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

Exosome-derived circ-001422 promote tumor-associated macrophage M2 polarization to accelerate the progression of glioma | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Exosome-derived circ-001422 promote tumor-associated macrophage M2 polarization to accelerate the progression of glioma Lei Shan, Wenpeng Cao, Zhirui Zeng, JianFei Sun, Yunhua Chen, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4616289/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version 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. 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 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction 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. Results 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. 1 A). Among them, through qRT-PCR experiments, we found that circ-001422 was elevated most significantly (Fig. 1 B). Therefore, we focused on circ-001422. Through performed qRT-PCR (Fig. 1 C) and ISH (Fig. 1 E-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. 1 D). 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. 1 G). Furthermore, circ-001422 was found to locate in cytoplasm mostly (Fig. 1 H-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. 2 A). Through performing chip-PCR in LN229 and U87, we found that using STAT3 antibody significantly enriched the beads of WHSC1 (Fig. 2 B). 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. 2 C), a total of 4 potential binding sites for STAT3 in the promoter of WHSC1 was found (Fig. 2 D). 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. 2 E). 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. 2 F). 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. 2 G). It is interesting to found that overexpression of STAT3 in LN229 and U87 cells significantly elevated the levels of WHSC1 pre-mRNA (Fig. 2 H) and circ-001422 (Fig. 2 J), while the mRNA levels of WHSC1 were reduced (Fig. 2 I). 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. 3 A and Fig. 3 C) and weight (Fig. 3 B), while those derived with circ-001422 knockdown group exhibited lower tumor volume (Fig. 3 A and Fig. 3 C) and weight (Fig. 3 C). 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. 3 D), while knockdown of circ-001422 induced the oppositive effects (Fig. 3 D). 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. 3 E). 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. 3 F). 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. 4 A). 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. 4 B). Conversely, the macrophages co-culturing with glioma cells contained circ-001422 knockdown exhibited higher M1 biomarkers levels and lower M2 biomarkers (Fig. 4 B). 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. 4 C). 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. 4 D). 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. 4 D). 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. 5 A-B). Interesting, western blotting results indicated that circ-001422 overexpression increased the expression of exosome protein markers including HSP70, CD81, CD9 and TSG101 (Fig. 5 C). In addition, we found the extracellular levels of circ-001422 were reduced in medium after removing exosomes (Fig. 5 D). 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. 5 E). 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. 5 F). 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. 5 G). 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. 5 H). 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. 5 J), colony formation (Fig. 5 I) and invasion (Fig. 5 K) 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. 6 A). Similarly, results of RIP experiment using anti-p300 and anti-STAT3 antibodies indicated that both p300 and STAT3 significantly enriched the circ-001422 (Fig. 6 B). Moreover, we found that circ-001422 was mostly co-located with STAT3 and p300 in macrophage like-THP-1 (Fig. 6 C). 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. 6 D). These cells also increased the expression of STAT3 and NF-κB p65 in nucleus (Fig. 6 D). Reduced the expression of circ-001422 induced the opposite effects in macrophage like-THP-1 (Fig. 6 D). Furthermore, we found that circ-001422 knockdown also inhibited the re-localization of STAT3 and NF-κB p65 in nucleus (Fig. 6 E-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. 7 A).Transfection of STAT3 plasmids in macrophage like-THP-1 had significant reversed effects (Fig. 7 A). Interesting, we found that reversed effects of STAT3-K685 plasmids were significantly stronger than wildtype STAT3 plasmids and STAT3-K685R plasmids (Fig. 7 A). 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. 7 B). 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. 7 B). Among them, STAT3-K685Q plasmids exhibited the strongest effects (Fig. 7 B). 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. 7 C). 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. 7 D). 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. 7 E-F). Taken together, The activate of STAT3/NF-κB signaling induced by circ-001422 containing exosomes is STAT3-K685 acetylation dependent. Discussion 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. Materials and methods 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% CO 2 . 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 2×10 6 glioma cells mixed with 1×10 5 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% CO 2 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 × 10 6 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. Declarations 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. References Wang LM, Englander ZK, Miller ML, Bruce JN. Malignant Glioma. Adv Exp Med Biol. 2023; 1405:1-30. Dal Bello S, Martinuzzi D, Tereshko Y, Veritti D, Sarao V, Gigli GL, et al. The Present and Future of Optic Pathway Glioma Therapy. Cells. 2023; 12(19):2380. Varela ML, Comba A, Faisal SM, Argento A, Franson A, Barissi MN, et al. Gene Therapy for High Grade Glioma: The Clinical Experience. Expert Opin Biol Ther. 2023; 23(2):145-161. Bilotta MT, Antignani A, Fitzgerald DJ. Managing the TME to improve the efficacy of cancer therapy. Front Immunol. 2022; 13:954992. Yunna C, Mengru H, Lei W, Weidong C. Macrophage M1/M2 polarization. Eur J Pharmacol. 