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

doi:10.21203/rs.3.rs-4616289/v1

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

https://doi.org/10.21203/rs.3.rs-4616289/v1

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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. Co-culturing macrophages with glioma cells knockdown of circ-001422 significantly reduced cell proliferation and invasion. 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.

Biological sciences/Cancer/CNS cancer

Biological sciences/Molecular biology/Transcription

Biological sciences/Genetics/Gene expression

Glioma, a primary cranial malignant tumor originating from glial cells, exhibits a global incidence rate of 4.6 to 5.7 per 100,000 individuals, with a notable increase in recent years (1). This disease is characterized by a high mortality rate coupled with a low cure rate. The initial treatment options for glioma encompass surgical intervention, radiotherapy, and chemotherapy (2, 3). However, the median survival rate remains unsatisfactory. Therefore, exploring biomarkers of glioma progression may contribute to the therapy of glioma.

The tumor microenvironment (TME) is comprised of cytokines, growth factors, and various tumor-induced immune cells that contribute significantly to tumor progression (4). Tumor-associated macrophages (TAMs) have the ability to differentiate into two distinct phenotypes, M1 and M2, in response to environmental cues and their activation status. M1 macrophages typically release pro-inflammatory cytokines to combat tumor cells, whereas M2 macrophages tend to produce elevated levels of anti-inflammatory cytokines that support tumor cell advancement (5). Elevated levels of M2 macrophages were detected in glioma tissues as opposed to neighboring non-tumor tissues, with the potential of M2 macrophages to enhance glioma cell proliferation, metastasis, and resistance to drugs (6, 7). Nevertheless, the precise molecular mechanisms underlying the upregulation of M2 macrophages in glioma tissues remain largely unexplored.

Circular RNAs, a novel category of endogenous non-coding RNAs, possess closed circular structures unlike linear RNAs (8, 9). Research has indicated that dysregulated expression of circRNAs is evident in various types of cancer, including glioma (10). For instance, Yan et al. observed elevated levels of circRNA-104718 in glioma tissues compared to adjacent tissues, with patients exhibiting high levels of circRNA-10718 showing a decreased overall survival rate (11). Additionally, Chen et al. demonstrated that circPTN promotes proliferation and stemness in glioma by acting as a sponge for miR-145-5p/miR-330-5p (12). Our prior research demonstrated that circ-001422 is situated on chromosome 4, spans 1703 bp, and is produced through the circularization of exons 2–7 of the NSD2 gene (also known as WHSC1). Circ-001422 exhibited the potential to enhance the advancement and metastasis of osteosarcoma cells (13). Nevertheless, the function of circ-001422 in glioma remained unexplored.

In the present investigation, it was demonstrated that circ-001422 exhibited up-regulation in glioma cells treated with IL-6 and in glioma tissues. Moreover, heightened levels of circ-001422 were correlated with unfavorable prognosis. Glioma cells were found to release circ-001422 to macrophages through the exosomal pathway, subsequently inducing M2 polarization of macrophages by interacting with STAT3 and p300 and activating the NF-κB pathway. This process ultimately enhanced the macrophages' capacity to facilitate the proliferation and metastasis of glioma cells. Thus, circ-001422 may serve as a crucial mediator in the interaction between glioma cells and macrophages, and targeting circ-001422 may contribute the glioma therapy.

IL-6 induced the up-regulation of circ-001422 in glioma cells

IL-6 is a key cytokine in the microenvironment of glioma tissues, which exhibited significant effects on glioma cell progression (14). Therefore, we used IL-6 to treat glioma cell U87, and analyzed the change of circRNAs. Through differentially expressed analysis, total 6 circRNAs (circ-0114230, circ-0009581, circ-0002983, circ-0009677, circ-0006955, and circ-001422) was found to increased significantly in cells treated with IL-6 (Fig. 1A). Among them, through qRT-PCR experiments, we found that circ-001422 was elevated most significantly (Fig. 1B). Therefore, we focused on circ-001422.

