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. 2022 May 20;38(8):761–771. doi: 10.1002/kjm2.12555

M2 macrophage facilitated angiogenesis in cutaneous squamous cell carcinoma via circ_TNFRSF21/miR‐3619‐5p/ROCK axis

Jun Ma 1, Lei Huang 1, Yan‐Bin Gao 1, Min‐Xiong Li 1, Liang‐Long Chen 1, Lei Yang 1,✉
PMCID: PMC11896439  PMID: 35593591

Abstract

In recent years, the role of circular RNA in cancer cells has been studied broadly; however, the functional significance of circular RNA in the regulation of the tumor microenvironment (TME) is not fully understood. In this study, we aimed to reveal the role of circ_TNFRSF21 in M2 macrophage‐induced cutaneous squamous cell carcinoma (cSCC) angiogenesis. Quantitative polymerase chain reaction and Western blotting were performed to determine the levels of the indicated genes. Direct binding between circ_TNFRSF21 and miR‐3619‐5p, miR‐3619‐5p, and ROCK2 was verified by dual‐luciferase activity. The migration and invasion of human umbilical vein endothelial cells were evaluated by wound healing and transwell assays. Tube formation was performed to detect in vitro angiogenesis. Circ_TNFRSF21 and ROCK2 were upregulated in cSCC tissue, while miR‐3619‐5p was downregulated. Circ_TNFRSF21 negatively regulated the expression of miR‐3619‐5p, while miR‐3619‐5p negatively regulated the expression of ROCK2. miR‐3619‐5p suppressed tube formation by inhibiting ROCK signaling. M2 macrophages facilitated tube formation via the circ_TNFRSF21/miR‐3619‐5p/ROCK2 axis. Our present study revealed that circ_TNFRSF21 was elevated in M2 macrophages and mediated M2 macrophage‐induced tube formation in vitro.

Keywords: angiogenesis, circ_TNFRSF21, miR‐3619‐5p, ROCK2, tumor‐associated macrophages

Abbreviations

cSCC

cutaneous squamous cell carcinoma

ROCK2

Rho‐associated kinase 2

TNFRSF21

TNF receptor superfamily member 21

TAM

tumor‐associated macrophage

TME

tumor microenvironment

qRT–PCR

reverse transcription followed by quantitative real‐time PCR

WB

Western blot

1. INTRODUCTION

Cutaneous squamous cell carcinoma (cSCC) is the second most common type of cutaneous cancer worldwide. 1 cSCC originates from the stratum corneum of the skin and is mainly found in areas that often experience long‐term exposure to physical and chemical factors; these areas include the head, face, and neck. 2 The invasive phenotype of cSCC increases the risk of metastasis, leading to a significant decrease in the survival rate. The 5‐year survival rate of cSCC patients with lymph node metastasis is only 26%–34%. 3 Therefore, it is very important to find new potential biomarkers and prognostic factors for cSCC.

The tumor microenvironment (TME) is a complex system consisting of extracellular matrix and stromal cells, including tumor cells, immune cells, fibroblasts, and endothelial cells. The extracellular matrix (ECM) influences tumor development mainly through the release of extracellular molecules by cells in a paracrine and autocrine manner. 4 Tumor‐associated macrophages (TAMs) are macrophages derived from peripheral blood mononuclear cells that infiltrate solid tumor tissue and account for a large proportion of stromal cells. In recent years, an increasing number of studies have shown that TAMs are involved in promoting tumor development and stimulating tumor cell growth and metastasis through tumor angiogenesis, lymphangiogenesis, immunosuppression and other processes. 5 These findings suggested that TAMs might be a new target for intervention in the treatment of cancer.

