Circular RNA CircCSPP1 Promotes the Occurrence and Development of Colon Cancer by Sponging miR-431 and Regulating the Expressions of ROCK1 and ZEB1

Colon cancer is a common malignant tumor of the digestive tract, and its incidence is ranked third among gastrointestinal tumors. The present study investigated the role of a novel circular RNA (circCSPP1) in colon cancer and explored the possible underlying molecular mechanisms. Bioinformatics analysis and reverse transcription-quantitative PCR were used to detect the expression levels of circCSPP1 in colon cancer specimens and cell lines. The effects of circCSPP1 on the behavior of colon cancer cells were investigated using CCK-8, Transwell and clonogenic assays. Bioinformatics analysis along with luciferase, uorescence in situ hybridization and RNA pull-down assays were used to reveal the interaction between circCSPP1, microRNA (miR)-431, Rho associated coiled-coil containing protein kinase 1 (ROCK1) and zinc nger E-box binding homeobox 1 (ZEB1). It was found that circCSPP1 expression was signicantly upregulated in colon cancer tissues and cell lines. The overexpression of circCSPP1 signicantly promoted the proliferation, migration and invasion of colon cancer cells, whereas the silencing of circCSPP1 exerted opposite effects. Mechanistically, circCSPP1 was found to bind with miR-431. In addition, ROCK1 and ZEB1 were identied as the target genes of miR-431. Rescue experiments further conrmed the interaction between circCSPP1, miR-431, ROCK1 and ZEB1. Moreover, circCSPP1 promoted the expression levels of ROCK1, cyclin D1, cyclin-dependent kinase 4, ZEB1 and Snail, and lowered the E-cadherin expression level. Notably, circCSPP1 from SW620 cells was transferred to macrophages via exosomes and enhanced the colon cancer microenvironment. Taken together, the ndings of the present study indicated that circCSPP1 may functions as a competing endogenous RNA in the progression of colon cancer by regulating the miR-431/ROCK1 and miR-431/ZEB1 signaling axes.


Introduction
Colon cancer is a common malignant tumor of the digestive tract, and its incidence is ranked third among gastrointestinal tumors (1). Over the past few decades, the rapid development of molecular biology has enriched the theory of colorectal cancer carcinogenesis (1)(2)(3)(4). In addition, immense progress has been made in diagnostic and treatment strategies for colorectal cancer; the 5-year survival rate of patients with localized disease is 90.1% (5). However, following the metastasis of colorectal cancer to adjacent organs or lymph nodes, the 5-year survival rate of patients decreases to 69.2%. Of note, only 39% of patients with colorectal cancer are diagnosed at the localized stage of the disease, prior to metastasis (6, 7). Therefore, further in-depth investigations of the pathogenesis of colorectal cancer, as well as the identi cation of more effective early diagnostic and treatment strategies for colorectal cancer, are of utmost importance.
MicroRNAs (miRNAs/miRs) are small endogenous single-stranded RNA molecules composed of ~20 nucleotides, which act mostly on the 3'UTR of target mRNAs and either degrade or inhibit multiple transcripts (8, 9). Previous studies have demonstrated that miRNAs play a critical regulatory role in the initiation and progression of human cancers (10,11). Circular RNAs (circRNAs) are newly discovered non-coding RNAs with a covalently closed ring structure, which are widely found in a variety of cells (12)(13)(14)(15). They are produced by the reverse splicing of precursor mRNAs and are characterized by a stable structure, a conserved sequence and tissue speci city. Recent studies have indicated that circRNAs can act as miRNA sponges to inhibit the activity of targeted miRNAs (16,17). In addition, circRNAs can regulate gene transcription by binding with RNA binding proteins, or can be translated to produce proteins (18). Thus, circRNAs play a vital role during the progression of tumors, and may provide a novel direction for tumor diagnosis and therapy (19,20).
In the present study, the commonly differentially expressed circRNAs between colon cancer tissues and adjacent normal tissue in two datasets were screened with the aim of identifying novel molecular targets for the treatment of colon cancer. It was found that circCSPP1 was signi cantly upregulated in cancer tissues. In addition, the role of circCSPP1 in colon cancer was examined in vitro and in vivo.