Through performed qRT-PCR (Fig. 1C) and ISH (Fig. 1E-F) in 48 pair glioma tissues and adjacent tissues, higher levels of circ-001422 was observed in glioma tissues in comparison to adjacent non-tumor tissues. Similarly, elevated levels of circ-001422 were found in glioma cells (T98G, LN229, LN18, U251, U87 and SHG-44) in comparison to normal human astrocyte NHA cells (Fig. 1D). Moreover, we found that patient with high levels of circ-001422 had lower overall survival rate in comparison to those with lower levels of circ-001422 (Fig. 1G). Furthermore, circ-001422 was found to locate in cytoplasm mostly (Fig. 1H-I). These evidences indicated that IL-6 can induce the up-regulation of circ-001422 in glioma cells, which was elevated in glioma tissues and predicted poor prognosis.

STAT3 transcriptionally upregulates circ-001422 expression in glioma cells

Previous studies indicated that circ-001422 is generated by circularization of exons 2–7 of the host gene WHSC1 (13). Therefore, to determine the mechanism of circ-001422 up-regulation under IL-6, we predicted potential transcription factors via performing bioinformatics analysis in PROMO, ChipBase and Human TFDB online database. A total of 7 transcription factors was predicted to have potential to bind to the promoter of WHSC1, including YY1, IRF1, STAT3, SPI1, ELF1, E2F1 and ETS1 (Fig. 2A). Through performing chip-PCR in LN229 and U87, we found that using STAT3 antibody significantly enriched the beads of WHSC1 (Fig. 2B).

As previous studies indicated that IL6 can activate STAT3 to induce the increasing of the transcription of targets genes, we considered that whether host gene WHSC1 is a target of STAT3. Through obtaining motif of STAT3 in JASPAR (Fig. 2C), a total of 4 potential binding sites for STAT3 in the promoter of WHSC1 was found (Fig. 2D). In order to analyze the specific binding sites in the promoter of WHSC1 to STAT3, fluorescein reporter plasmids containing wildtype WHSC1 promoter sequence and those containing promoter sequence with binding site mutation were constructed. Results indicated that only binding site 4 mutation can block the elevation of fluorescence intensity induced by STAT3 overexpression in LN229 and U87 cells (Fig. 2E). Similarly, through performing chip-PCR experiments, we found that STAT3 antibody significantly enriched the sequence of binding site 4 of WHSC1 promoter, especially in the cells with STAT3 overexpression (Fig. 2F).

Since circ-001422 and host gene WHSC1 promoter are common, in order to further explore whether STAT3 promoted the transcription of circ-001422 or host gene WHSC1, we designed amplification primers specifically targeting WHSC1 pre-mRNA, WHSC1 mRNA and circ-001422, respectively (Fig. 2G). It is interesting to found that overexpression of STAT3 in LN229 and U87 cells significantly elevated the levels of WHSC1 pre-mRNA (Fig. 2H) and circ-001422 (Fig. 2J), while the mRNA levels of WHSC1 were reduced (Fig. 2I). Knockdown of STAT3 induced the reversed effects. These evidences may indicate that STAT3 transcriptionally upregulates circ-001422 expression in glioma cells.

Circ-001422 promoted the proliferation and mobility of glioma cells while co-culturing with macrophage-like THP-1.

To determine the biological functions of circ-001422, subcutaneous tumor formation experiments in nude mice were performed to detect the proliferation of circ-001422 overexpression, circ-001422 knockdown and NC cells which co-culturing with macrophage-like THP-1. Results indicated that tissues derived with circ-001422 overexpressed group exhibited the higher tumor volume (Fig. 3A and Fig. 3C) and weight (Fig. 3B), while those derived with circ-001422 knockdown group exhibited lower tumor volume (Fig. 3A and Fig. 3C) and weight (Fig. 3C). Similarly, in situ model indicated that, under suspend with macrophage-like THP-1, LN229 cells with circ-001422 overexpression exhibited faster growth rate in comparison to NC cells (Fig. 3D), while knockdown of circ-001422 induced the oppositive effects (Fig. 3D).

Moreover, we found that circ-001422 overexpressed LN229 and U87 cells culturing with macrophage-like THP-1 exhibited elevated EDU positive rate, while circ-001422 knockdown cells culturing with macrophage-like THP-1 had lower EDU positive rate (Fig. 3E). Furthermore, via performing transwell assay, we found that, under co-culturing with macrophage-like THP-1, elevated invasion ability was observed in circ-001422 overexpressed cells, while cells with circ-001422 knockdown exhibited lower invasion ability (Fig. 3F). These results indicated that circ-001422 promoted the proliferation and mobility of glioma cells while co-culturing with macrophage-like THP-1.