As a novel noncoding RNA, in recent years, circular RNAs have been indicated to regulate TME in multiple ways. For instance, circ‐CCAC1 was found to facilitate angiogenesis in cholangiocarcinoma by elevating the expression of SH3GL2. 6 It was also demonstrated that circ‐Erbin substantially enhanced angiogenesis in colorectal cancer by increasing the expression of hypoxia‐induced factor (HIF‐1α). 7 Several studies have also indicated the critical role of circular RNA in macrophage polarization. As shown by Zhang et al., circPPM1F enhanced the M1 polarization of macrophages in type 1 diabetes mellitus. 8 In addition, circHECTD1 was also found to regulate macrophage activation in silicosis. 9 However, the regulatory effect of circular RNA on TAMs remains largely unknown. circ_TNFRSF21 (hsa_circ_0001610) is a novel circular RNA derived from the TNFRSF21 gene and was reported to be upregulated in endometrial carcinoma 10 and cSCC. 11 Our previous work revealed that circ_TNFRSF21 facilitated M2 polarization to promote cSCC metastasis. However, whether circ_TNFRSF21 is involved in TAM‐induced angiogenesis in the TME remains elusive.

In this study, our results demonstrated that circ_TNFRSF21 was elevated in M2 macrophages and mediated M2 macrophage‐induced tube formation in vitro. Mechanistically, circ_TNFRSF21 served as a sponge of miR‐3619‐5p to upregulate ROCK2 and facilitate tube formation. Our work might provide new evidence to identify effective biomarkers and prognostic factors for cSCC.

2. MATERIALS AND METHODS

2.1. Clinic sample collection and ethics statement

To compare the differential expression of circ_TNFRSF2, miR‐3619‐5p, ROCK2, CD163, and CD31, clinic cSCC tissues and adjacent tissues were obtained from 28 patients who underwent the surgical resection of tumors at Nanfang Hospital, Southern Medical University. This study was approved by the Medical Ethics Committee of Nanfang Hospital, Southern Medical University. Tissue specimens used for immunohistochemistry (IHC) analysis were embedded in paraffin, cut into 5‐μm‐thick sections, and probed with anti‐CD163 (Abcam, ab182422) and anti‐CD31 (Abcam, ab76533) antibodies overnight at 4 °C. Then, tissue sections were incubated with a secondary antibody at room temperature for 30 min. The immunostaining of each section was analyzed by an experienced pathologist who was blinded to the experiment.

2.2. Cell culture

The endothelial human umbilical vein endothelial cell (HUVECs) and monocytic leukemia THP‐1 cells were purchased from the Cell Bank of the Chinese Academy (Shanghai, China). A431 and SCL‐1 cells were cultured in DMEM (Invitrogen, USA) containing 10% fetal bovine serum (FBS) (Gibco, USA), and HUVECs and THP‐1 cells were cultured in RPMI 1640 (Invitrogen) with 10% FBS.

2.3. THP‐1 differentiated macrophage

For M0 macrophages, THP‐1 cells were incubated with 100 ng/ml phorbol myristate acetate (PMA) (Sigma–Aldrich) for 48 h. Then, M0 macrophages were incubated with 100 ng/ml lipopolysaccharide (LPS) (Sigma–Aldrich) and 100 ng/ml tumor necrosis factor α (TNF‐α) for 24 h and polarized into M1 macrophages; M0 macrophages were incubated with 20 ng/ml interleukin 4 (IL‐4) for 24 h and polarized into M2 macrophages. For TAM cells, THP‐1 cells were cultured in 50% RPMI 1640 containing 10% FBS and 50% conditioned medium obtained from A431 or SCL‐1 cells serum‐starved culture for 48 h. Flow‐cytometry analysis was then performed to analyze the surface markers of M0 macrophages (CD68), M1 macrophages (CD86), and M2 macrophages (CD206) using a FACSCalibur flow cytometer (BD Biosciences, USA).

2.4. Plasmid construction and transfection

The shCirc_TNFRSF21 for the silencing of TNFRSF21, the miR‐3619‐5p mimics for the overexpression of miR‐3619‐5p, the miR‐3619‐5p inhibitor for the inhibition of miR‐3619‐5p, and their negative controls were synthesized by Shanghai GenePharma. Cells were transfected with the above plasmid via Lipofectamine 3000 (Invitrogen) for 48 h.

