Inhibition of Angiogenesis by MiR-524-5p through Suppression of AKT and ERK Activation by Targeting CXCR7 in Colon Cancer Cells

Increasing evidence shows that alterations in microRNA (miRNA) expression are involved in the occurrence and development of various malignant tumors, including colon cancer. MiRNA-524-5p has been reported to have anticancer activity in colon cancer. This study explored the influence of the miRNA-524-5p/CXCR7 axis on angiogenesis using colon cancer cells and further studied the mechanisms involved. We found that changing the expression of miRNA-524-5p can affect colonic proliferation, migration, and angiogenesis. Furthermore, angiogenesis induced by miRNA-524-5p overexpression was reversed by overexpression of CXCR7 in HT-29 cells, while the opposite was observed in Caco-2 cells. Furthermore, miRNA-524-5p inhibited the activation of AKT and ERK signaling by targeting CXCR7. Overall, our results indicated that the miRNA-524-5p/CXCR7 axis regulated angiogenesis in colon cancer cells through the AKT and ERK pathways.


Introduction
Colorectal cancers (CRCs) are the third most common malignancy in the world [1,2]. In the past 30 years, the global incidence and mortality due to colon cancer have been high [3]. Although trends in the incidence and mortality of colorectal cancer vary between countries, the global burden of this disease is projected to increase over the next decade [1]. Terefore, there is an urgent need to study new targeting factors in colon cancer. Tere are several studies showing that the inhibition of blood vessel formation can play an important role in cancer progression [4,5]. Many factors are involved in angiogenesis, such as vascular endothelial growth factor (VEGF). Most tumors are associated with the overexpression of VEGF, especially the VEGF-A/VEGFR2 axis, which plays a key role in angiogenesis [6]. Terefore, it is very important to better understand the mechanism of angiogenesis-related factors in colon cancer.
MicroRNA (miRNA) are short RNA molecules of 19-25 nucleotides in size that can silence target genes after transcription [7,8]. A single miRNA can target hundreds of mRNAs and afect the expression of many genes involved in various interaction pathways [9][10][11]. In nonsmall cell lung cancer (NSCLC), the knockdown of LINC00184 inhibits cell proliferation, migration, and accelerates apoptosis, which are closely related to the regulation of the miR-524-5p/ HMGB2 axis [12]. Zhao et al. [13] showed that miR-524 has an inhibitory efect on glioma cells and targets C-myc, which binds to its promoter region and activates the expression of the epidermal growth factor receptor (EGFR). Te high expression of LncRNA TUG1 in oral squamous cell carcinoma (OSCC) mediates the expression of distal homeobox 1 (DLX1) through competitive binding to miR-524-5p [14]. Terefore, dysregulation of miRNA-524-5p can be observed in a wide range of diseases, including colon cancer [15]. However, its molecular mechanism for promoting angiogenesis in colon cancer needs further study.
Growing clinical data on colon cancer suggests that mRNA and protein levels of the CXC chemokine receptor 7 (CXCR7) were up-regulated in tumor tissues of colon cancer patients with lymph node metastases compared to nonmetastatic tumors [16]. CXCR7 is attached to chemokinespecifc seven transmembrane guanosine-bindingproteincoupled receptors [17]. Several studies suggested that, except for embryonic neurons, fetal heart tissues, and certain hematopoietic cells, CXCR7 is absent in most normal tissues in humans but is highly expressed in the human endometrium and several types of malignancies, including colon cancer [18][19][20]. Tis new chemokine receptor is defned as a high-afnity receptor for the CXC chemokine ligand 12 (CXCL12) that can also bind CXCL11 [21]. Increasing CXCR7 expression accelerated the growth and metastasis capacity of various malignant tumors, which was accompanied by the regulation of angiogenesis and immunity [22]. Terefore, to study the molecular mechanism of miR-524-5p that regulates CXCR7 expression in colon cancer, we respectively regulated the expression of miR-524-5p and CXCR7 in HT-29 and Caco-2 cells to evaluate the infuence of the miR-524-5p/CXCR7 axis on colon cancer angiogenesis.

