Exosome-derived circ-001422 promotes tumor-associated macrophage M2 polarization to accelerate the progression of glioma

Abstract

Cytokines, tumor cells, and tumor-associated macrophages play crucial roles in the composition of glioma tissue. Studies have demonstrated that certain cytokines can induce M2 polarization of tumor-associated macrophages and contribute to the progression of glioma. Nonetheless, the intricate molecular interactions among cytokines, glioma cells, and tumor-associated macrophages remain largely unexplored. To investigate this cross-talk, a combination of RNA-sequencing, chromatin immunoprecipitation, immunoprecipitation, exosome isolation, and biological experiments were employed. Treatment with IL-6 significantly increased circ-001422 expression in glioma cells. A poorer prognosis was associated with elevated levels of circ-001422 in glioma tissues. Circ-001422 was transcribed directly by STAT3 through binding to its promoter. Circ-001422 exerted cancer-promoting functions when co-cultured with M2 macrophages. Furthermore, glioma cells were found to transfer circ-001422 to macrophages via an exosomal pathway, promoting M2 polarization. Mechanically, circ-001422 interacted with p300, resulting in STAT3 acetylation, thus promoting nuclear localization and transcriptional activity of STAT3/NF-κB and M2 macrophage polarization. In conclusion, glioma cells released exosomes enriched with circ-001422, which in turn induce M2 macrophage polarization by activating the STAT3/NF-κB pathway, thereby enhancing the aggressive characteristics of glioma cells. Targeting circ-001422 may represent a potential therapeutic approach for glioma.

Fig. 1. Circ-001422 was up-regulated in glioma cells treatment with IL-6 and glioma tissues.

A RNA-sequencing was performed to detect the differentially expressed circRNAs induced by IL-6 in U87 cells. B qRT-PCR was performed to detect the expression of differentially expressed circRNAs in U87 cells after IL-6 treatment. Data were expressed as mean ± SD for triplicate experiments. C qRT-PCR experiments were used to detect the expression of circ-001422 in 47 pair glioma tissues and adjacent normal tissue. D qRT-PCR was used to detect the expression of circ-001422 in NHA, T98G, LN229, LN18, U251, U87 and SHG-44 cell. Data were expressed as mean ± SD for triplicate experiments. E, F ISH was performed to detect the expression of circ-001422 in 47 pair glioma tissues and adjacent normal tissue. G KM-plot was used to analyze the survival difference between glioma patients with high and low circ-001422 expression. H, I Circ-001422 was mostly located in nuclear of glioma cells. *P < 0.05; **P < 0.01. Data were expressed as mean ± SD for triplicate experiments.

Fig. 2. STAT3 transcriptionally increased circ-001422 in glioma cells.

A Three online tool including PROMO, ChipBase and Human TFDB was used to analyzed the transcription factor of WHSC1 (host gene of circ-001422). B Chip-qPCR was used to analyzed the binding between potential transcription factor and WHSC1. Data were expressed as mean ± SD for triplicate experiments. C Motif of STAT3 was obtained from JASPAR. D Potential binding sites between STAT3 and WHSC1 were predicted by JASPAR. E Mutation of binding site 4 significantly reduced the binding with STAT3 and WHSC1. Data were expressed as mean ± SD for triplicate experiments. F Chip-qPCR indicated that STAT3 significantly bind with the binding site 4 of WHSC1. Data were expressed as mean ± SD for triplicate experiments. G Design specific primers for amplifying pre-mRNA of WHSC1, WHSC1 mRNA and circ-001422. H STAT3 elevated the levels of pre-mRNA of WHSC1, while knockdown of STAT3 reduced the levels. Data were expressed as mean ± SD for triplicate experiments. I STAT3 reduced the levels of mRNA of WHSC1, while knockdown of STAT3 increased the mRNA levels of WHSC1. Data were expressed as mean ± SD for triplicate experiments. J STAT3 elevated the levels of circ-001422, while knockdown of STAT3 reduced the levels. Data were expressed as mean ± SD for triplicate experiments. *P < 0.05; **P < 0.01.

Fig. 3. Circ-001422 promotes proliferation, invasion and metastasis ability of glioma cells under co-culture with macrophages.

A Photographs of the subcutaneous xenografts derived from U87 cells transfected as indicated and mixed with THP-1 cells (n = 5/group). B, C Tumor weight and volume of tissues derived from U87 cells transfected as indicated and mixed with THP-1 cells (n = 5/group). D In situ model indicated the xenografts derived from U87 cells transfected as indicated and mixed with THP-1 cells (n = 5/group). E EDU assays indicated that overexpression of circ-001422 increased cell proliferation under macrophages co-culture, while knockdown of circ-001422 induced the opposite effects. Data were expressed as mean ± SD for triplicate experiments. F Transwell assays indicated that overexpression of circ-001422 increased cell invasion under macrophages co-culture, while knockdown of circ-001422 induced the opposite effects. Data were expressed as mean ± SD for triplicate experiments. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 4. Circ-001422 promotes macrophages M2 polarization in co-culturing with glioma cells.