2020; 877:173090. Ren J, Xu B, Ren J, Liu Z, Cai L, Zhang X, et al. The Importance of M1-and M2-Polarized Macrophages in Glioma and as Potential Treatment Targets. Brain Sci. 2023; 13(9):1269. Grabowski MM, Sankey EW, Ryan KJ, Chongsathidkiet P, Lorrey SJ, Wilkinson DS, et al. Immune suppression in gliomas. J Neurooncol. 2021; 151(1):3-12. Chen LL, Yang L. Regulation of circRNA biogenesis. RNA Biol. 2015;12(4):381-8. Xue C, Li G, Zheng Q, Gu X, Bao Z, Lu J, et al. The functional roles of the circRNA/Wnt axis in cancer. Mol Cancer. 2022; 21(1):108. Peng D, Luo L, Zhang X, Wei C, Zhang Z, Han L. CircRNA: An emerging star in the progression of glioma. Biomed Pharmacother. 2022; 151:113150. Yan Y, Wang H, Hu J, Guo T, Dong Q, Yin H, et al. CircRNA-104718 promotes glioma malignancy through regulation of miR-218-5p/HMGB1 signalling pathway. Metab Brain Dis. 2023; 38(5):1531-1542. Chen J, Chen T, Zhu Y, Li Y, Zhang Y, Wang Y, et al. circPTN sponges miR-145-5p/miR-330-5p to promote proliferation and stemness in glioma. J Exp Clin Cancer Res. 2019; 38(1):398. Yang B, Li L, Tong G, Zeng Z, Tan J, Su Z, et al. Circular RNA circ_001422 promotes the progression and metastasis of osteosarcoma via the miR-195-5p/FGF2/PI3K/Akt axis. J Exp Clin Cancer Res. 2021; 40(1):235. Wang Y, Chen X, Tang G, Liu D, Peng G, Ma W, et al. AS-IL6 promotes glioma cell invasion by inducing H3K27Ac enrichment at the IL6 promoter and activating IL6 transcription. FEBS Lett. 2016; 590(24):4586-4593. Zhai W, Ye X, Wang Y, Feng Y, Wang Y, Lin Y, et al. CREPT/RPRD1B promotes tumorigenesis through STAT3-driven gene transcription in a p300-dependent manner. Br J Cancer. 2021; 124(8):1437-1448. Galbraith K, Snuderl M. Molecular Pathology of Gliomas. Surg Pathol Clin. 2021; 14(3):379-386. Ludwig K, Kornblum HI. Molecular markers in glioma. J Neurooncol. 2017; 134(3):505-512. Iwami K, Natsume A, Wakabayashi T. Cytokine networks in glioma. Neurosurg Rev. 2011; 34(3):253-63. Okada H, Pollack IF. Cytokine gene therapy for malignant glioma. Expert Opin Biol Ther. 2004; 4(10):1609-20. Cao F, Zhang Q, Chen W, Han C, He Y, Ran Q, et al. IL-6 increases SDCBP expression, cell proliferation, and cell invasion by activating JAK2/STAT3 in human glioma cells. Am J Transl Res. 2017; 9(10):4617-4626. Wang Q, He Z, Huang M, Liu T, Wang Y, Xu H, et al. Vascular niche IL-6 induces alternative macrophage activation in glioblastoma through HIF-2α. Nat Commun. 2018; 9(1):559. Pegtel DM, Gould SJ. Exosomes. Annu Rev Biochem. 2019; 88:487-514. Li B, Cao Y, Sun M, Feng H. Expression, regulation, and function of exosome-derived miRNAs in cancer progression and therapy. FASEB J. 2021; 35(10):e21916. Zhang J, Li S, Li L, Li M, Guo C, Yao J, Mi S. Exosome and exosomal microRNA: trafficking, sorting, and function. Genomics Proteomics Bioinformatics. 2015 Feb;13(1):17-24. Xu Z, Chen Y, Ma L, Chen Y, Liu J, Guo Y, et al. Role of exosomal non-coding RNAs from tumor cells and tumor-associated macrophages in the tumor microenvironment. Mol Ther. 2022; 30(10):3133-3154. Tian Y, Gao X, Yang X, Chen S, Ren Y. Glioma-derived exosome Lncrna Agap2-As1 promotes glioma proliferation and metastasis by mediating Tgf-β1 secretion of myeloid-derived suppressor cells. Heliyon. 2024; 10(9):e29949. Li J, Wang K, Yang C, Zhu K, Jiang C, Wang M, et al. Tumor-Associated Macrophage-Derived Exosomal LINC01232 Induces the Immune Escape in Glioma by Decreasing Surface MHC-I Expression. Adv Sci (Weinh). 2023; 10(17):e2207067. Dorrington MG, Fraser IDC. NF-κB Signaling in Macrophages: Dynamics, Crosstalk, and Signal Integration. Front Immunol. 2019; 10:705. Yunna C, Mengru H, Lei W, Weidong C. Macrophage M1/M2 polarization. Eur J Pharmacol. 2020; 877:173090. Zhao T, Zeng J, Xu Y, Su Z, Chong Y, Ling T, et al. Chitinase-3 like-protein-1 promotes glioma progression via the NF-κB signaling pathway and tumor microenvironment reprogramming. Theranostics. 2022; 12(16):6989-7008. Xu Y, Liao C, Liu R, Liu J, Chen Z, Zhao H, et al. IRGM promotes glioma M2 macrophage polarization through p62/TRAF6/NF-κB pathway mediated IL-8 production. Cell Biol Int. 2019; 43(2):125-135. Qian M, Wang S, Guo X, Wang J, Zhang Z, Qiu W, et al. Hypoxic glioma-derived exosomes deliver microRNA-1246 to induce M2 macrophage polarization by targeting TERF2IP via the STAT3 and NF-κB pathways. Oncogene. 2020; 39(2):428-442. Ni J, Shen Y, Wang Z, Shao DC, Liu J, Kong YL, et al. P300-dependent STAT3 acetylation is necessary for angiotensin II-induced pro-fibrotic responses in renal tubular epithelial cells. Acta Pharmacol Sin. 2014; 35(9):1157-66. Wang Y, Zhou C, Gao H, Li C, Li D, Liu P, et al. Therapeutic effect of Cryptotanshinone on experimental rheumatoid arthritis through downregulating p300 mediated-STAT3 acetylation. Biochem Pharmacol. 2017; 138:119-129. Ge W, Li YA, Ruan Y, Wu N, Ma P, Xu T, et al. IL-17 induces NSCLC cell migration and invasion by elevating MMP19 gene transcription and expression through the interaction of p300-dependent STAT3-K631 acetylation and its Y705-phosphorylation. Oncol Res. 2024; 32(4):625-641. Additional Declarations There is NO conflict of interest to disclose. Supplementary Files SupplementalMaterial.pdf TableS1.xlsx Supplemental Table S1. Primers used in the current study. TableS2.xlsx Supplement Table S2. Binding protein with circ-001422. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4616289","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":319056351,"identity":"86769579-7e76-4db7-8707-d4bc78ba0e3e","order_by":0,"name":"Lei Shan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1UlEQVRIiWNgGAWjYBACPgbGBmYgLQfhshGhhQ2qxZgULQwMIC2JDcRrYW9u/lxQcyd9ftjhBwwfyg4z8M9uIKCF52Cb9Ixjz3I33k4zYJxx7jCDxJ0DBLRIJLYx87Adzt04O4eBmbftMIOBRAIBLfIPmz/z/DucbgjS8pcoLRKMDdJAwxPkpYFaGInSwpPYJs3bd9hwg3SawcGec+k8EjcIaOFnP/74M8+3w/Lys5MfPvhRZi3HP4OAFjgwOMDAAEQMPESqBwL5BuLVjoJRMApGwQgDABiiPxZpKH6UAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0003-3789-4186","institution":"School of Basic Medical Sciences, Guizhou Medical University","correspondingAuthor":true,"prefix":"","firstName":"Lei","middleName":"","lastName":"Shan","suffix":""},{"id":319056352,"identity":"e49e66a8-7283-400c-bb85-13640671ac33","order_by":1,"name":"Wenpeng Cao","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Wenpeng","middleName":"","lastName":"Cao","suffix":""},{"id":319056353,"identity":"16697a8d-78ca-4038-8aa4-fb99f31028ed","order_by":2,"name":"Zhirui