2.5. Wound‐healing assay

A431 cell culture supernatant was obtained and used as HUVEC conditioned medium. HUVECs were cultured in 50% conditioned medium and 50% RPMI 1640 with 10% FBS for 24 h. A 200‐μl tip was equipped to draw beelines on the cell surface to form wounds, and then fresh RPMI 1640 medium was used instead of conditioned medium. The wound width was measured at 0 and 24 h.

2.6. Transwell invasion assay

Treated HUVECs were harvested and resuspended in RPMI 1640 without FBS. A total of 1 × 105 cells were seeded into transwell chamber‐coated Matrigel (BD Biosciences). After 48 h, the Matrigel membrane was abraded, and the invasive cells were stained and calculated.

2.7. Tube formation assay on Matrigel

Matrigel was held at 4°C overnight to liquefy, and 10‐μl liquid Matrigel was added to the μ‐slide platform chamber (Ibidi, Martinsried, Germany). A 50‐μl suspension containing 104 HUVECs was seeded into the μ‐slide chamber coated with polymerization Matrigel. After 12 h of incubation, tube‐like structures were captured under a microscope.

2.8. RNA extraction and quantitative real‐time PCR analysis

Total RNA of cSCC tissues or cells was extracted using TRIzol (Invitrogen), and cDNA was synthesized using a reverse transcription kit (TaKaRa, Japan). Next, gene expression differences were measured by quantitative PCR using an RT–PCR kit (Invitrogen). Data were analyzed using 2−ΔΔCt method.

2.9. Western blot assay

After the infected HUVECs were cultured in a conditioned medium, total protein lysates were extracted using RIPA reagent (Dingguo, Beijing). Total protein was separated on 10% SDS–PAGE. Primary antibodies specific for ROCK2 (Abcam, ab228000) and VEGFA (Abcam, ab214424) were used and incubated with PVDF membranes. Subsequently, the membranes were covered with secondary antibodies, and the protein beads were visualized and qualified by ImageJ software.

2.10. Luciferase reporter assay

The potential correlation between Circ_TNFRSF21 or ROCK2 and miR‐3619 was explored through StarBase. The full‐length wild‐type and mutant 3′UTRs of Circ_TNFRSF21 or ROCK2 were synthesized and cloned into the pmirGLO luciferase reporter vector (Gene Pharma) to construct the pmirGLO‐ROCK2‐WT, pmirGLO‐ROCK2‐MUT, pmirGLO‐Circ_TNFRSF21‐WT, and pmirGLO‐Circ_TNFRSF21‐MUT vectors. The vectors were cotransfected with miR‐3619 mimics or miR‐3619 inhibitors separately in HUVECs. The luciferase activities were determined via a Double‐Luciferase Reporter Assay Kit (Promega, USA).

2.11. RNA immunoprecipitation assay

An EZ Magna RIP kit (Merck Millipore, USA) was used for the RNA immunoprecipitation (RIP) assay. HUVEC cell lysates were extracted using RIP buffer and then coupled with Ago2 antibody (Abcam, ab32381) or IgG antibody (Abcam, ab172730) and incubated with magnetic beads for 4 h. Then, mixtures were placed in the magnet to obtain RNA immunoprecipitates, and TRIzol reagent was used to extract RNA.

2.12. Statistical analysis

GraphPad Prism 8.0 was used to perform the statistical analysis. Data were obtained from separate experiments performed in triplicate and are presented as the means ± SDs. Comparisons among groups were assessed using Student's t test or one‐way analysis of variance (ANOVA), and a value of p < 0.05 was considered meaningful.