Circ-001422 promoted the M2 polarization in the co-culture condition of macrophage-like THP-1 and glioma cells

We then analyzed the effects of circ-001422 in the cross-talk between glioma cells and macrophages. Interesting, macrophages co-culturing with LN229 and U87 cells with circ-001422 overexpression exhibited higher CD206 rate, while those co-cultured with glioma cells with circ-001422 knockdown had lower CD206 rate (Fig. 4A). These results indicated that circ-001422 in glioma cells may had potential to promote macrophage-like THP-1 into M2 macrophage.

To further verify this guess, we then determined the expression of M1 biomarker and M2 biomarker in macrophages co-culturing with glioma cells. Through performing qRT-PCR, higher mRNA levels of M2 biomarkers including IL10, ARG1 and TGFB1 were observed in the macrophages co-culturing with glioma cells contained circ-001422 overexpression, while the mRNA levels of M1 biomarkers including IL2A, TNF and NOS2 were reduced (Fig. 4B). Conversely, the macrophages co-culturing with glioma cells contained circ-001422 knockdown exhibited higher M1 biomarkers levels and lower M2 biomarkers (Fig. 4B). Similarly, using western blotting, we found that macrophages co-culturing with glioma cells contained circ-001422 overexpression exhibited higher protein levels of M2 biomarkers (IL10, ARG1 and TGFB1), while those co-culturing with glioma cells contained circ-001422 knockdown reduced the expression of these protein levels (Fig. 4C).

Furthermore, IHC was performed in the tumor tissues from subcutaneous tumor formation experiments, higher levels of M2 biomarker (CD68, CD163 and CD206) and proliferation biomarkers (PCNA and KI67) were observed in the tumor tissues derived from glioma cells contained circ-001422 overexpression co-culturing with macrophage-like THP-1 (Fig. 4D). Conversely, tumor tissues derived from glioma cells contained circ-001422 knockdown co-culturing with macrophage-like THP-1 exhibited lower levels of M2 biomarkers and proliferation biomarkers (Fig. 4D). Taken together, these evidences indicated that circ-001422 promoted the M2 polarization in the co-culture condition of macrophage-like THP-1 and glioma cells.

Exosome-mediated transfer of circ-001422 promoted macrophage M2 polarization and stimulates PC cell proliferation and metastasis

To analyzed how circ-001422 affect the macrophage M2 polarization, exosomes secreted from LN229 and U87 cells were extracted (Fig. 5A-B). Interesting, western blotting results indicated that circ-001422 overexpression increased the expression of exosome protein markers including HSP70, CD81, CD9 and TSG101 (Fig. 5C). In addition, we found the extracellular levels of circ-001422 were reduced in medium after removing exosomes (Fig. 5D). Macrophage like-THP-1 was treated with exosomes extracted from medium of NC cells and circ-001422 cells. qRT-PCR results indicated that circ-001422 levels were significantly elevated in macrophage like-THP-1 treatment with exomes from circ-001422 overexpressed cells, but no in those treated with exomes from NC cells (Fig. 5E). Incubation of macrophage like-THP-1 with GBM exosomes was performed after they had been isolated and labeled with membrane phospholipid dye. Increased green fluorescence signaling was observed in macrophage like-THP-1 indicating that GBM cell-derived exosomes were taken up by macrophage like-THP-1 (Fig. 5F).

qRT-PCR results indicated that levels of M2 biomarkers including IL-10, ARG1 and TGFB1 were increased in macrophage like-THP-1 treatment with exosomes from circ-001422 overexpressed cells, while M1 biomarkers including IL-10, ARG1 and TGFB1 were all reduced (Fig. 5G). Similarly, flow cytometry results indicated that the M2 biomarker CD206 was elevated in macrophage like-THP-1 treatment with exosomes from circ-001422 overexpressed cells (Fig. 5H). Furthermore, through constructing a co-culture condition, we found that macrophage like-THP-1 treatment with exosomes from circ-001422 overexpressed cells had significant potential to promote the proliferation (Fig. 5J), colony formation (Fig. 5I) and invasion (Fig. 5K) of glioma cells, while those treatment with exosomes from NC cells had no significant effects. These evidences indicated that exosome-mediated transfer of circ-001422 can promote macrophage M2 polarization and stimulate PC cell proliferation and metastasis.