Zeng","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Zhirui","middleName":"","lastName":"Zeng","suffix":""},{"id":319056354,"identity":"6861f7e0-4be4-4f3f-bce9-441f262eb47a","order_by":3,"name":"JianFei Sun","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"JianFei","middleName":"","lastName":"Sun","suffix":""},{"id":319056355,"identity":"41ebeffc-673a-4ca8-875d-b2d0003a1363","order_by":4,"name":"Yunhua Chen","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Yunhua","middleName":"","lastName":"Chen","suffix":""},{"id":319056356,"identity":"2302c7ea-3e21-4093-80b0-c8856c419d32","order_by":5,"name":"FaGuang Kuang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"FaGuang","middleName":"","lastName":"Kuang","suffix":""},{"id":319056357,"identity":"55e7776a-fc1d-4424-9b6f-f37563221415","order_by":6,"name":"Shipeng Luo","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Shipeng","middleName":"","lastName":"Luo","suffix":""},{"id":319056358,"identity":"f6eed447-ff9b-46e9-85f0-da36df5494e3","order_by":7,"name":"Jinzhi Lan","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Jinzhi","middleName":"","lastName":"Lan","suffix":""}],"badges":[],"createdAt":"2024-06-21 09:10:31","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4616289/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4616289/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":60466296,"identity":"9a1a3434-2a49-495b-bd63-7853c31e0949","added_by":"auto","created_at":"2024-07-17 05:23:12","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":696288,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCirc-001422 was up-regulated in glioma cells treatment with IL-6 and glioma tissues.\u003c/strong\u003e(A) RNA-sequencing was performed to detect the differentially expressed circRNAs induced by IL-6 in U87 cells. (B) RT-qPCR was performed to detect the expression of differentially expressed circRNAs in U87 cells after IL-6 treatment. (C) qRT-PCR experiments were used to detect the expression of circ-001422 in 48 pair glioma tissues and adjacent non-tumor tissues. (D) qRT-PCR was used to detect the expression of circ-001422 in NHA, T98G, LN229, LN18, U251, U87 and SHG-44 cell. (E-F) ISH was performed to detect the expression of circ-001422 in 48 pair glioma tissues and adjacent non-tumor tissues. (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\u0026lt;0.05; **, P\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4616289/v1/77ec3ce44f60251245d6d2d5.png"},{"id":60466300,"identity":"c586e3be-68dd-4f0d-b622-9b1c5cc051cb","added_by":"auto","created_at":"2024-07-17 05:23:12","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":196927,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSTAT3 transcriptionally increased circ-001422 in glioma cells.\u003c/strong\u003e(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. (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. (F) Chip-qPCR indicated that STAT3 significantly bind with the binding site 4 of WHSC1. (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. (I) STAT3 reduced the levels of mRNA of WHSC1, while knockdown of STAT3 increased the mRNA levels of WHSC1. (J) STAT3 elevated the levels of circ-001422, while knockdown of STAT3 reduced the levels. *, P\u0026lt;0.05; **, P\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4616289/v1/7d0fbcf522f212a968dd4635.png"},{"id":60466293,"identity":"9965a9e8-7edb-4acf-bbcc-c07412a5e765","added_by":"auto","created_at":"2024-07-17 05:23:12","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":260725,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCirc-001422 promotes proliferation, invasion and metastasis ability of glioma cells under co-culture with macrophages.\u003c/strong\u003e (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. (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. *, P\u0026lt;0.05; **, P\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4616289/v1/a220f1aa451fa1f2495f1092.png"},{"id":60466303,"identity":"11fa6cf3-0ae9-4ccc-8dd3-dfd8902a7d03","added_by":"auto","created_at":"2024-07-17 05:23:12","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":17162711,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCirc-001422 promotes macrophages M2 polarization in co-culturing with glioma cells. \u003c/strong\u003e(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. (B-C) qRT-PCR and western blotting 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. (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\u0026lt;0.05; **, P\u0026lt;0.01; ***, P\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4616289/v1/7ed6f044cbc728c27e84362d.png"},{"id":60466683,"identity":"39c6f188-4d91-4693-8a15-9c7d7c4717fc","added_by":"auto","created_at":"2024-07-17 05:31:12","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":244515,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExosome-mediated transfer of circ-001422 promoted macrophage M2 polarization and stimulates PC cell proliferation and metastasis. \u003c/strong\u003e(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. (E) qRT-PCR was used to detect the levels of circ-001422 in macrophage-like THP-1 after treatment with exosomes from NC glioma cells and 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. (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. (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. (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. *, P\u0026lt;0.05; **, P\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4616289/v1/6ef265c540f2707cd3714b54.png"},{"id":60467046,"identity":"f318f87b-14d6-4060-b891-d357011edb7b","added_by":"auto","created_at":"2024-07-17 05:39:12","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":261749,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCirc-001422 interacts with p300 and STAT3 and activates STAT3/NF-κB signaling. \u003c/strong\u003e(A) Protein profile analysis of circ-001422 binding protein, especially for the p300 and STAT3. (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 glioma cells. (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 ofSTAT3, 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. *, P\u0026lt;0.05; **, P\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-4616289/v1/5e032d60fd1bbef90fba8246.png"},{"id":60466686,"identity":"c35e0e14-5681-421a-be5e-cda0050f6ff6","added_by":"auto","created_at":"2024-07-17 