3. RESULTS

3.1. Circ_TNFRSF21 and ROCK2 were upregulated in cSCC tissue, while miR‐3619‐5p was downregulated

We first investigated the abnormal expression of the involved molecules in cSCC tissue and adjacent tissue. As shown in Figure 1A–C, the expression levels of circ_TNFRSF21 and ROCK2 were substantially elevated in cSCC tissue compared to the adjacent tissue. However, the expression of miR‐3619‐5p showed the opposite trend, with lower expression in cSCC tissue. Pearson correlation analysis revealed that in cSCC tissue, the expression levels of circ_TNFRSF21 and ROCK2 were negatively correlated with that of miR‐3619‐5p, while the expression of circ_TNFRSF21 was positively correlated with that of ROCK2 (Figure 1D–F). TAMs were proven to be a major regulator of angiogenesis in the TME. 12 We next investigated the markers of TAMs (CD163) and vascular endothelial cells (CD31) in the collected cSCC tissue. As expected, the expression levels of CD163 and CD31 were both elevated in cSCC tissue (Figure 1G,H). Additionally, we confirmed that the expression of CD163 was positively correlated with that of CD31 in tumor tissue (Figure 1I). Consistently, the IHC analysis showed that the expression of CD163 and CD31 (Figure 1J) in cSCC tissues was significantly higher than that in adjacent tissue samples. Additionally, correlation analysis revealed that the protein expression levels of CD163 and CD31 in sSCC tissues were positively correlated (Figure 1K). Moreover, in sSCC tissue samples, the expression of circTNFRSF21 was positively correlated with the mRNA expression levels of both CD163 (Figure 1L) and CD31 (Figure 1M). The expression level of miR‐3619‐5p was negatively correlated with the mRNA level of CD31 (Figure 1N), while the level of ROCK2 was positively correlated with the mRNA expression level of CD31 (Figure 1O). Collectively, these results indicated that circ_TNFRSF21, ROCK2, CD163, and CD31 were upregulated in tumor tissue, while miR‐3619‐5p was downregulated.

FIGURE 1.

FIGURE 1

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circ_TNFRSF21 and ROCK2 were upregulated in cSCC tissue, while miR‐3619‐5p was downregulated. (A–C) The expression of circ_TNFRSF21, ROCK2, and miR‐3619‐5p in cSCC tissue was evaluated by qPCR. (D–F) Correlations between circ_TNFRSF21 and miR‐3619‐5p and between miR‐3619‐5p and ROCK2 were analyzed by Pearson correlation analysis. (G and H) mRNA expression of CD163 and CD31 in cSCC tissues was determined by qPCR. (I) Correlation between CD163 and CD31 was analyzed by Pearson correlation analysis. (J) Protein expression of CD163 and CD31 in cSCC tissues was detected by IHC. (K) Correlation of CD163 and CD31 protein expression in cSCC tissues was analyzed by Pearson correlation analysis. (L) Correlation of circ_TNFRSF21 and mRNA expression of CD163 in cSCC tissues was analyzed by Pearson correlation analysis. (M) Correlation of circ_TNFRSF21 and mRNA expression of CD31 in cSCC tissues was analyzed by Pearson correlation analysis. (N) Correlation of miR‐3619‐5p and mRNA expression of CD31 in cSCC tissues was analyzed by Pearson correlation analysis. (O) Correlation of ROCK2 and mRNA expression of CD31 in cSCC tissues was analyzed by Pearson correlation analysis. *p < 0.05; **p < 0.01; ***p < 0.001.