A Overexpression of circ-001422 in glioma cells increased the expression of CD206 in macrophages under co-culture condition, while knockdown of circ-001422 induced the opposite effects. Data were expressed as mean ± SD for triplicate experiments. B qRT-PCR results indicated that overexpression of circ-001422 in glioma cells increased the levels of M2 biomarkers including IL-10, ARG1 and TGFB1, and reduced the levels of M1 biomarkers including IL2A, TNF and NOS2; knockdown of circ-001422 induced the contrary effects. C Western blotting results indicated that overexpression of circ-001422 in glioma cells increased the levels of M2 biomarkers including IL-10, ARG1 and TGFB1; knockdown of circ-001422 induced the contrary effects. D The expression of CD68, CD206, CD163, KI67 and PCNA in subcutaneous xenografts derived from indicated U87 cells (circ-001422-overexpression, circ-001422 knockdown, and NC) mixed with macrophages. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 5. Exosome-mediated transfer of circ-001422 promoted macrophage M2 polarization and stimulates GBM cell proliferation and metastasis.

A Electron microscope observation of exosomes from glioma cells. B Electron microscope observation for the exosomes from glioma cells. C Western blotting was used to detect the biomarkers of exosomes from glioma cells including HSP70, CD81, CD9 and TSG101. D qRT-PCR was used to detect the expression of circ-001422 in the cell culture medium with or without exosomes. Data were expressed as mean ± SD for triplicate experiments. E qRT-PCR was used to detect the levels of circ-001422 in macrophage-like THP-1 after treatment with exosomes from NC LN229, U87 cells with circ-001422 overexpression. F Immunofluorescence to detect the internalization of glioma cell derived exosomes in THP-1 cells. G qRT-PCR was used to detect the levels of M1 biomarkers and M2 biomarkers in the THP-1 cells after treatment with exosomes from glioma cells with circ-001422 overexpression and NC cells. H Expression of CD206 was detected in the THP-1 cells treatment with exosomes from glioma cells with circ-001422 overexpression and NC cells. Data were expressed as mean ± SD for triplicate experiments. I Colony formation assay was used to perform detect the colony formation ability of glioma cells treatment with exosomes from glioma cells with circ-001422 overexpression and NC cells under THP-1 co-culture. Data were expressed as mean ± SD for triplicate experiments. J EDU assay was used to perform detect the proliferation ability of glioma cells treatment with exosomes from glioma cells with circ-001422 overexpression and NC cells under THP-1 co-culture. Data were expressed as mean ± SD for triplicate experiments. K Transwell assay was used to detect the invasion ability of glioma cells treatment with exosomes from glioma cells with circ-001422 overexpression and NC cells under THP-1 co-culture. Data were expressed as mean ± SD for triplicate experiments. *P < 0.05; **P < 0.01.

Fig. 6. Circ-001422 interacts with p300 and STAT3 and activates STAT3/NF-κB signaling.

A Protein profile analysis of circ-001422 binding protein, especially for the p300 and STAT3 in the THP-1 cells. B RIP assays indicated that p300 and STAT3 bind with circ-001422. C Circ-001422 was co-located with p300 and STAT3 in nuclear of THP-1 cells. Data were expressed as mean ± SD for triplicate experiments. D Western blot and immunoprecipitation experiments demonstrate that circ-001422 overexpression in THP-1 cells enhances p300 and STAT3 acetylation, leading to STAT3/NF- κB nuclear translocation. Knockdown of circ-001422 in THP-1 cells induced the opposite effects. E Immunofluorescence analysis shows reduced expression of circ-001422 decreased the co-localization of STAT3, and p300 in macrophage like THP-1 cells. F Luciferase reporter assay confirmed the impact of circ-001422 on the transcriptional activity of STAT3 and NF-κB in the THP-1 cells. Data were expressed as mean ± SD for triplicate experiments. *P < 0.05; **P < 0.01.

Fig. 7. The activate of STAT3/NF-κB signaling induced by circ-001422 containing exosomes is STAT3-K685 acetylation dependent.

A Western blotting experiments demonstrated that circ-001422 activated STAT3/NF-κB-dependent STAT3 acetylation modification. B Western blotting indicated that exosomal circ-001422 was shown to activate STAT3/NF-κB-dependent STAT3 acetylation. C Immunofluorescence assays revealed that exosome-derived circ-001422 modulated STAT3 through acetylation and nuclear translocation of NF-κB. D Double fluorescein reporter experiments confirmed that exosome-derived circ-001422 regulated the transcriptional activity of STAT3 and NF-κB via STAT3 acetylation. Data were expressed as mean ± SD for triplicate experiments. E Flow cytometry results indicated that exosome-derived circ-001422 increased the positive rate of CD206 in THP-1 cells via STAT3 acetylation. Data were expressed as mean ± SD for triplicate experiments. F qRT-PCR results indicated that exosome-derived circ-001422 increased M2 biomarkers and reduced M1 biomarkers in THP-1 cells via STAT3 acetylation. *P < 0.05; **P < 0.01. (1) NC. (2) sh-#1 U87 Exo; (3) sh-#1 U87 Exo + STAT3-K685R; (4) sh-#1 U87 Exo + STAT3-K685Q; (5) sh-#1 U87 Exo + STAT3-WT.