05:31:12","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":258602,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe activate of STAT3/NF-κB signaling induced by circ-001422 containing exosomes is STAT3-K685 acetylation dependent. \u003c/strong\u003e(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) \u0026nbsp;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. (E) Flow cytometry results indicated that exosome-derived circ-001422 increased the positive rate of CD206 in THP-1 cells via STAT3 acetylation. (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\u0026lt;0.05; **, P\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-4616289/v1/e30fbff8dba9cb8bc4650c25.png"},{"id":61097454,"identity":"c111aa7b-6618-4f9c-aa83-e3e8c3f56e4c","added_by":"auto","created_at":"2024-07-25 14:21:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":32106695,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4616289/v1/f9e9e2df-961c-4e32-8629-5fbee1d74d87.pdf"},{"id":60466684,"identity":"9983e75a-cc88-4fb6-af91-4dcbbf40120e","added_by":"auto","created_at":"2024-07-17 05:31:12","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2608044,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalMaterial.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4616289/v1/aac0fd4dd0f46897d9a484ed.pdf"},{"id":60467045,"identity":"c64b1357-cdfc-4f5b-9fc7-f410d225e31e","added_by":"auto","created_at":"2024-07-17 05:39:12","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":11956,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplemental Table S1. Primers used in the current study.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"TableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4616289/v1/26cf59e7180904c4001d244d.xlsx"},{"id":60466298,"identity":"6e228a0e-a5bb-4c83-877f-aad689426917","added_by":"auto","created_at":"2024-07-17 05:23:12","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":22391,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplement Table S2. Binding protein with circ-001422.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"TableS2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4616289/v1/954377fc684789887e5a7f2a.xlsx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose.","formattedTitle":"Exosome-derived circ-001422 promote tumor-associated macrophage M2 polarization to accelerate the progression of glioma","fulltext":[{"header":"Introduction","content":"\u003cp\u003eGlioma, 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 (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). 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 (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). However, the median survival rate remains unsatisfactory. Therefore, exploring biomarkers of glioma progression may contribute to the therapy of glioma.\u003c/p\u003e \u003cp\u003eThe tumor microenvironment (TME) is comprised of cytokines, growth factors, and various tumor-induced immune cells that contribute significantly to tumor progression (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). 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 (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). 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 (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Nevertheless, the precise molecular mechanisms underlying the upregulation of M2 macrophages in glioma tissues remain largely unexplored.\u003c/p\u003e \u003cp\u003eCircular RNAs, a novel category of endogenous non-coding RNAs, possess closed circular structures unlike linear RNAs (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Research has indicated that dysregulated expression of circRNAs is evident in various types of cancer, including glioma (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). For instance, Yan \u003cem\u003eet al.\u003c/em\u003e 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 (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Additionally, Chen \u003cem\u003eet al.\u003c/em\u003e demonstrated that circPTN promotes proliferation and stemness in glioma by acting as a sponge for miR-145-5p/miR-330-5p (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Our prior research demonstrated that circ-001422 is situated on chromosome 4, spans 1703 bp, and is produced through the circularization of exons 2\u0026ndash;7 of the NSD2 gene (also known as WHSC1). Circ-001422 exhibited the potential to enhance the advancement and metastasis of osteosarcoma cells (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). Nevertheless, the function of circ-001422 in glioma remained unexplored.\u003c/p\u003e \u003cp\u003eIn 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.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eIL-6 induced the up-regulation of circ-001422 in glioma cells\u003c/h2\u003e \u003cp\u003eIL-6 is a key cytokine in the microenvironment of glioma tissues, which exhibited significant effects on glioma cell progression (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). 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.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Among them, through qRT-PCR experiments, we found that circ-001422 was elevated most significantly (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Therefore, we focused on circ-001422.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThrough performed qRT-PCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC) and ISH (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE-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.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). 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.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). Furthermore, circ-001422 was found to locate in cytoplasm mostly (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH-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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eSTAT3 transcriptionally upregulates circ-001422 expression in glioma cells\u003c/h2\u003e \u003cp\u003ePrevious studies indicated that circ-001422 is generated by circularization of exons 2\u0026ndash;7 of the host gene WHSC1 (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). 