3.2. Circ_TNFRSF21 knockdown in M2 macrophages impaired in vitro tube formation

We next verified whether M2 macrophages promoted in vitro tube formation by releasing circ_TNFRSF21. Macrophage surface markers, including CD68 (M0 macrophage marker; Figure 2A), CD86 (M1 macrophage marker; Figure 2B), and CD206 (M2 macrophage marker; Figure 2C), were validated in treated THP‐1 cells by flow cytometry. As shown in Figure 2D, circ_TNFRSF21 was dramatically increased in M2 macrophages and TAMs. The expression of circ_TNFRSF21 showed a similar pattern in conditioned medium (CM) from M1, M2, and TAM culture systems (Figure 2E). To knockdown circ_TNFRSF21 in M0 macrophages, we transfected shRNA into M0 macrophages. Figure 2F shows that the expression of circ_TNFRSF21 was decreased in M0 macrophages transfected with shRNA targeting circ_TNFRSF21. Then, the transfected M0 macrophages were stimulated with IL‐4 to induce M2 polarization. Quantitative polymerase chain reaction (qPCR) showed that IL‐4 stimulation significantly increased the expression of M0 circ_TNFRSF21 in the CM, while circ_TNFRSF21 knockdown compromised this elevation (Figure 2G). CM from different groups was applied to treat vascular endothelial cells. As shown by wound‐healing assay and transwell assay, CM from M2 macrophages (IL‐4) substantially promoted the migrative and invasive ability of vascular endothelial cells (Figure 2H,I). Moreover, CM from M2 macrophages dramatically enhanced the tube formation of vascular endothelial cells, as indicated by an increased number of branches (Figure 2J). However, circ_TNFRSF21 knockdown impaired the migration, invasion and tube formation promoted by M2 CM. Collectively, these results indicated that circ_TNFRSF21 knockdown in M2 macrophages impaired in vitro tube formation.

FIGURE 2.

FIGURE 2

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circ_TNFRSF21 knockdown in M2 macrophages impaired in vitro tube formation. Flow‐cytometry analysis of treated THP‐1 cells was performed for the M0 macrophage marker CD68 (A), M1 macrophage marker CD86 (B), and M2 macrophage marker CD206 (C). (D and E) Expression of circ_TNFRSF21 was determined by qPCR in macrophages (D) and CM from macrophages (E). TAMs (tumor‐associated macrophages) were THP‐1 cells cultured in 50% RPMI 1640 containing 10% FBS and 50% conditioned medium obtained from A431 or SCL‐1 cell serum‐starved cultures for 48 h. (F) Knockdown efficiency of circ_TNFRSF21 in macrophages was determined by qPCR. (G–J) THP‐1 cells polarized into M2 macrophages by IL‐4 treatment. (G) Expression of circ_TNFRSF21 in CM after circ_TNFRSF21 knockdown was detected by qPCR. (H) Wound healing was applied to determine the migration of HUVECs. (I) HUVEC invasion was determined by transwell assay. (J) Tube formation assay was applied to evaluate in vitro angiogenesis of HUVECs. *p < 0.05; **p < 0.01; ***p < 0.001.

3.3. circ_TNFRSF21 negatively regulated the expression of miR‐3619‐5p

To investigate the potential mechanism of action of circ_TNFRSF21, we used starBase 2.0 to predict candidate miRNAs. As shown in Figure 3A, CM from M2 macrophages notably downregulated the expression of miR‐3619‐5p, while circ_TNFRSF21 knockdown recovered the expression (Figure 3A). miR‐3619‐5p mimics and inhibitor were transfected into vascular endothelial cells to overexpress or knockdown miR‐3619‐5p (Figure 3B). The potential binding site between circ_TNFRSF21 and miR‐3619‐5p was illustrated in Figure 3C. RIP assays were performed to detect the enrichment of circ_TNFRSF21 and miR‐3619‐5p in AGO2. The results suggested that both circ_TNFRSF21 and miR‐3619‐5p were enriched in AGO2 (Figure 3D). In addition, a dual luciferase assay illustrated that when the wild‐type binding site of circ_TNFRSF21 was cotransfected with the miR‐3619‐5p inhibitor, the relative luciferase activity was elevated. In contrast, when the wild‐type binding site of circ_TNFRSF21 was cotransfected with miR‐3619‐5p mimics, the relative luciferase activity was decreased. However, the mutated (MUT) binding site of circ_TNFRSF21 cotransfected with either miR‐3619‐5p mimics or miR‐3619‐5p inhibitor did not significantly influence the relative luciferase activity compared to the respective NC group (Figure 3E). Taken together, these results validated that circ_TNFRSF21 sponged miR‐3619‐5p.

FIGURE 3.

