

Results: CircRNA_0013936 enriched in BCa-derived exosomes could promote the expression of FATP2 and inhibit the expression of RIPK3 in PMN-MDSCs. Mechanistically, circRNA_0013936 promoted the expression of FATP2 and inhibited the expression of RIPK3 expression via sponging miR-320a and miR-301b, which directly targeted JAK2 and CREB1 respectively. Ultimately, circRNA_0013936 significantly inhibited the functions of CD8⁺ T cells by up-regulating FATP2 through the circRNA_0013936/miR-320a/JAK2 pathway, and down-regulating RIPK3 through the circRNA_0013936/miR-301b/CREB1 pathway in PMN-MDSCs.

Conclusions: BCa-derived exosomal circRNA_0013936 promotes suppressive immunity by up-regulating FATP2 through the circRNA_0013936/miR-320a/JAK2 pathway and down-regulating RIPK3 through the circRNA_0013936/miR-301b-3p/CREB1 pathway in PMN-MDSCs. These findings help to find new targets for clinical treatment of human bladder cancer.

Keywords: Bladder cancer; CircRNA; FATP2; PMN-MDSCs; RIPK3.

Fig. 1

PMN-MDSCs infiltrating the tumor microenvironment with overexpression of FATP2 and low expression of RIPK3. (A) Detection of PMN-MDSCs (CD11b⁺LOX-1⁺) (yellow) in bladder cancer tissues and controls (adjacent tissues) by immunofluorescence (bar = 200 μm). (B and C) Immunohistochemical detection of RIPK3 and FATP2 in bladder cancer tissues and adjacent tissues (bar = 200 μm). (D and E) Correlation analysis of expression of RIPK3 or FATP2 and PMN-MDSCs infiltration in bladder tumor tissue. (F) Immunohistochemical detection of LOX-1, RIPK3 and FATP2 expression in human bladder cancer tissues (n = 18). (G) The expression of RIPK3 and FATP2 in bladder cancer patients with different clinical stages. (* P < 0.05)

Fig. 2

BCa-derived exosomes up-regulated FATP2 and down-regulated RIPK3 in PMN-MDSCs. (A) Morphological characteristics of BCa-derived exosomes revealed by transmission electron microscopy (bar = 100 nm). (B) Immunofluorescence showed that the BCa-derived exosomes (green) were gradually taken up by PMN-MDSCs. (C and D) Detection of the expression of RIPK3 and FATP2 in PMN-MDSCs using Western blotting. (E) Detection of Prostaglandin E2 (PGE2), Arachidonic Acid (AA), and Triglycerides (TG) in PMN-MDSCs by LC/MS. (F) ELISA detected the levels of immunosuppressive molecules (IL-10, INOS, Arg-1) in PMN-MDSCs. (*P < 0.05)

Fig. 3

Identifcation and characterization of circRNA_0013936. (A) The heatmap showed the differentially expressed circRNAs (> 4-fold) in BCa-exosomes and SV-HUC-1-exosomes. (B) Schematic diagram of the composition of circ_0013936. (C) qRT-PCR was used to detect the expression of circ_0013936 in exosomes derived from three bladder cancer cell lines (T24, UMUC-3 and BIU-87) and normal urothelial cell (SV-HUC-1). (D) Sanger sequencing and agarose gel electrophoresis were used to verify the back splicing sequence and RT-PCR product of circ_0013936. (E) Detection of circRNA_0013936 expression by real-time PCR in T24 and UMUC3 cells treated with or without RNase R. (F) FISH confirmed circRNA_ 0013936 is mainly located in the cytoplasm. (*P < 0.05)

Fig. 4

CircRNA_ 0013936 up-regulated FATP2 and down-regulated RIPK3 in PMN-MDSCs. (A and B) Western blotting detection of the expression of FATP2 and RIPK3 in PMN-MDSCs transfected with the cirRNA_0013936 mimics. (C) LC-MS detection of AA, PGE2, and TG in PMN-MDSCs. (D) ELISA detection of the immunosuppressive molecules (IL-10, INOS, Arg-1) in PMN-MDSCs. (* P < 0.05)