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.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Through performing chip-PCR in LN229 and U87, we found that using STAT3 antibody significantly enriched the beads of WHSC1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAs 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.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), a total of 4 potential binding sites for STAT3 in the promoter of WHSC1 was found (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). 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.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). 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.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003eSince 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.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). It is interesting to found that overexpression of STAT3 in LN229 and U87 cells significantly elevated the levels of WHSC1 pre-mRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH) and circ-001422 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ), while the mRNA levels of WHSC1 were reduced (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI). Knockdown of STAT3 induced the reversed effects. These evidences may indicate that STAT3 transcriptionally upregulates circ-001422 expression in glioma cells.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCirc-001422 promoted the proliferation and mobility of glioma cells while co-culturing with macrophage-like THP-1.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo 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.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC) and weight (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), while those derived with circ-001422 knockdown group exhibited lower tumor volume (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC) and weight (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). 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.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD), while knockdown of circ-001422 induced the oppositive effects (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMoreover, 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.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). 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.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). These results indicated that circ-001422 promoted the proliferation and mobility of glioma cells while co-culturing with macrophage-like THP-1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eCirc-001422 promoted the M2 polarization in the co-culture condition of macrophage-like THP-1 and glioma cells\u003c/h2\u003e \u003cp\u003eWe 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.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). These results indicated that circ-001422 in glioma cells may had potential to promote macrophage-like THP-1 into M2 macrophage.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo 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.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Conversely, the macrophages co-culturing with glioma cells contained circ-001422 knockdown exhibited higher M1 biomarkers levels and lower M2 biomarkers (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). 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.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eFurthermore, 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.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). 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.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). 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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eExosome-mediated transfer of circ-001422 promoted macrophage M2 polarization and stimulates PC cell proliferation and metastasis\u003c/h2\u003e \u003cp\u003eTo analyzed how circ-001422 affect the macrophage M2 polarization, exosomes secreted from LN229 and U87 cells were extracted (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-B). Interesting, western blotting results indicated that circ-001422 overexpression increased the expression of exosome protein markers including HSP70, CD81, CD9 and TSG101 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). In addition, we found the extracellular levels of circ-001422 were reduced in medium after removing exosomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). 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.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). 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.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eqRT-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.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG). 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.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH). 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.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eJ), colony formation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI) and invasion (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eK) 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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eCirc-001422 interacts with p300 and STAT3 and activates STAT3/NF-κB signaling\u003c/h2\u003e \u003cp\u003eTo 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.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Similarly, results of RIP experiment using anti-p300 and anti-STAT3 antibodies indicated that both p300 and STAT3 significantly enriched the circ-001422 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Moreover, we found that circ-001422 was mostly co-located with STAT3 and p300 in macrophage like-THP-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). 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.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). These cells also increased the expression of STAT3 and NF-κB p65 in nucleus (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). Reduced the expression of circ-001422 induced the opposite effects in macrophage like-THP-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). Furthermore, we found that circ-001422 knockdown also inhibited the re-localization of STAT3 and NF-κB p65 in nucleus (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE-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.