Te 5-Ethynyl-20-Deoxyuridine (EdU) Assay. HT-29 and
Caco-2 cells were seeded in a 24-well plate at a density of 2 × 10 5 cells per well and cultured in the normal growth stage. Cells were transfected with the miRNA-524-5p mimic and the miRNA-524-5p inhibitor for 48 hours, then 50 μM EdU was added, and cells were incubated at 37°C for 2 hours. Subsequently, cells were stained using cell light EdU DNA imaging (RiboBio).

Cell Migration Assay.
Te transfected cells (4 × 10 5 cells/ well) described above were seeded in the lower chamber and cultured until the cells adhered to the plate. HUVEC (2 × 10 5 cells/well) was seeded in the upper chamber and incubated at 37°C for 12 h. After fxation and staining, the migrating cells were photographed and counted using a microscope.

Tube Formation Assay.
A 200 μl volume of Matrigel (BD Biosciences) was added to each well in the lower chamber of the 24-well plate and incubated at 37°C for 30 minutes. HUVEC was seeded in the upper chamber at 2 × 10 5 cells per well. Subsequently, the transfected cells (4 × 10 5 cells/well) were resuspended in 200 μL of complete medium and added to the upper chamber. After incubation for 6 hours at 37°C, the number of junctions was counted after images were acquired with a microscope.

Enzyme-Linked Immunosorbent Assay (ELISA).
An ELISA kit (R&D Systems) was used to estimate the concentrations of CXCL11, CXCL12, and VEGF according to the manufacturer's instructions. Te OD value at 450 nm was measured using a microplate reader.

Immunohistochemical Analysis.
Te tumor tissues obtained were fxed, embedded, and sliced (thickness, 6 μm) and then subjected to immunohistochemical experiments. Tumor sections were incubated with the primary antibodies Ki67, CXCR7, AKT, p-AKT, ERK, p-ERK, VEGF, PDGF, and CD34 at 4°C overnight, and then incubated with secondary antibodies. Te sections were stained with diaminobenzidine. Immunopositive proteins were observed under an optical microscope at 200x and 400x magnifcation.

Statistical Analysis.
All data were expressed as means ± standard deviation (SD). All statistical analyses were performed using SPSS version 20.0 software (IBM Corp., Armonk, NY, USA). Statistical diferences between groups were calculated using one-way analysis of variance or the Student's t-test. Te p-value ＜0.05 was considered statistically signifcant.

Efects of MiR-524-5p on the Migration and Tube Formation of HUVECs.
To explore whether miR-524-5p is associated with tumor angiogenesis, we cocultured colon cancer cells transfected with miR-524-5p with HUVECs and used transwell and lumen formation experiments to detect the migration and angiogenesis abilities of HUVECs. As shown in Figures 2(a) and 2(b), the number of migrating cells in cocultured HUVECs and HT-29 cells transfected with miR-524-5p mimic decreased signifcantly. Te number of cocultured HUVECs with migrating cells and Caco-2 cells transfected with the miR-524-5p inhibitor increased signifcantly. After coculturing with HT-29 cells transfected with the miR-524-5p mimic, the number of HUVECs junctions was signifcantly reduced. In contrast, coculturing of Caco-2 cells transfected with the miR-524-5p inhibitor signifcantly increased the number of HUVECs junction (Figures 2(c) and 2(d)).

MiR-524-5p Infuences VEGF Expression.
Since VEGF is an important factor involved in angiogenesis, we detected   Te results showed that the concentration of VEGF in HT-29 cells transfected with miR-524-5p mimic was signifcantly reduced, while the concentration of VEGF in Caco-2 cells transfected with the miR-524-5p inhibitor was signifcantly increased (Figure 3(c)).