Materials And Methods
Specimen collection. Cancer tissues and adjacent normal tissues were collected from 25 patients (14 male, 11 female), who diagnosed with colon cancer at the First A liated Hospital of Soochow University (Suzhou, China) (August, 2020 to July, 2021). These patients received no treatment before and age of them was range from 37 to 72 old. The tissues were stored in liquid nitrogen immediately after resection. The present study was approved by the Ethics Committee of the First A liated Hospital of Soochow University (No. FAHSU20200719) and written informed consent was obtained from each patient.
Gene Expression Omnibus (GEO) data analysis. The present study analyzed the GSE121895 and GSE126094 datasets from the GEO database. The expression levels in each group were normalized. The threshold value of differentially expressed genes was set at two of different multiples and P<0.05.
Hybridizations were performed according to the manufacturer's instructions provided with the uorescence in situ hybridization kit. The cell nuclei were stained with DAPI at room temperature for 20 min. Subsequently, images were visualized using a uorescence microscope (200x) as previously described (24).
Luciferase assay. The luciferase assay was performed using the dual-luciferase reporting system psiCHECK (Thermo Fisher Scienti c, Inc.). The wild-type (WT) or mutant-type (mut) sequences of circCSPP1, ROCK1 and ZEB1 were cloned into the psiCHECK2 plasmid. 293T cells (ATCC, 2x10 4 cells/well) were cultured overnight in 24-well plates. The cells were transfected with the WT or mut reporter vector along with miR-431 mimics (10 nM) or mimics control (10 nM) using Lipofectamine® 3000 (Thermo Fisher Scienti c, Inc.). Finally, the luciferase activity of cells was detected with a Dual-Luciferase Detection kit (Promega Corporation) after 48 h of transfection. The data were quanti ed by normalizing to Renilla luciferase activity. RNA pull-down assay. Biotin labeled miR-431 and the control probes were synthesized by Sangon Biotech (Shanghai) Co., Ltd. Probe-coated beads were generated by co-incubation with streptavidin-coated beads (Thermo Fisher Scienti c, Inc.) at 25˚C for 2 h. The SW620 and LOVO cells were collected, lysed and incubated with miR-431 probes overnight at 4˚C. Thereafter, the beads were eluted, and the complex was puri ed using TRIzol® reagent (Takara Biotechnology Co., Ltd.). The levels of circCSPP1, ROCK1 and ZEB1 were then analyzed using RT-qPCR.
RNA immunoprecipitation (RIP) assay. RIP assay was performed using the EZ-Magna RIP RNA-Binding Protein Immunoprecipitation kit (MilliporeSigma). Brie y, magnetic beads conjugated with negative control normal IgG (cat.no. AB21-KC, 1:5,000) or anti-Ago2 (cat.no. 03-110, 1:5,000) antibody (MilliporeSigma) were co-incubated with the cell lysates for 4 h at room temperature. To investigate the enrichment of the binding targets, the immunoprecipitated RNAs were extracted and subjected to RT-qPCR.
Xenograft tumor model. Nude mice (n=24, 4-6 weeks old, 20-22 g) were obtained from the Animal center of Soochow University and randomly divided into four groups (shRNA2 ctrl, circCSPP1 shRNA2, pcDNA3.1 ctrl and pcDNA3.1-circCSPP1). All mice were housed in a SPF-grade animal room (temperature 18-22˚C; humidity 40-60%; light/dark cycle 12/12 h each day) and had free access to food and water. The subcutaneous injection of colon cancer cells was performed after 3 days of adaptive breeding. Each mouse was subcutaneously injected with 3x10 6 colon cancer cells (100 µl in PBS). Tumor size was measured every 2 days, and the major axis (a) and minor axis (b) of the tumor were measured. The tumor volume was calculated using the following formula: ab 2 /2. At the end of the experiment, the mice were sacri ced using a 40% volume/min CO 2  Isolation and characterization of exosomes. SW680 cells were cultured in DMEM supplemented with 10% FBS. Following 48 h of incubation at 37℃, the media were collected and centrifuged at 650 x g for 15 min, followed by 155,00 x g for 20 min at 4˚C (FBS was depleted of exosomes). The supernatants were then ltered (0.22 µm; MilliporeSigma) and centrifuged at 120,000 x g for 60 min at 4˚C. After washing with PBS, the exosome pellets were again centrifuged at 120,000 x g for 60 min at 4˚C. The quantity of exosomes was detected with BCA Protein Assay kit (Beyotime Institute of Biotechnology). Additionally, the particle size distribution of the exosomes was detected using NanoSight (Malvern Panalytical).