FIGURE 3

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circ_TNFRSF21 negatively regulated the expression of miR‐3619‐5p. (A) THP‐1 cells polarized into M2 macrophages by IL‐4 treatment. The expression of miR‐3619‐5p in HUVECs was evaluated by qPCR after M2 CM treatment. (B) Transfection efficiency of miR‐3619‐5p mimics, inhibitor or negative control was determined by qPCR. (C) Illustration of the binding site between circ_TNFRSF21 and miR‐3619‐5p. (D) Enrichment of circ_TNFRSF21 and miR‐3619‐5p in AGO2 was determined by RIP. (E) A dual luciferase assay was performed to validate the binding of circ_TNFRSF21 and miR‐3619‐5p. *p < 0.05; **p < 0.01; ***p < 0.001.

3.4. miR‐3619‐5p targeted ROCK2 in vascular endothelial cells

We next investigated the downstream target of miR‐3619‐5p. The RhoA/ROCK2 signaling pathway is a well‐known pathway that mediates angiogenesis in cancer. 13 As shown in Figure 4A, CM from M2 macrophages substantially upregulated the expression of ROCK2, while circ_TNFRSF21 knockdown decreased the expression of ROCK2. miR‐3619‐5p mimics reduced the expression of ROCK2, while the miR‐3619‐5p inhibitor showed the opposite effect on the expression of ROCK2 (Figure 4B,C). The potential binding site between ROCK2 and miR‐3619‐5p was shown in Figure 4D. Dual‐luciferase assays illustrated that when the wild‐type binding site of ROCK2 was cotransfected with miR‐3619‐5p inhibitor, the relative luciferase activity was elevated. In contrast, when the wild‐type binding site of ROCK2 was cotransfected with miR‐3619‐5p mimics, the relative luciferase activity was decreased. However, the mutated (MUT) binding site of ROCK2 cotransfected with either miR‐3619‐5p mimics or miR‐3619‐5p inhibitor did not significantly influence the relative luciferase activity compared to the respective NC group (Figure 4E). Collectively, these results validated that miR‐3619‐5p targeted ROCK2 and decreased the expression of ROCK2.

FIGURE 4.

FIGURE 4

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miR‐3619‐5p targeted ROCK2 in vascular endothelial cells. (A) THP‐1 cells polarized into M2 macrophages by IL‐4 treatment. The expression of ROCK2 in HUVECs treated with M2 CM was evaluated by qPCR. (B and C) The expression of ROCK2 in HUVECs transfected with miR‐3619‐5p mimics or inhibitor was evaluated by qPCR (B) and Western blot assay (C). (D) Illustration of the binding site between ROCK2 and miR‐3619‐5p. (E) A dual‐luciferase assay was performed to validate the binding of ROCK2 and miR‐3619‐5p. *p < 0.05; **p < 0.01; ***p < 0.001.

3.5. miR‐3619‐5p suppressed tube formation by inhibiting ROCK signaling

To investigate whether miR‐3619‐5p regulates tube formation of vascular endothelial cells through ROCK signaling, we simultaneously used the miR‐3619‐5p inhibitor and ROCK pathway inhibitor Y‐27632 to treat cells. As shown in Figure 5A,B, miR‐3619‐5p overexpression by mimics attenuated the migration and invasion of cells. In contrast, the miR‐3619‐5p inhibitor accelerated the migration and invasion of vascular endothelial cells. Interestingly, the addition of Y‐27632 compromised the promotion of migration and invasion. Similarly, miR‐3619‐5p overexpression inhibited the tube formation of vascular endothelial cells, while miR‐3619‐5p inhibition facilitated the tube formation of vascular endothelial cells. However, Y‐27632 reduced the miR‐3619‐5p inhibitor and elevated the number of tube branches (Figure 5C). Additionally, we also evaluated the expression of VEGFA, a major regulator of angiogenesis. As shown in Figure 5D, miR‐3916‐5p mimics attenuated, while miR‐3619‐5p inhibitor elevated, the expression of VEGFA. Treatment with Y‐27632 reversed the increase in VEGFA expression induced by the miR‐3619‐5p inhibitor. Taken together, our results indicated that miR‐3619‐5p suppressed tube formation by inhibiting ROCK signaling.