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

To analyze the molecular mechanisms of circ-001422 involved in the macrophage M2 polarization, RNA pulldown assays were performed. Through performing mass spectrometry analysis and verification, we found that circ-001422 significantly bound with STAT3 and p300 (Fig. 6A). Similarly, results of RIP experiment using anti-p300 and anti-STAT3 antibodies indicated that both p300 and STAT3 significantly enriched the circ-001422 (Fig. 6B). Moreover, we found that circ-001422 was mostly co-located with STAT3 and p300 in macrophage like-THP-1 (Fig. 6C). Western blotting results indicated that overexpression of circ-001422 in macrophage like-THP-1 elevated acetylated levels of STAT3 in lysine 685 site (Ace-STAT3-K685), phosphorylated levels of STAT3 at tyrosine 705 site (p-STAT3-Y705) and phosphorylated levels of NF-κB p65 at serine 536 site (p-NF-κB-p65-S536) (Fig. 6D). These cells also increased the expression of STAT3 and NF-κB p65 in nucleus (Fig. 6D). Reduced the expression of circ-001422 induced the opposite effects in macrophage like-THP-1 (Fig. 6D). Furthermore, we found that circ-001422 knockdown also inhibited the re-localization of STAT3 and NF-κB p65 in nucleus (Fig. 6E-F). These evidences indicated that circ-001422 promotes the binding between STAT3 and p300, and activates the STAT3/ NF-κB pathway in macrophage like-THP-1.

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

Previous studies suggested that activated STAT3 recruits p300 to the promoters of STAT3-targeted genes, thereby facilitating transcription through STAT3-K685 acetylation (15). Therefore, to investigate whether STAT3-K685 acetylation was necessary for circ-001422-containing exosome-mediated activation of STAT3/ NF-κB pathway, wildtype STAT3 plasmids and plasmids with K685 activate mutation (K685Q) and inactivate mutation (K685R) was constructed. Western blotting experiments indicated that ace-STAT3-K685, p-STAT3-Y705 and p-NF-κB-p65-S536 was significantly reduced in macrophage like-THP-1 after circ-001422 knockdown, while expression of STAT3 and NF-κB-p65 in nuclear was also reduced (Fig. 7A).Transfection of STAT3 plasmids in macrophage like-THP-1 had significant reversed effects (Fig. 7A). Interesting, we found that reversed effects of STAT3-K685 plasmids were significantly stronger than wildtype STAT3 plasmids and STAT3-K685R plasmids (Fig. 7A). Similarly, lower levels of ace-STAT3-K685, p-STAT3-Y705 and p-NF-κB-p65-S536 were observed in macrophage like-THP-1 cells treatment with exosomes from circ-001422 knockdown glioma cells compared with those treatment with exosomes from NC glioma cells, as well as lower nuclear location of STAT3 and NF-κB-p65 (Fig. 7B). Transfection of STAT3 plasmids in macrophage like-THP-1 cells treatment with exosomes from circ-001422 knockdown glioma cells significantly increased the levels of ace-STAT3-K685, p-STAT3-Y705 and p-NF-κB-p65-S536, and nuclear location of STAT3 and NF-κB-p65 (Fig. 7B). Among them, STAT3-K685Q plasmids exhibited the strongest effects (Fig. 7B).

Moreover, immunofluorescence results indicated that macrophage like-THP-1 cells treatment with exosomes from circ-001422 knockdown glioma cells exhibited lower co-location of STAT3 and NF-κB-p65 in nuclear, overexpression of STAT3 plasmids, especially STAT3-K685Q plasmids, significantly increased the co-location of STAT3 and NF-κB-p65 in nuclear in those cells (Fig. 7C). In addition, lower STAT3 and NF-κB-p65 transcriptional activity was observed in macrophage like-THP-1 cells treating with circ-001422 low exosomes, while this effect was reversed by STAT3-K685Q plasmids compared with STAT3-K685R or STAT3-WT plasmids (Fig. 7D). Furthermore, after detecting the phenotype of macrophages, in contrast to STAT3-K685R or STAT3-WT plasmids, exosomes with low expression of circ-001422 had significant effects on macrophage M2 polarization, and this effect could be reversed by STAT3-K685Q plasmids (Fig. 7E-F). Taken together, The activate of STAT3/NF-κB signaling induced by circ-001422 containing exosomes is STAT3-K685 acetylation dependent.