CXCR7
Acted as a Target of miR-524-5p. We studied the targeting relationship between miR-524-5p and CXCR7. Te luciferase reporter vector CXCR7 3′UTR wild-type (WT) that we constructed contained the complementary sequence to miR-524-5p, and the CXCR7 3′UTR mutated (MUT) reporter plasmid was used as a control (Figure 4(a)). Te results of the double luciferase reporter gene assay indicated that in the presence of the miR-524-5p mimic, the luciferase activity of CXCR7 3′UTR WT was reduced in HT-29 and Caco-2 cells. However, the miR-524-5p mimic did not reduce the CXCR7 3′UTR MUT luciferase activity in HT-29 and Caco-2 cells (Figures 4(b) and 4(c)). As shown in Figures 4(d) and 4(e), the expression of CXCR7 mRNA and protein in HT-29 cells transfected with miR-524-5p mimic was signifcantly reduced, and the expression of CXCR7 mRNA and protein in Caco-2 cells transfected with miR-524-5p inhibitor increased signifcantly.

MiR-524-5p Regulated Angiogenesis by Activating AKT/ ERK Signaling in HT-29 Cells.
We manipulated the expression of miR-524-5p and CXCR7 in colon cancer cell lines to study their relationship with angiogenesis. Te CCK-8 assay revealed that the reduced proliferation capacity of the miR-524-5p mimic could be reversed by overexpression of CXCR7 in HT-29 cells (Figure 5(a)). Simultaneously, the release of CXCL11, CXCL12, and VEGF could also be reversed (Figures 5(b)-5(d)). Te coculture of HT-29 cells transfected with miR-524-5p mimic and HUVEC cells reduced the number of migrating cells and the number of junctions, which was reversed by the overexpression of CXCR7 (Figures 5(e)-5(g)). Furthermore, CXCR7, VEGF, and PDGF expression and phosphorylation of AKT and ERK also increased after overexpression of CXCR7 in HT-29 cells transfected with miR-524-5p mimic ( Figure 5(h)).

MiR-524-5p Regulated Angiogenesis by Activating AKT/ ERK Signaling in Caco-2 Cells.
Te CCK-8 results showed that the increase in proliferation capacity of the miR-524-5p inhibitor could be reversed by silencing CXCR7 expression in Caco-2 cells (Figure 6(a)), and at the same time, the release of CXCL11, CXCL12, and VEGF could also be reversed (Figures 6(b)-6(d)). Coculture of Caco-2 cells transfected with the miR-524-5p inhibitor and HUVEC cells increased the number of migrating cells and the number of junctions, which was reversed by the silencing of CXCR7 expression (Figures 6(e)-6(g)). Furthermore, CXCR7, VEGF, and PDGF expression and phosphorylation of AKT and ERK were also inhibited after silencing CXCR7 expression in Caco-2 cells transfected with the miR-524-5p inhibitor (Figure 6(h)).

Efects of MiR-524-5p on Tumor Growth in Vivo.
Finally, we injected HT-29 cells subcutaneously into BALB/c nude mice to study the efects of miR-524-5p overexpression on colon cancer growth ( Figure S1). After injection, tumor size was tested every 7 days, and miR-524-5p agomir was injected into the tumor every 3 days. Nude mice were euthanized 28 days after tumor formation and weighed. Te size of the transplanted tumor injected with miR-524-5p agomir was signifcantly reduced compared to the control group (Figure 7(a)). Te size and weight of the transplanted tumor also decreased signifcantly (Figures 7(b) and 7(c)). Te results of immunohistochemical staining confrmed that the expressions of Ki67, CXCR7, VEGF, CD34, p-AKT, and p-ERK in miR-524-5p agomir xenografts were signifcantly inhibited (Figures 7(e) and 7(f )). In addition, the expression of miR-524-5p in the miR-524-5p agomir xenograft was signifcantly increased (Figure 7(d)). In contrast, the expression of CXCR7 mRNA as well as the protein expression of CXCR7, VEGF, PDGF, p-AKT, and p-ERK was signifcantly suppressed (Figures 7(g) and 7(h)).