FIGURE 5.

FIGURE 5

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miR‐3619‐5p suppressed tube formation by inhibiting ROCK signaling. HUVECs were transfected with miR‐3619‐5p mimics or simultaneously treated with miR‐3619‐5p inhibitor and ROCK pathway inhibitor Y‐27632. (A) Wound healing was applied to determine the migration of HUVECs. (B) HUVEC invasion was determined by transwell assay. (C) Tube formation assay was applied to evaluate in vitro angiogenesis of HUVECs. (D) VEGFA expression was determined by Western blot assay. *p < 0.05; **p < 0.01; ***p < 0.001.

3.6. M2 macrophages facilitated tube formation via the circ_TNFRSF21/miR‐3619‐5p/ROCK axis

The aforementioned results demonstrated that M2 macrophages facilitated tube formation via circ_TNFRSF21. In the last part, we investigated whether M2 macrophages facilitated tube formation via the circ_TNFRSF21/miR‐3619‐5p/ROCK axis. Vascular endothelial cells were transfected with miR‐3619‐5p mimics or Y27632 before treatment with CM from IL‐4‐induced M2 macrophages. As shown in Figure 6A,B, CM treatment promoted the migration and invasion of vascular endothelial cells. Interestingly, transfection of miR‐3619‐5p mimics or addition of Y‐27632 compromised the promoted migration and invasion. Similarly, CM treatment facilitated tube formation of vascular endothelial cells. However, miR‐3619‐5p mimics or Y‐27632 reduced the CM‐induced increase in the number of tube branches (Figure 6C). As shown in Figure 6D, CM treatment elevated the expression of VEGFA. Treatment with Y‐27632 or miR‐3619‐5p mimics reversed the increase in VEGFA expression induced by CM treatment. Collectively, these results indicated that M2 macrophages facilitated tube formation via the circ_TNFRSF21/miR‐3619‐5p/ROCK axis.

FIGURE 6.

FIGURE 6

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M2 macrophages facilitated tube formation via the circ_TNFRSF21/miR‐3619‐5p/ROCK axis. THP‐1 cells polarized into M2 macrophages by IL‐4 treatment. Vascular endothelial cells were transfected with miR‐3619‐5p mimics or Y27632 before treatment with CM from IL‐4‐induced M2 macrophages. (A) Wound healing was applied to determine the migration of HUVECs. (B) HUVEC invasion was determined by transwell assay. (C) Tube formation assay was applied to evaluate in vitro angiogenesis of HUVECs. (D) VEGFA expression was determined by Western blot. *p < 0.05; **p < 0.01; ***p < 0.001.

4. DISCUSSION

Recently, numerous reports have revealed the critical role of circular RNA in cancer progression. 14 In this study, we found that circ_TNFRSF21 was upregulated in cSCC and regulated cell–cell interactions in the TME. circ_TNFRSF21 was involved in angiogenesis induced by M2 macrophages. Mechanistically, circ_TNFRSF21 in CM from M2 macrophages facilitated angiogenesis by sponging miR‐3619‐5p and thus upregulating ROCK2 signaling.

Although the role of circular RNA in cancer cells has been studied broadly, the functional significance of circular RNA in the regulation of the TME is not fully understood. It was reported that hsa_circ_0005519 was involved in tumor immunity via regulation of the cytokines IL‐13 and IL‐6 in CD4+ T cells. 15 Moreover, cancer‐induced circular RNAs have also been proven to regulate NK cell activity. 16 CircASAP1 was revealed as a key regulator of TAM infiltration by targeting miR‐326/miR‐532‐5p. 17 In addition, circ_CCAC1 was proven to contribute to the progression of cholangiocarcinoma by inducing angiogenesis in the TME. 6 Circular RNA MYLK was also verified as a regulator of angiogenesis in bladder cancer by facilitating VEGFA/VEGFR2 signaling. 18 In this study, we revealed a novel regulatory work of circ_TNFRSF21 involved in M2 macrophage‐induced angiogenesis in the TME. Moreover, we demonstrated that circ_TNFRSF21 was upregulated in M2 macrophages and TAMs, which was consistent with our previous work showing that circ_TNFRSF21 facilitates M2 polarization in cSCC.