Fig. 5

CircRNA_ 0013936 functions as a sponge for miR-320a and miR-301b-3p. (A) Evaluation of the effects of 9 candidate miRNAs on the luciferase activity of circ_0013936 using luciferase reporter gene assay. (B and C) The RNA pull-down assay was performed in PMN-MDSCs using circ_0013936 and negative probe. (D) The basic expressions of miR-320a and miR-301b-3p in PMN-MDSCs vs. PMNs. (E) Schematic diagram of mutant (Mut) or wild-type (Wt) circ_0013936 luciferase reporter vectors carrying miR-320a or miR-301b-3p binding sites. (F and G) The relative expressions of miR-320a and miR-301b-3p detected by qRT-PCR in 293T cells transfected with mutant (Mut) or wild-type (Wt) circ_0013936. (* P < 0.05)

Fig. 6

JAK2 is the direct target gene for miR-320a, and CREB1 is the direct target gene for miR-301b-3p. (A and G) Venn diagram showing the predicted target genes of miR-320a and 301b-3p. (B and H) qRT-PCR detected changes in mRNA levels of predicted targets overexpressed or silenced by circ_0013936. (C and D) Relative luciferase protease activity measured by luciferase reporter gene assay in 293T cells transfected with JAK2 (Mut or Wt) and miR-320a mimics. (E and F) Detection of JAK2 expression in PMN-MDSCs transfected with miR-320a inhibitors or miR-320a mimics using Western blotting and qRT-PCR. (J and K) Relative luciferase activity measured by luciferase reporter gene assay in 293T cells transfected with CREB1 (Mut or Wt) and miR-301b-3p mimics. (L and M) Detection of CREB1 expression in PMN-MDSCs transfected with miR-301p-3p inhibitors or miR-301b-3p mimics using Western blotting and qRT-PCR (*P < 0.05)

Fig. 7

CircRNA_0013936 up-regulated FATP2 and down-regulated RIPK3 through the miR-320a/JAK2 and miR-301b-3p/CREB1 pathway in PMN-MDSCs. (A, B and C) The expressions of JAK2 and FATP2 in PMN-MDSCs after over-expressing or silencing circ_0013936 were detected by qRT-PCR and Western blotting. (D, E and F) The FATP2 expression in PMN-MDSCs after over-expressing or silencing of JAK2 were detected by qRT-PCR and Western blotting. (G) Detection of TG, AA, and PGE2 in PMN-MDSCs transfected with vector, lv-circ_0013936, mimic-miR-320a or mimic-nc using LC-MS. (H, I and J) Detection of CREB1 and RIPK3 expression in PMN-MDSCs after over-expressing or silencing circ_0013936 using Western blotting and qRT-PCR. (K, L and M) Detection of RIPK3 expression in PMN-MDSCs after silencing or over-expressing CREB1 using qRT-PCR and Western blotting. (N) Detection of immunosuppressive molecules (IL-10, INOS, Arg-1) in PMN-MDSCs transfected with vector, lv-circ_0013936, mimic-miR-301b-3p or mimic-nc using ELISA. (*P < 0.05)

Fig. 8

Exosomal circRNA_0013936 promotes suppressive immunity by up-regulating FATP2 through miR-320a/JAK2 pathway and down-regulating RIPK3 through miR-301b-3p/CREB1 pathway in PMN-MDSCs. (A and B) Detection of mRNA expression of JAK2, pSTAT5, and FATP2 in PMN-MDSCs co cultured with BCa derived exosomes or circRNA_0013936-KO-BCa derived exosomes using qRT-PCR and Western blotting. (C) Detection of TG, AA, and PGE2 in PMN-MDSCs using LC-MS. (D and E) Detection of mRNA expression of CREB1 and RIPK3 in PMN-MDSCs using qRT-PCR and Western blot. (F) Detection of immunosuppressive molecules (IL-10, INOS, Arg-1) in PMN-MDSCs using ELISA. (G) Flow cytometry was performed to evaluate the effect of PMN-MDSCs cocultured with BCa-derived exosomes or circRNA_0013936-KO-BCa-derived exosomes on the IFN-γ production of CD8⁺ T cells. (H) CFSE analysis was performed to evaluate the effect of PMN-MDSCs cocultured with BCa-derived exosomes or circRNA_0013936-KO-BCa-derived exosomes on the proliferation function of CD8⁺ T cells. (*P < 0.05)