For observing the structure of the exosomes, transmission electron microscopy was used. Brie y, the exosomes were xed with 2% paraformaldehyde and stained with 2% phosphotungstic acid (Beyotime) at 4℃ for 2 min. The samples were then observed using a transmission electron microscope (Hitachi, Ltd.).
In addition, uorescent PKH67 dye (Thermo Fisher Scienti c, Inc.) was used to label the exosomes at 4℃ overnight and uorescent Phalloidin dye (Thermo Fisher Scienti c, Inc.) was used to label the cytoskeleton at room temperature for 2 h.
Cell cycle distribution analysis. SW620 or LOVO cells (5x10 5 ) were xed using with 75% ethanol for 20 min on ice. Then, cells were permeabilized with 0.25% Triton X-100 and stained with PI/RNase (Sigma Aldrich). Results circCSPP1 is highly expressed in colon cancer. To explore novel molecular targets for the treatment of colon cancer, the differentially expressed circRNAs in cancer and adjacent normal tissues were rst analyzed using two GEO datasets (GSE121895 and GSE126094) (Fig. 1A). Intersection analysis of the two omics data identi ed a total of 161 differentially expressed circRNAs (Fig. 1B). Further veri cation at the tissue level revealed that hsa_circ_0001806 (circCSPP1) was signi cantly upregulated in colon cancer ( Fig. 1C and D). Consistently, compared with the HFC cells, the circCSPP1 level was found to be upregulated in colon cancer cells (Fig. 1E). In addition, circRNA circularization data indicated that circCSPP1 was spliced by exons 8-11 of the CSPP1 transcript, which was con rmed by Sanger sequencing (Fig. 1F).
Subsequently, the distribution of circCSPP1 in the cells was detected using FISH assay and RT-qPCR. The data revealed that circCSPP1 was mainly located in the cytoplasm (Fig. 1G and H). Compared with linear RNA, circCSPP1 was more resistant to actinomycin or RNase R treatment (Fig. 1I and J). These data thus indicated that circCSPP1 had higher stability and a longer half-life.
Knockdown of circCSPP1 signi cantly inhibits the tumorigenesis of colon cancer. In order to investigate the role of circCSPP1 in colon cancer, cell proliferation, invasion and migration were detected. First, the expression of circCSPP1 was knocked down in colon cancer cells using shRNA1 and shRNA2. The results of RT-qPCR revealed that both these shRNAs effectively suppressed the level of circCSPP1 in the cells (Fig.  2A). In addition, the results of the CCK-8 assay demonstrated that the knockdown of circCSPP1 signi cantly inhibited the proliferation of colon cancer cells (Fig. 2B). Consistently, circCSPP1 knockdown notably decreased the colony-forming ability of the cells (Fig. 2C). Moreover, Transwell assay revealed that the knockdown of circCSPP1 inhibited the invasive and migratory ability of colon cancer cells (Fig. 2D and E). These data thus suggested that the knockdown of circCSPP1 signi cantly inhibited the progression of colon cancer. circCSPP1 promotes colon cancer tumor growth and metastasis in vivo. With the purpose of con rming the biological function of circCSPP1 in colon cancer, an animal experiment was performed. The results of the animal experiment revealed that the overexpression of circCSPP1 signi cantly promoted tumor growth, whereas the knockdown of circCSPP1 inhibited tumor growth (Fig. 3A-D). Moreover, the metastasis of colon cancer in vivo was assessed. The results indicated that circCSPP1 knockdown notably decreased the metastasis of colon cancer, whereas circCSPP1 overexpression promoted metastasis ( Fig. 3E and F). In addition, the level of circCSPP1 in tumor tissues was inhibited by circCSPP1 shRNA2, while it was upregulated by circCSPP1 (Fig. 3G). On the whole, these data indicated that circCSPP1 promoted colon cancer tumor growth and metastasis in vivo. circCSPP1 sponges with miR-431 in colon cancer cells. To explore the potential target of circCSPP1, Circinteractome (https://circinteractome.irp.nia.nih.gov/) was used. A total of six miRNAs (miR-197, miR-324-5p, miR-375, miR-431, miR-324-5p and miR-486-3p) were predicted to be the candidate targets of circCSPP1 (Fig. S1A). Luciferase reporter assay was then used to screen the candidate binding miRNAs. The data revealed that the relative luciferase activities of the cells were notably inhibited by miR-431 mimics or miR-486-3p mimics (Fig. S1A). Based on these data, miR-431 was demonstrated to be the most likely candidate target. The binding site between circCSPP1 and miR-431 is presented in Fig. S1B.