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eThe activate of STAT3/NF-κB signaling induced by circ-001422 containing exosomes is STAT3-K685 acetylation dependent\u003c/h2\u003e \u003cp\u003ePrevious studies suggested that activated STAT3 recruits p300 to the promoters of STAT3-targeted genes, thereby facilitating transcription through STAT3-K685 acetylation (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). 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.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA).Transfection of STAT3 plasmids in macrophage like-THP-1 had significant reversed effects (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Interesting, we found that reversed effects of STAT3-K685 plasmids were significantly stronger than wildtype STAT3 plasmids and STAT3-K685R plasmids (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). 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.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). 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.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). Among them, STAT3-K685Q plasmids exhibited the strongest effects (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMoreover, 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.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). 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.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). 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.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE-F). Taken together, The activate of STAT3/NF-κB signaling induced by circ-001422 containing exosomes is STAT3-K685 acetylation dependent.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe 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 (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). 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 (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). There were strong evidences that soluble factors, specifically chemokines, regulate tumorigenesis of glioma (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). 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 (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Investigating the molecular mechanisms of IL-6 may offer insights into potential therapeutic strategies for glioma.\u003c/p\u003e \u003cp\u003eThe 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.\u003c/p\u003e \u003cp\u003eIt 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 (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). 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 (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). Previous research has suggested that non-coding RNAs within exosomes serve as crucial mediators between different cells in the tumor microenvironment (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). For instance, Tian \u003cem\u003eet al.\u003c/em\u003e 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 (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Li et al. indicated that TAM-derived exosomal LINC01232 promoted the immune escape of glioma cells (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). 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.\u003c/p\u003e \u003cp\u003eIn 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 (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). A previous report showed that activating NF-κB promotes tumor growth by regulating M2 polarization genes (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). Numerous factors within tumors can modulate the activation of NF-κB, consequently impacting M2 polarization (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). A previous investigation demonstrated that acetyltransferase p300 facilitated the acetylation of STAT3, thereby enhancing its activation (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). A disruption of the STAT3 acetylation site or suppression of histone deacetylase activity prevents it from dimerizing and controlling transcription (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). 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.\u003c/p\u003e \u003cp\u003eIn 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.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eClinical samples\u003c/h2\u003e \u003cp\u003eA 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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eqRT-PCR assays\u003c/h2\u003e \u003cp\u003eTotal 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\u003csup\u003e\u0026minus;ΔΔCT\u003c/sup\u003e method for expression level analysis with β-actin as the internal control. The primers employed in this study are detailed in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e of the supplementary data.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eWestern blotting\u003c/h2\u003e \u003cp\u003eAn 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, 30\u0026micro;g 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 0.45\u0026micro;m (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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eRNA in situ hybridization (ISH)\u003c/h2\u003e \u003cp\u003eISH 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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry (IHC) experiments\u003c/h2\u003e \u003cp\u003eThe 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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eCell culture and transfection\u003c/h2\u003e \u003cp\u003eHuman 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\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. 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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eNuclear and cytoplasmic separation\u003c/h2\u003e \u003cp\u003eFollowing the manufacturer's instructions, nuclear and cytoplasmic RNAs were isolated using the PARISTM Kit (Thermo Fisher Scientific, USA). Centrifugation at 4 \u0026ordm;C 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 \u0026ordm;C, the RNA fractions were passed through a filter cartridge.