Discussion
In recent years, the role of miRNA in tumors has been extensively studied, including colon cancer [22]. Chen et al. showed that LINC00662 overexpression regulates colon cancer development through competitive binding to miR-340-5p [23]. Yan et al. showed that the overexpression of miR-182-5p in colon cancer cells signifcantly inhibited the carcinogenicity of SW620 cells and the angiogenesis and lymphangiogenesis of xenograft tumors in nude mice [24]. In this study, we examined changes in the proliferation, migration, and luminal formation of colon cancer cells (HT-29 and Caco-2) after forced up-or down-regulation of miR-524-5p. Overexpression of miR-524-5p inhibited the proliferation, migration, and luminal formation of colon cancer cells. Te opposite was observed in the absence of miR-524-5p. We further tested the expression of VEGF, and the results showed that overexpression of miR-524-5p inhibited the expression of VEGF. In contrast, the lack of miR-524-5p increased the expression of VEGF. Altogether, these fndings indicated that miR-524-5p was closely related to angiogenesis. To further investigate the molecular mechanisms of miR-524-5p in colon cancer angiogenesis, we detected altered CXCR7 expression in transfected HT-29 and Caco-2 cells. Recently, the identifcation of CXCR7, formerly called the orphan receptor RDC1, was confrmed [18]. As a highafnity receptor for CXCL12 and a low-afnity receptor for CXCL11 and CXCR7 serves as the key factor regulating cell survival, growth, and migration, rather than typical chemokine responses, such as calcium mobilization mediated by G protein-coupled receptors [21,25]. Recently, several studies have shown the tumorigenic role of CXCR7 in polytype cancers, such as breast carcinoma and lung tumors, with stimulative growth and migration [18,26]. Based on these fndings, we analyzed CXCR7 expression after artifcially engineering miR-524-5p expression. We found that CXCR7 expression was negatively regulated by miR-524-5p through target binding. Furthermore, knockdown of CXCR7 could decrease angiogenesis caused by loss of miR-524-5p. Tese results indicated that CXCR7 was an important downstream molecule of miR-524-5p. Recent investigations have reported that CXCR7 can promote Akt phosphorylation, and the ERK pathway has also been found to play an important role in angiogenesis       [27,28]. In the present study, co-transfection with the miR-524-5p inhibitor and the CXCR7 siRNA was carried out in Caco-2 cells and co-transfection with the miR-524-5p mimic and CXCR7OE in HT-29 cells to investigate changes in angiogenesis, VEGF and PDGF production, and in Akt and ERK phosphorylation. Our data indicated that the angiogenetic ability of HUVECs was signifcantly enhanced following coincubation with the miR-524-5p inhibitor-transfectedCaco-2 cells, an efect that could be reversed by CXCR7 knockdown. Meanwhile, VEGF and PDGF production by miR-524-5p inhibitor-transfectedCaco-2 cells also increased and was accompanied by elevated phosphorylation levels of Akt and ERK. However, the angiogenetic ability of HUVECs was signifcantly reduced following incubation with miR-524-5p mimic-transfected HT-29 cells, and this efect could be reversed by CXCR7 overexpression. Meanwhile, VEGF and PDGF generation in transfected HT-29 cells treated with miR-524-5p mimic also decreased, which was accompanied by reduced phosphorylation levels of Akt and ERK. Terefore, we inferred that miR-524-5p/ CXCR7 signaling regulated angiogenesis through AKT and ERK phosphorylation in colon cancer cells. Finally, the potential signifcance of increasing miR-524-5p expression was studied in transplanted tumor nude mice. Our fndings provide evidence that the injection of miR-524-5p agomir reduced the expression of CXCR7, VEGF, PDGF, Ki67, CD34, and the levels of p-AKT and p-ERK, proving that miR-524-5p could inhibit the growth and angiogenesis of a colon cancer tumor. In conclusion, our results indicated that the miRNA-524-5p/CXCR7 axis regulated angiogenesis in colon cancer cells through the AKT and ERK pathways. CXCR7 will be a new target for future treatment and research in colon cancer.

Data Availability
Te datasets are available from the corresponding author upon reasonable request.

Conflicts of Interest
Te authors declare that they have no conficts of interest. Journal of Oncology 13