Subsequently, luciferase reporter assay con rmed that circCSPP1 was able to bind to miR-431 (Fig. S1C). In addition, the results of FISH assay revealed the co-localization of circCSPP1 with miR-431 in the cytoplasm of the cells (Fig. S1D). Moreover, RIP and RNA pull-down assays revealed that circCSPP1 could directly bind with miR-431 and RT-qPCR data suggested knockdown or overexpression of circCSPP1 did not affect the expression of miR-431 in cell (Fig. S1E, F and G). Thus, these data suggested that circCSPP1 sponged miR-431 in colon cancer cells.
RT-qPCR data then revealed that ROCK1 expression was notably upregulated in colon cancer tissues compared with adjacent normal tissues (Fig. S2B). The potential complementary pairing sequence between miR-431 and the 3'-UTR of ROCK1 is presented in Fig. S2C. In addition, luciferase reporter assay indicated that the luciferase activity of the cells carrying the WT ROCK1 3'-UTR was signi cantly reduced by miR-431 mimics (Fig. S2D). Consistently, the results of RIP and RNA pull-down assays revealed the direct interaction between miR-431 and ROCK1 (Fig. S2E and F). On the whole, these data con rmed that ROCK1 was a target gene of miR-431 in colon cancer cells.
Knockdown of ROCK1 reverses the tumor-promoting effects of circCSPP1. To further con rm the interaction among circCSPP1, miR-431 and ROCK1, rescue experiments were performed. The results of RT-qPCR revealed that the overexpression of circCSPP1 promoted ROCK1 expression, whereas this effect was reversed by transfection with miR-431 mimics (Fig. 4A). In addition, the data of CCK-8 and colony formation assays indicated that circCSPP1 signi cantly increased colon cancer cell proliferation, which was reversed by transfection with miR-431 mimics or by ROCK1 knockdown (Fig. 4B and C). Consistently, the Transwell assay results revealed that circCSPP1 notably promoted the migration and invasion of colon cancer cells, whereas these effects were reversed by miR-431 mimics or by ROCK1 knockdown (Fig. 4D and E). Additionally, the effects of circCSPP1, sh-ROCK1 or sh-ZEB1 on their target genes in cells were detected with RT-qPCR, respectively (Fig. S4A, B and C). Meanwhile, miR-431 mimics signi cantly increased the level of miR-431, while miR-431 inhibitor exhibited completely opposite effect ( Fig. S4D and   F). Taken together, these ndings demonstrated that the knockdown of ROCK1 reversed the tumorpromoting effects of circCSPP1. miR-431 targets ZEB1 in colon cancer cells. The present study then explored the interaction among circCSPP1, miR-431 and ZEB1. The potential complementary pairing sequence between miR-431 and the 3'-UTR of ZEB1 is presented in Fig. 5A. The results of luciferase reporter experiment indicated that miR-431 mimics signi cantly reduced the luciferase activity of cells carrying the WT ZEB1 3'-UTR (Fig. 5B). In addition, the results of RT-qPCR and western blot analyses revealed that miR-431 mimics notably decreased ZEB1 expression at the mRNA and protein level in the cells; by contrast, miR-431 inhibitor increased ZEB1 expression (Fig. 5C-E). Moreover, RIP assay using the antibody against Ago2 con rmed the interaction between miR-431 and ZEB1 (Fig. 5F).
Subsequently, the interaction between circCSPP1 and ZEB1 was investigated using RT-qPCR. The data revealed that the overexpression of circCSPP1 promoted the level of ZEB1; however, this phenomenon was completely reversed by transfection with miR-431 mimics or by ZEB1 knockdown (Fig. 5G). Similarly, the promoting effects of circCSPP1 on cell invasion and migration were signi cantly inhibited by ZEB1 knockdown (Fig. 5H and I). Thus, these data illustrated miR-431 targeted ZEB1 in colon cancer cells. circCSPP1 activates the cyclin D1/CDK4/Rb signaling pathway in colon cancer. To further examine the role of the circCSPP1 in colon cancer, the levels of cell cycle-related proteins were detected using western blot analysis. The results indicated that the overexpression of circCSPP1 increased the expression of ROCK1, cyclin D1, p-CDK4 and p-Rb in the cells; however, these phenomena were reversed by transfection with miR-431 mimics or by ROCK1 knockdown (Fig. 6A). Moreover, it was found that circCSPP1 overexpression also increased the expression of ZEB1 and Snail, and downregulated the E-cadherin level.