The pathogenesis of glioma involves an intricate series of steps, encompassing cellular neoplastic transformation, resistance to programmed cell death, self-sustaining growth signaling, development of a vascular network, evasion of immune detection, and acquisition of invasive and metastatic capabilities (16, 17). The presence of soluble factors in the tumor microenvironment, originating from neoplastic cells, stromal cells, immune cells, plays a crucial role in regulating various aspects of these neoplastic processes (18). There were strong evidences that soluble factors, specifically chemokines, regulate tumorigenesis of glioma (19). One of these, IL-6 has been extensively researched in the context of glioma and has been shown to play a role in the proliferation, metastasis, and immune evasion of glioma cells (20, 21). Investigating the molecular mechanisms of IL-6 may offer insights into potential therapeutic strategies for glioma.

The findings in this study indicate that IL-6 upregulates the expression of circ-001422 in glioma cells. Circ-001422 is highly expressed in glioma tissues and is associated with poor patient outcomes. In addition, overexpression of circ-001422 significantly facilitated glioma cell progression and metastasis in a co-culture setting with macrophages like THP-1. These evidences indicated that circ-001422 was a key mediator link to the cross talk between macrophages and glioma cells.

It is believed that exosomes may contribute to the pathogenesis of many cancers because they are small extracellular membrane vesicles with a diameter between 30 and 150 nm (22). These vesicles are rich in proteins, double-stranded DNA, single-stranded DNA, messenger RNAs, and microRNAs, which are transported to specific target cells where they play a role in reshaping the tumor microenvironment (23, 24). Previous research has suggested that non-coding RNAs within exosomes serve as crucial mediators between different cells in the tumor microenvironment (25). For instance, Tian et al. illustrated that the glioma-derived exosomal long non-coding RNA Agap2-As1 facilitates glioma progression by modulating the secretion of TGF-β1 by myeloid-derived suppressor cells (26). Li et al. indicated that TAM-derived exosomal LINC01232 promoted the immune escape of glioma cells (27). In the present study, we exhibited that circ-001422 was exosome-enriched non-coding RNA. Glioma cells were found to release circ-001422 to macrophages through the exosomal pathway, subsequently inducing M2 polarization of macrophages.

In order for M1/M2 phenotypes and the transition between these polarized macrophage states to remain balanced, a complex network of receptors and signaling pathways must be activated. This pathway modulates macrophage behavior in cancer in a manner that balances immunosuppressive, tumor-promoting activity with pro-inflammatory, protective functions undertaken by tumor-associated macrophages (28, 29). A previous report showed that activating NF-κB promotes tumor growth by regulating M2 polarization genes (30). Numerous factors within tumors can modulate the activation of NF-κB, consequently impacting M2 polarization (31, 32). A previous investigation demonstrated that acetyltransferase p300 facilitated the acetylation of STAT3, thereby enhancing its activation (33). A disruption of the STAT3 acetylation site or suppression of histone deacetylase activity prevents it from dimerizing and controlling transcription (34, 35). Our current research revealed that circ-001422 interacts with both p300 and STAT3. Upregulation of circ-001422 augmented the interaction between p300 and STAT3, as well as increasing the acetylation of STAT3.

In conclusion, increased levels of circ-001422 in glioma cells and tissues were linked to poor prognosis. Glioma cells released circ-001422 to macrophages via exosomes, promoting M2 polarization by interacting with STAT3 and p300 and activating the NF-κB pathway, thus enhancing their ability to support glioma cell growth and metastasis. Circ-001422 maybe a key target for blocking the cross-talk between macrophages and glioma cells, and contribute to the therapy of glioma.

Clinical samples

A cohort of 48 pairs of human glioma tissues and adjacent non-tumor tissue samples was procured from the Pathology department at the Affiliated Hospital of Guizhou Medical University in Guiyang, China. The glioma diagnosis of all cancer tissue specimens was validated through postoperative pathological examination, and clinicopathological data was collected at the time of sample acquisition. Approval for all experiments involving human specimens was obtained from the Human Ethics Review Committee of the Affiliated Hospital of Guizhou Medical University.

qRT-PCR assays

Total RNA was extracted using TRIzol reagent (Thermo Fisher Scientific, USA) and subjected to cDNA synthesis with a Quantscript RT Kit (Thermo Fisher Scientific, USA). Transcript quantification was performed with a SYBR RT-PCR kit (Thermo Fisher Scientific, USA) and specific primers, utilizing the 2^−ΔΔCT method for expression level analysis with β-actin as the internal control. The primers employed in this study are detailed in Supplementary Table S1 of the supplementary data.