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eFluorescence in situ hybridization (FISH)\u003c/h2\u003e \u003cp\u003eAn 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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eChromatin immunoprecipitation (ChIP)\u003c/h2\u003e \u003cp\u003eA 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 \u0026ordm;C. PCR was used to analyze the purified DNA fragments resulting from the antibody-DNA complex.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eLuciferase Assay\u003c/h2\u003e \u003cp\u003eTo 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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eMacrophage induction\u003c/h2\u003e \u003cp\u003eMonocyte-like THP-1 cells were cultured in six-well plates at a density of 1 \u0026times; 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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometry\u003c/h2\u003e \u003cp\u003eMacrophage-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 \u0026micro;l 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.\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003e5-ethynyl-2\u0026rsquo;-deoxyuridine (EDU) Assay\u003c/h2\u003e \u003cp\u003eThe EDU assay was performed using a BeyoClick\u0026trade; 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 \u0026micro;M EDU. After a 2.5-hour incubation at 37\u0026deg;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, 500\u0026micro;l 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.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eColony formation\u003c/h2\u003e \u003cp\u003eThe study examined the colony formation abilities of LN229 and U87 cells following treatment. Colonies exceeding 75 \u0026micro;m 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.\u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eTranswell assay\u003c/h2\u003e \u003cp\u003eOne 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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003eIn vivo mice model\u003c/h2\u003e \u003cp\u003eThe 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\u0026times;10\u003csup\u003e6\u003c/sup\u003e glioma cells mixed with 1\u0026times;10\u003csup\u003e5\u003c/sup\u003e 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\u0026thinsp;=\u0026thinsp;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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003eExosome extraction\u003c/h2\u003e \u003cp\u003eCulture dishes containing DMEM-supplemented medium were incubated at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e 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.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003eRNA pulldown assay\u003c/h2\u003e \u003cp\u003eT7 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\u0026ndash;protein binding complex with the PierceTM Magnetic RNA\u0026ndash;protein Pull-Down Kit (Thermo Fisher), specific protein targets were identified using mass spectrometry.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003eRNA binding protein immunoprecipitation (RIP) assay\u003c/h2\u003e \u003cp\u003eWhole cell lysis was obtained from 2 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e 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\u0026deg;C overnight after centrifugation at 1000g for 10 minutes. Finally, qRT-PCR was used to quantify the purified RNA.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec30\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical 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\u0026thinsp;\u0026plusmn;\u0026thinsp;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.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors state that there are no potential conflicting interests\u0026nbsp;in their work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contribution\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eShan Lei,\u0026nbsp;Wenpeng Cao, and\u0026nbsp;Zhirui Zeng\u0026nbsp;designed the experiments and wrote the manuscript;\u0026nbsp;Wenpeng Cao,\u0026nbsp;Yunhua Chen, Jinzhi Lan, FaGuang Kuang, Shipeng Luo, JianFei Sun\u0026nbsp;conducted and processed the data. The final version of the manuscript was reviewed by all of the writers, and they granted their approval.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was funded by\u0026nbsp;the Continuous support fund for the Department of Education of Guizhou Province (Guizhou Teaching and Technology (2023) 015)\u0026nbsp;and the\u0026nbsp;National Natural Science Foundation of China\u0026nbsp;(82360522).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eWang LM, Englander ZK, Miller ML, Bruce JN. Malignant Glioma. Adv Exp Med Biol. 2023; 1405:1-30.\u003c/li\u003e\n \u003cli\u003eDal Bello S, Martinuzzi D, Tereshko Y, Veritti D, Sarao V, Gigli GL, et al. The Present and Future of Optic Pathway Glioma Therapy. Cells. 2023; 12(19):2380.\u003c/li\u003e\n \u003cli\u003eVarela ML, Comba A, Faisal SM, Argento A, Franson A, Barissi MN, et al. Gene Therapy for High Grade Glioma: The Clinical Experience. Expert Opin Biol Ther. 2023; 23(2):145-161.\u003c/li\u003e\n \u003cli\u003eBilotta MT, Antignani A, Fitzgerald DJ. Managing the TME to improve the efficacy of cancer therapy. Front Immunol. 2022; 13:954992.\u003c/li\u003e\n \u003cli\u003eYunna C, Mengru H, Lei W, Weidong C. Macrophage M1/M2 polarization. Eur J Pharmacol. 2020; 877:173090.\u003c/li\u003e\n \u003cli\u003eRen J, Xu B, Ren J, Liu Z, Cai L, Zhang X, et al. The Importance of M1-and M2-Polarized Macrophages in Glioma and as Potential Treatment Targets. Brain Sci. 2023; 13(9):1269.\u003c/li\u003e\n \u003cli\u003eGrabowski MM, Sankey EW, Ryan KJ, Chongsathidkiet P, Lorrey SJ, Wilkinson DS, et al. Immune suppression in gliomas. J Neurooncol. 2021; 151(1):3-12.\u003c/li\u003e\n \u003cli\u003eChen LL, Yang L. Regulation of circRNA biogenesis. RNA Biol. 2015;12(4):381-8.\u003c/li\u003e\n \u003cli\u003eXue C, Li G, Zheng Q, Gu X, Bao Z, Lu J, et al. The functional roles of the circRNA/Wnt axis in cancer. Mol Cancer. 2022; 21(1):108.\u003c/li\u003e\n \u003cli\u003ePeng D, Luo L, Zhang X, Wei C, Zhang Z, Han L. CircRNA: An emerging star in the progression of glioma. Biomed Pharmacother. 2022; 151:113150.