Similarly, the effects of circCSPP1 overexpression on these proteins were reversed by transfection with miR-431 mimics or by ZEB1 knockdown (Fig. 6B). In addition, circCSPP1 knockdown induced G1 arrest, while circCSPP1 overexpression promoted G1 arrest in colon cancer cells (Fig. S3A and B). The potential mechanism through which circCSPP1 regulates the progression of colon cancer are presented in Fig. 6C.
The schematic diagram illustrates that circCSPP1 promotes the progression of colon cancer by regulating the miR-431/ROCK1 and miR-431/ZEB1 pathways.
Transfer of circCSPP1 from SW620 cells to macrophages cells via exosomes. As is known, tumor-derived exosomes play vital roles in cancer progression (27). Therefore, in the present study, exosomes were isolated from SW620 cells (with or without circCSPP1 overexpression). As demonstrated in Fig. 7A and B, the tumor cell-derived exosomes were round, cup-shaped particles ranging from 50 to 150 nm in diameter.
In addition, the exosomal protein markers, CD81 and CD9, were highly expressed in the exosomes derived from the SW620 cells (Fig. 7C). Moreover, the level of circCSPP1 in exosomes derived from the SW620 cells overexpressing circCSPP1 was signi cantly unregulated compared with that from the control SW620 cells (Fig. 7D).
To explore cell-to-cell crosstalk by transmitting circCSPP1 between SW620 cells and macrophages (PMAstimulated THP-1 monocytes), macrophages were incubated with SW620 cell-derived exosomes (PKH26labeled) for 48 h. The results indicated that the PKH26 lipid dye could be observed in the cytoplasm of the macrophages, suggesting that the SW620 cell-derived exosomes were transferred to the macrophages (Fig. 7E). On the whole, these data suggested circCSPP1 could be transferred from SW620 cells to macrophages via exosomes. SW620 cell-derived exosomes promote macrophage M2 polarization. The present study then examined the effects of exosomes derived from SW620 cells (with or without circCSPP1 overexpression) on macrophage M2 polarization. As demonstrated in Fig. 8A, exosomes derived from SW620 cells (with or without circCSPP1 overexpression) signi cantly increased the rate of CD206-positive macrophages. In addition, the levels of the M2 macrophage-associated cytokines, arginase-1 and IL-10, were signi cantly increased when the macrophages incubated with exosomes derived from SW620 cells (with or without circCSPP1 overexpression) (Fig. 8B). Moreover, the invasiveness and migratory ability of the SW620 cells was notably promoted when the cells were co-incubated with exosome-treated macrophages (Fig. 8C).
These data suggested that SW620 cell-derived exosomes promoted macrophage M2 polarization.

Discussion
Increasing evidence has uncovered the critical role of circRNAs in the progression of human cancers, including colon cancer (19,20). A previous study reported that circCCDC66 expression was upregulated in colon cancer and a high expression of circCCDC66 was associated with a poor prognosis of the patients (28). In the present study, the dysregulated circRNAs were analyzed using the GEO database. A novel circRNA back-splicing 8-11 exons of the CSPP1 gene, termed circCSPP1, was found, which plays a tumorpromoting role in colon cancer.
Subsequently, the mechanisms through which circCSPP1 regulates the progression of colon cancer were explored. circRNA can function as a sponge of various miRNAs and as a competing endogenous RNA. In colon cancer, hsa_circ_0055625 has been shown to increase cell proliferation by sponging miR-106b-5p (1). circRNA CBL.11 has been shown to suppress cell proliferation by sponging miR-6778-5p (29). Similar to these reports, the present study found that circCSPP1 promoted the progression of colon cancer by sponging miR-431 and regulating the expression of ROCK1 and ZEB1. ROCK1/2 are Rho-GTPase effectors that control vital aspects of the actin cytoskeleton. The RhoA/ROCK pathway is activated in a variety of tumors and exerts a direct regulatory effect on the mobility of tumor cells (30)(31)(32). Previous research has indicated that G1/S progression requires ROCK (33). The role of ROCK1 in the regulation of the cell cycle may explain its effect on the proliferation of colon cancer cells.