Western blotting

An analysis of the concentration of protein extracted from glioma cells using RIPA reagent containing 5% PMSF protease inhibitor was performed using the BCA method. Subsequently, 30µg of proteins per line were loaded and separated by 10% SDS-PAGE for 120 minutes. The proteins were then transferred onto PVDF membranes with a pore diameter of 0.45µm (Millipore, USA), blocked in 5% BSA for 30 minutes, and incubated with primary antibodies including TGF-β1 (1:1000, Cat No : 21898-1-AP, Proteintech), ARG1 (1:5000, Cat no. 16001-1-AP, Proteintech), IL-10 (1:2000, Cat no. 60269-1-Ig), HSP70 (1:5000, Cat no. 10995-1-AP, Proteintech), CD81 (1:1000, Cat no. 66866-1-Ig, Proteintech), CD9 (1:1000, Cat no : 20597-1-AP, Proteintech), TSG101 (1:2000, Cat no. 28283-1-AP, Proteintech), ace-STAT3 (1:1000, Cat no. #2523, CST), p-STAT3 (Y705) (1:2000, Cat no. #9145, CST), STAT3 (1:2000, Cat No : 10253-2-AP, Proteintech), NF-κB-p65 (1:5000, Cat no. 80979-1-RR, Proteintech), p300 (1:500, Cat no. 20695-1-AP, Proteintech), NF-κB-p65 (S536) (1:1000, Cat no. #3033, CST), and GAPDH (1:50000, Cat no. 60004-1-Ig, Proteintech) for 16 h at 4℃. A high sensitivity ECL reagent was used in the MultiImager to visualize the blots, and Image J software was used to quantify the relative protein expression.

RNA in situ hybridization (ISH)

ISH probes were inserted into glioma tissues after fixing and treating with pepsin, followed by hybridization and wash steps before digoxin antibodies were injected. The Aperio ImageScope system was used to capture ISH images after DAB was applied to detect signals.

Immunohistochemistry (IHC) experiments

The tissue sections underwent dewaxing, rehydration, and incubation in a repair solution at 96˚C. Following cooling, the sections were subjected to inactivation of endogenous enzymes and blocked with a 10% goat serum solution. Subsequently, primary antibodies including CD68 (1:1000, Cat no. 28058-1-AP, Proteintech), CD206 (1:10000, Cat no. 28058-1-AP, Proteintech), CD163 (1:1000, cat no, 16646-1-AP, Proteintech), KI67 (1:2000, Cat no. 16646-1-AP, Proteintech), PCNA (1:3000, 10205-2-AP, Proteintech) were applied and incubated overnight at 4˚C, followed by the addition of secondary antibodies. The sections were stained with 3,3'-diaminobenzidine (DAB) and hematoxylin, dehydrated, and sealed with gum before being examined under a microscope.

Cell culture and transfection

Human normal glial cells (NHA), monocyte THP-1, and glioma cell lines (T98G, LN229, LN18, U251, U87, and SHG-44) were procured from ATCC (USA) and cultured in DMEM (Invitrogen, USA) and 10% FBS at 37°C with 5% CO₂. After cloning STAT3 cDNA into pCDNA3 vectors, site-directed mutagenesis was used to create plasmids containing STAT3 (K685Q) and STAT (K685R). GeneChem (Shanghai, China) verified the authenticity of all plasmids. Circum-001422 lentiviral plasmid was acquired from GeneChem (Shanghai, China) and transfected using Polybrene (Thermo Fisher, USA) according to the manufacturer's instructions.

Nuclear and cytoplasmic separation

Following the manufacturer's instructions, nuclear and cytoplasmic RNAs were isolated using the PARISTM Kit (Thermo Fisher Scientific, USA). Centrifugation at 4 ºC for 5 minutes was used to separate the nuclear and cytoplasmic RNA fractions after cell lysis. Afterward, the supernatant, which contains cytoplasmic RNA, was transferred to an RNase-free test tube, and the pellet, which contains nuclear RNA, was lysed in 500 mL of cell destruction buffer. Following separation of nuclear and cytoplasmic RNA fractions, washing, eluting, and storing at -80 ºC, the RNA fractions were passed through a filter cartridge.