MiR‐3619‐5p was proven to be a tumor suppressive miRNA in several cancer types. Liu et al. proved that miR‐3619‐5p prohibited gastric adenocarcinoma by targeting TBC1D10B. 19 A previous study also indicated that miR‐3619‐5p inhibited the stemness and chemoresistance of gastric cancer by targeting the AMPK/PGC1‐α/CEBPB axis. 20 Moreover, miR‐3619‐5p was also proven to impair the cell growth of prostate cancer via upregulation of CDKN1A expression. 21 In hepatocellular carcinoma, miR‐3619‐5p was found to exert tumor suppressive effects by targeting ARL2. 22 A previous study also verified that miR‐3619‐5p was downregulated in cisplatin‐resistant cSCC cells and inhibited the proliferation and cisplatin resistance of cSCC. 23 Our present study revealed that miR‐3619‐5p was downregulated in cSCC tissue, which was consistent with a previous report. Moreover, we revealed for the first time that miR‐3619‐5p regulated tube formation in HUVECs, indicating that miR‐3619‐5p was involved in the regulatory work of the TME.

Rho GTPase belongs to the RAS superfamily and is involved in cell migration, phagocytosis, contraction, and adhesion. Rho‐associated kinase (ROCK) is the downstream target effector molecule of Rho. Rho‐ROCK pathway is a key regulator in TME. Accumulated evidence has shown that Rho‐ROCK signaling enhances modified ECM composition and increases the migration of cancer‐associated fibroblasts and lymphocytes. 24 Regarding angiogenesis in the TME, Rho‐ROCK signaling was verified to be a crucial mediator in VEGFA‐mediated angiogenic processes. 25 Targeting Rho‐ROCK signaling by noncoding RNAs might be an effective way to modulate angiogenesis in the TME. 26 , 27 In this study, we revealed that circ_TNFRSF21 facilitated tube formation of HUVECs by sponging miR‐3619‐5p and upregulating ROCK2. Moreover, our results indicated that ROCK2 was directly prohibited by miR‐3619‐5p, which was consistent with a previous report. However, we should also note that another study also indicated a contractive role of ROCK1/2 in angiogenesis, which suggested that the function of Rho‐ROCK signaling in angiogenesis might be an alternative in different diseases. 28

In conclusion, our results demonstrated that circ_TNFRSF21 was elevated in M2 macrophages and mediated M2 macrophage‐induced tube formation in vitro. Mechanistically, circ_TNFRSF21 served as a sponge of miR‐3619‐5p to upregulate ROCK2 and facilitate tube formation. Our work might provide new evidence to identify effective biomarkers and prognostic factors of cSCC.

CONFLICT OF INTEREST

All authors declare no conflict of interest.

ETHICS STATEMENT

This study was approved by the Medical Ethics Committee of Nanfang Hospital, Southern Medical University.

CONSENT FOR PUBLICATION

The informed consent was obtained from study participants.

ACKNOWLEDGMENTS

We would like to give our sincere gratitude to the reviewers for their constructive comments.

Ma J, Huang L, Gao Y‐B, Li M‐X, Chen L‐L, Yang L. M2 macrophage facilitated angiogenesis in cutaneous squamous cell carcinoma via circ_TNFRSF21/miR‐3619‐5p/ROCK axis. Kaohsiung J Med Sci. 2022;38(8):761–771. 10.1002/kjm2.12555

Funding informationThis work was supported by the Natural Science Foundation of Guangdong Province, No. 2020A151501107; the Guangdong Province Key Field R&D Program, No. 2020B1111150001; the Science and Technology Innovation Project of Guangdong Province, No. 2018KJYZ005; and the Natural Science Foundation of Tibet Autonomous Region, No. XZ2017ZR‐ZY021

Jun Ma and Lei Huang are co‐first authors.

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