\u003c/li\u003e\n \u003cli\u003eYan Y, Wang H, Hu J, Guo T, Dong Q, Yin H, et al. CircRNA-104718 promotes glioma malignancy through regulation of miR-218-5p/HMGB1 signalling pathway. Metab Brain Dis. 2023; 38(5):1531-1542.\u003c/li\u003e\n \u003cli\u003eChen J, Chen T, Zhu Y, Li Y, Zhang Y, Wang Y, et al. circPTN sponges miR-145-5p/miR-330-5p to promote proliferation and stemness in glioma. J Exp Clin Cancer Res. 2019; 38(1):398.\u003c/li\u003e\n \u003cli\u003eYang B, Li L, Tong G, Zeng Z, Tan J, Su Z, et al. Circular RNA circ_001422 promotes the progression and metastasis of osteosarcoma via the miR-195-5p/FGF2/PI3K/Akt axis. J Exp Clin Cancer Res. 2021; 40(1):235.\u003c/li\u003e\n \u003cli\u003eWang Y, Chen X, Tang G, Liu D, Peng G, Ma W, et al. AS-IL6 promotes glioma cell invasion by inducing H3K27Ac enrichment at the IL6 promoter and activating IL6 transcription. FEBS Lett. 2016; 590(24):4586-4593.\u003c/li\u003e\n \u003cli\u003eZhai W, Ye X, Wang Y, Feng Y, Wang Y, Lin Y, et al. CREPT/RPRD1B promotes tumorigenesis through STAT3-driven gene transcription in a p300-dependent manner. Br J Cancer. 2021; 124(8):1437-1448.\u003c/li\u003e\n \u003cli\u003eGalbraith K, Snuderl M. Molecular Pathology of Gliomas. Surg Pathol Clin. 2021; 14(3):379-386.\u003c/li\u003e\n \u003cli\u003eLudwig K, Kornblum HI. Molecular markers in glioma. J Neurooncol. 2017; 134(3):505-512.\u003c/li\u003e\n \u003cli\u003eIwami K, Natsume A, Wakabayashi T. Cytokine networks in glioma. Neurosurg Rev. 2011; 34(3):253-63.\u003c/li\u003e\n \u003cli\u003eOkada H, Pollack IF. Cytokine gene therapy for malignant glioma. Expert Opin Biol Ther. 2004; 4(10):1609-20.\u003c/li\u003e\n \u003cli\u003eCao F, Zhang Q, Chen W, Han C, He Y, Ran Q, et al. IL-6 increases SDCBP expression, cell proliferation, and cell invasion by activating JAK2/STAT3 in human glioma cells. Am J Transl Res. 2017; 9(10):4617-4626.\u003c/li\u003e\n \u003cli\u003eWang Q, He Z, Huang M, Liu T, Wang Y, Xu H, et al. Vascular niche IL-6 induces alternative macrophage activation in glioblastoma through HIF-2\u0026alpha;. Nat Commun.\u0026nbsp;2018; 9(1):559.\u003c/li\u003e\n \u003cli\u003ePegtel DM, Gould SJ. Exosomes. Annu Rev Biochem. 2019; 88:487-514.\u003c/li\u003e\n \u003cli\u003eLi B, Cao Y, Sun M, Feng H. Expression, regulation, and function of exosome-derived miRNAs in cancer progression and therapy. FASEB J. 2021; 35(10):e21916.\u003c/li\u003e\n \u003cli\u003eZhang J, Li S, Li L, Li M, Guo C, Yao J, Mi S. Exosome and exosomal microRNA: trafficking, sorting, and function. Genomics Proteomics Bioinformatics. 2015 Feb;13(1):17-24.\u003c/li\u003e\n \u003cli\u003eXu Z, Chen Y, Ma L, Chen Y, Liu J, Guo Y, et al. Role of exosomal non-coding RNAs from tumor cells and tumor-associated macrophages in the tumor microenvironment. Mol Ther. 2022; 30(10):3133-3154.\u003c/li\u003e\n \u003cli\u003eTian Y, Gao X, Yang X, Chen S, Ren Y. Glioma-derived exosome Lncrna Agap2-As1 promotes glioma proliferation and metastasis by mediating Tgf-\u0026beta;1 secretion of myeloid-derived suppressor cells. Heliyon. 2024; 10(9):e29949.\u003c/li\u003e\n \u003cli\u003eLi J, Wang K, Yang C, Zhu K, Jiang C, Wang M, et al. Tumor-Associated Macrophage-Derived Exosomal LINC01232 Induces the Immune Escape in Glioma by Decreasing Surface MHC-I Expression. Adv Sci (Weinh). 2023; 10(17):e2207067.\u003c/li\u003e\n \u003cli\u003eDorrington MG, Fraser IDC. NF-\u0026kappa;B Signaling in Macrophages: Dynamics, Crosstalk, and Signal Integration. Front Immunol. 2019; 10:705.\u003c/li\u003e\n \u003cli\u003eYunna C, Mengru H, Lei W, Weidong C. Macrophage M1/M2 polarization. Eur J Pharmacol. 2020; 877:173090.\u003c/li\u003e\n \u003cli\u003eZhao T, Zeng J, Xu Y, Su Z, Chong Y, Ling T, et al. Chitinase-3 like-protein-1 promotes glioma progression via the NF-\u0026kappa;B signaling pathway and tumor microenvironment reprogramming. Theranostics. 2022; 12(16):6989-7008.\u003c/li\u003e\n \u003cli\u003eXu Y, Liao C, Liu R, Liu J, Chen Z, Zhao H, et al. IRGM promotes glioma M2 macrophage polarization through p62/TRAF6/NF-\u0026kappa;B pathway mediated IL-8 production. Cell Biol Int. 2019; 43(2):125-135.\u003c/li\u003e\n \u003cli\u003eQian M, Wang S, Guo X, Wang J, Zhang Z, Qiu W, et al. Hypoxic glioma-derived exosomes deliver microRNA-1246 to induce M2 macrophage polarization by targeting TERF2IP via the STAT3 and NF-\u0026kappa;B pathways. Oncogene. 2020; 39(2):428-442.\u003c/li\u003e\n \u003cli\u003eNi J, Shen Y, Wang Z, Shao DC, Liu J, Kong YL, et al. P300-dependent STAT3 acetylation is necessary for angiotensin II-induced pro-fibrotic responses in renal tubular epithelial cells. Acta Pharmacol Sin. 2014; 35(9):1157-66.\u003c/li\u003e\n \u003cli\u003eWang Y, Zhou C, Gao H, Li C, Li D, Liu P, et al. Therapeutic effect of Cryptotanshinone on experimental rheumatoid arthritis through downregulating p300 mediated-STAT3 acetylation. Biochem Pharmacol. 2017; 138:119-129.\u003c/li\u003e\n \u003cli\u003eGe W, Li YA, Ruan Y, Wu N, Ma P, Xu T, et al. IL-17 induces NSCLC cell migration and invasion by elevating MMP19 gene transcription and expression through the interaction of p300-dependent STAT3-K631 acetylation and its Y705-phosphorylation. Oncol Res. 2024; 32(4):625-641.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4616289/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4616289/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCytokines, 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.\u003c/p\u003e","manuscriptTitle":"Exosome-derived circ-001422 promote tumor-associated macrophage M2 polarization to accelerate the progression of glioma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-17 05:23:07","doi":"10.21203/rs.3.rs-4616289/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6737152e-ad99-4ea4-84f9-9b8655015027","owner":[],"postedDate":"July 17th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":33737122,"name":"Biological sciences/Cancer/CNS cancer"},{"id":33737123,"name":"Biological sciences/Molecular biology/Transcription"},{"id":33737124,"name":"Biological sciences/Genetics/Gene expression"}],"tags":[],"updatedAt":"2024-07-25T14:13:00+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-17 05:23:07","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4616289","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4616289","identity":"rs-4616289","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}