Fluorescence in situ hybridization (FISH)

An RNA-FISH experiment was performed to determine the subcellular distribution of circ-001422 in glioma cells. FITC-labeled probes for circ-001422 were synthesized by RiboBio (Guangzhou, China). A Fluorescence In Situ Hybridization Kit (RiboBio, China) was used to produce fluorescent signals, and an Olympus FV300 confocal laser scanning microscope (Japan) was used to capture cellular images.

Chromatin immunoprecipitation (ChIP)

A 1% formaldehyde solution was administered to cells for 10 minutes, followed by two washes with ice-cold PBS, then the cells were harvested and centrifuged. A protease inhibitor complex was injected into the cells, and they were sonicated. After this, either antibody or control IgG was added and the cells were incubated overnight at 4 ºC. PCR was used to analyze the purified DNA fragments resulting from the antibody-DNA complex.

Luciferase Assay

To confirm the binding of STAT3 to WHSC1, a dual luciferase reporter assay was conducted utilizing the online database JASPAR. Both wild-type and mutated WHSC1 promoter sequences were cloned into the psi-basic luciferase reporter vector (Promega, USA). Following cell seeding and overnight incubation, U87 and LN229 cells were placed in a 24-well plate. Luciferase reporter vectors, containing either the wildtype WHSC1 promoter sequence or a mutated sequence, were then introduced. These vectors were co-transfected with NC or STAT3 plasmids into glioma cells using Lipo2000. The luciferase activity of the cells was evaluated 24 hours after transfection.

Macrophage induction

Monocyte-like THP-1 cells were cultured in six-well plates at a density of 1 × 106 cells and incubated in a serum-free high-glycemic medium containing 100 ng/ml of phorbol 12-myristate 13-acetate (PMA, MCE) and 0.3% BSA for 72 hours to induce differentiation into macrophage-like THP-1 cells.

Flow cytometry

Macrophage-like THP-1 cells were cultured in 6-well plates at a density of 10^6 cells per well in DMEM medium supplemented with 5% FBS and specific stimuli. After a 48-hour incubation period, the macrophages were detached using Accutase (Sigma-Aldrich), washed twice with PBS, and incubated with a blocking buffer composed of 50 µl of PBS containing 2% FBS and 0.02% NaN3 for 30 minutes on ice. Subsequently, the cells were treated with a fluorescently conjugated anti-human CD206 antibody for 20 minutes on ice, followed by rinsing with a washing buffer and fixation with 1% paraformaldehyde. Flow cytometric analysis was then conducted using a BD FACSCalibur instrument and data analysis was performed in FlowJo software.

5-ethynyl-2’-deoxyuridine (EDU) Assay

The EDU assay was performed using a BeyoClick™ EdU-488 Proliferation Detection Kit (Beyotime, Suzhou, China). Glioma cell lines U87 and LN229 were cultured in 6-well plates, allowed to adhere, and then treated with fresh medium containing 10 µM EDU. After a 2.5-hour incubation at 37°C, the cells were fixed in 4% paraformaldehyde (Boster, Wuhan, China) for 15 minutes and permeabilized with 0.1% Triton X-100 (Boster, Wuhan, China) for 8 minutes. Subsequently, 500µl of Apollo dyeing reaction buffer was applied for 40 minutes in a light-free environment. This staining was followed by DAPI staining for a period of 10 minutes. A fluorescence microscope (Olympus, Japan) was used to visualize the EDU staining.

Colony formation

The study examined the colony formation abilities of LN229 and U87 cells following treatment. Colonies exceeding 75 µm in diameter or comprising more than 50 cells were classified as positive colonies. After seeding a total of 1000 cells into each well of a six-well culture plate, three wells were assigned to each sample. Following 2 weeks incubation, the cells were rinsed twice in PBS, then stained with crystal violet.

Transwell assay

One thousand U87 and LN229 cells were resuspended in 300 ml of FBS-free DMEM and seeded into matrigel-coated transwell chambers. DMEM medium with 10% FBS was used in the bottom transwell chambers. Invading cells were fixed and stained for 20 minutes with 0.5% crystal violet after 24 hours. A microscope was used to visualize and count invasive cells in five randomly selected fields (up, down, left, right, middle) of each chamber.