The other pathway is the induction of CDKI p16, which prevents the CDK4/6-mediated phosphorylation of Rb proteins, thereby blocking E2F-dependent transcription (34). The present study found that circCSPP1 promoted the expression of cyclin D1, p-CDK4 and p-Rb through the regulation of ROCK1 and miR-431. In addition, ZEB1 is well-known to be involved in the regulation of EMT in cancer cells (35). The present study found that circCSPP1 promoted EMT in colon cancer by modulating ZEB1. However, there are more experiments are required to explore the possible interaction between miR-431/ROCK1 and miR-431/ZEB1. Such as can inhibition of one or more signaling pathways in Figure 6C attenuate the growth, migration and invasion of the cancer cells, even with circCSPP1 overexpression? In addition, one limitation of current study is that circCSPP1/miR-431/ROCK1/Cyclin D/Rb axis was validated and the complex of cyclin D1 and CDK4 was not explored yet.
It has been reported that tumor cell-derived exosomes are closely associated with the progression of cancer (36), since exosomes can improve the microenvironment of cancer cells (37). Moreover, M2 macrophages have been reported to promote tumor metastasis and recurrence (38). In the present study, SW620 cell-derived exosomes increased M2 macrophage polarization, which in turn promoted the migration and invasion of colon cancer cells. However, a potential limitation of current study is that the properties of macrophage were not analyzed in the in vivo xenograft models.
In conclusion, the ndings of the present study demonstrated that circCSPP1 was upregulated in colon cancer and functioned as an oncogene. In addition, circCSPP1 promoted the progression of colon cancer functions as a competing endogenous RNA by the regulating miR-431/ROCK1 and miR-431/ZEB1 pathways. It was also found that circCSPP1 could be transferred from SW620 cells to macrophages via exosomes and, thus, enhanced the microenvironment of colon cancer. The ndings presented herein may provide novel insight into the pathogenesis of colon cancer. Figure 1 CircCSPP1 is highly expressed in colon cancer tissues.      MiR-431 targets ZEB1 in colon cancer cells.

Declarations
(A) The binding site between miR-431 and ROCK1 was predicted. (B) Luciferase reporter experiment was performed to detect the interaction between miR-431 and ZEB1. (C, D and E) colon cancer cells were treated with miR-431 mimics or miR-431 inhibitor for 24 h, the gene and protein level of ZEB1 was detected with RT-qPCR and WB, respectively. (F) RIP assay was performed to verify the binding between ZEB1 and miR-431. Colon cancer cells were treated with pcDNA3.1-circCSPP1, pcDNA3.1-circCSPP1 plus miR-431 mimics or pcDNA3.1-circCSPP1 plus ZEB1 shRNA (G). RT-qPCR was used to detect the expression of ZEB1. (H, I) Transwell assay was performed to assess the migration and invasion of colon cancer cells. **p<0.01; n = 3.  The potential mechanism by which circCSPP1 regulated the progression of colon cancer was presented. N = 3.

Figure 7
Isolation and characterization of exosomes.
(A) The morphology of isolated exosomes was observed by TEM. (B) The particle distribution of exosomes isolated from SW620 with or without pcDNA3.1-circCSPP1 (SW620 circCSPP1 Exo; SW620-Exo) was measured by NANOSIGHT. (C) The expressions of CD81 and CD9 in SW620-Exo or in SW620 circCSPP1 Exo were detected with western blot. (D) The level of -circCSPP1 in SW620-Exo or in SW620 circCSPP1 Exo was evaluated by RT-qPCR. (E) THP-1 (PMA-treated) cells were incubated with SW620-Exo for 24 h. Then, the cells dyed with PHK26, Phalloidin and DAPI and the morphology of cells was observed with uorescence microscope. **p<0.01 compared with SW620-Exo; n = 3.

Figure 8
CircCSPP1 was transferred from SW620 cells to macrophages cells via exosomes.
Macrophages were cultured with SW620 Exo, SW620 circCSPP1 Exo or 20 ng/ml IL-4/IL-13 for 24 h. (A) The CD206 positive macrophages were measured and quanti ed by ow cytometry. (B) The levels of Arginase-1 and IL-10 in macrophages were evaluated with RT-qPCR. (C) SW620 cells were co-cultured with macrophages (M0), Exo-treated macrophages or circCSPP1 Exo treated macrophages for 24 h, the cell migration and invasion ability was detected with transwell assays. **p<0.01 compared with SW620; n = 3.

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