In vivo mice model

The in vivo model was performed on female BALB/c nude mice obtained from the Animal Center of Guizhou Medical University. A subcutaneous injection of 2×10⁶ glioma cells mixed with 1×10⁵ macrophage-like THP-1 were administered either into the BALB/c mice's upper-right flank or into their in situ brain following adaptive feeding (n = 5 in each group). Guizhou Medical University's Animal Experimental Center approved the experiments on mice, monitoring health status daily and assessing tumor volume on a weekly basis.

Exosome extraction

Culture dishes containing DMEM-supplemented medium were incubated at 37°C with 5% CO₂ until 70% confluent. Subsequently, the cells underwent a medium change and were incubated for a period of 3 days. Supernatant was obtained through a series of low-speed and high-speed centrifugation steps, followed by filtration and ultracentrifugation for 90 minutes. The resulting supernatant was removed, and the cells were resuspended in PBS for secondary ultracentrifugation to collect and resuspend the exosome precipitation.

RNA pulldown assay

T7 High Yield RNA Transcription Kit (Vazyme, USA) and PierceTM RNA 3' End Desthiobiotinylation Kit (Thermo Fisher) were used to synthesize biotin-labeled circ-001422. Following isolation of the RNA–protein binding complex with the PierceTM Magnetic RNA–protein Pull-Down Kit (Thermo Fisher), specific protein targets were identified using mass spectrometry.

RNA binding protein immunoprecipitation (RIP) assay

Whole cell lysis was obtained from 2 × 10⁶ U87 and LN229 cells, followed by the addition of protease inhibitor cocktail and RNase inhibitor for a 5-minute incubation period. After incubation at room temperature for 30 minutes with IgG or antibodies, magnetic beads were coated with lysate from cell lysis and incubated at 4°C overnight after centrifugation at 1000g for 10 minutes. Finally, qRT-PCR was used to quantify the purified RNA.

Statistical analysis

Statistical analyses were conducted using the Student t test or one-way ANOVA to assess variances between two groups or multiple groups, respectively. Experiments were conducted thrice, with data presented as the mean ± SD. The overall survival rates were calculated using the Kaplan-Meier method, and log-rank tests were used for comparisons. Statistical significance was determined using GraphPad Prism 8 software.

Acknowledgements

Not applicable.

Conflict of Interest

The authors state that there are no potential conflicting interests in their work.

Author Contribution

Shan Lei, Wenpeng Cao, and Zhirui Zeng designed the experiments and wrote the manuscript; Wenpeng Cao, Yunhua Chen, Jinzhi Lan, FaGuang Kuang, Shipeng Luo, JianFei Sun conducted and processed the data. The final version of the manuscript was reviewed by all of the writers, and they granted their approval.

Ethics Statement

The Human Research Ethics Review Committee of Guizhou Medical University approved the application of these clinical samples, which was performed according to the tenets of the Declaration of Helsinki. The process of animal experiments was approved by Animal Ethics Committee of Guizhou Medical University.

Funding

This study was funded by the Continuous support fund for the Department of Education of Guizhou Province (Guizhou Teaching and Technology (2023) 015) and the National Natural Science Foundation of China (82360522).

Data Availability

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

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There is NO conflict of interest to disclose.

SupplementalMaterial.pdf
TableS1.xlsx
Supplemental Table S1. Primers used in the current study.
TableS2.xlsx
Supplement Table S2. Binding protein with circ-001422.

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

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Abstract

Figures

Introduction

Results

IL-6 induced the up-regulation of circ-001422 in glioma cells

STAT3 transcriptionally upregulates circ-001422 expression in glioma cells

Circ-001422 promoted the M2 polarization in the co-culture condition of macrophage-like THP-1 and glioma cells

Exosome-mediated transfer of circ-001422 promoted macrophage M2 polarization and stimulates PC cell proliferation and metastasis

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

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

Discussion

Materials and methods

Clinical samples

qRT-PCR assays

Western blotting

RNA in situ hybridization (ISH)

Immunohistochemistry (IHC) experiments

Cell culture and transfection

Nuclear and cytoplasmic separation

Fluorescence in situ hybridization (FISH)

Chromatin immunoprecipitation (ChIP)

Luciferase Assay

Macrophage induction

Flow cytometry

5-ethynyl-2’-deoxyuridine (EDU) Assay

Colony formation

Transwell assay

In vivo mice model

Exosome extraction

RNA pulldown assay

RNA binding protein immunoprecipitation (RIP) assay

Statistical analysis

Declarations

References

Additional Declarations

Supplementary Files

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