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

Abstract

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.

Figure 1

Effects of miR-524-5p on proliferation. (a) The relative miR-524-5p expression in colon cancer cells. (b) Relative miR-524-5p expression in HT-29 and Caco-2 cells after relevant transfection. CCK8 assay showing the proliferation of HT-29 (c) and Caco-2 (d) cells after the indicated transfection. (e) Relative E2F1 protein expression in HT-29 and Caco-2 cells after relevant transfection. Representative images of EdU-positive cells of HT-29 (f) and Caco-2 (g) cells after the indicated transfection. (h) Quantitative measurement of EdU-positive cells. Magnification 200x. ∗p < 0.05 vs. the indicated group.

Figure 2

Effects of miR-524-5p on the migration and tube formation of HUVECs. (a) Representative images of migration of HT-29 and Caco-2 cells after relevant transfection. (b) Quantitative measurement of migration cell number. (c) Representative image of tube formation in HT-29 and Caco-2 cells after relevant transfection. (d) Quantitative measurement of the junction number. Magnification 200x. ∗p < 0.05 vs. the indicated group.

Figure 3

Effects of miR-524-5p on VEGF expression. (a) The relative mRNA expression of VEGF in HT-29 and Caco-2 cells. (b) The relative protein expression of VEGF in HT-29 and Caco-2 cells. (c) The concentration of VEGF in HT-29 and Caco-2 cells. ^∗p < 0.05 vs. the indicated group.

Figure 4

CXCR7 acts as a target of miR-524-5p. (a) The binding region between miR-524-5p and CXCR7. (b, c) A dual-luciferase reporter assay to test the luciferase activity in HT-29 and Caco-2 cells. (d) The relative mRNA expression of CXCR7 in HT-29 and Caco-2 cells. (e) The relative protein expression of CXCR7 in HT-29 and Caco-2 cells. ^∗p < 0.05 vs. the indicated group.

Figure 5

MiR-524-5p regulates angiogenesis via AKT/ERK signaling in HT-29 cells. (a) Cell proliferation was measured by the CCK8 assay in HT-29 cells after cotransfection. The release of CXCL11 (b), CXCL12 (c), and VEGF (d) in HT-29 cells after cotransfection. (e) Representative images of migration and tube formation assays. (f) Quantification of migration ability. (g) Quantification of angiogenic ability. (h) Protein levels of CXCR7, AKT, p-AKT, ERK, p-ERK, VEGF, and PDGF in HT-29 cells after cotransfection.

Figure 6

MiR-524-5p regulates angiogenesis by AKT/ERK signaling in Caco-2 cells. (a) Cell proliferation is measured by the CCK8 assay in Caco-2 cells after cotransfection. The release of CXCL11 (b), CXCL12 (c), and VEGF (d) in Caco-2 cells after cotransfection. (e) Representative images of migration and tube formation assays. (f) Quantification of migration ability. (g) Quantification of angiogenic ability. (h) Protein levels of CXCR7, AKT, p-AKT, ERK, p-ERK, VEGF, and PDGF in Caco-2 cells after cotransfection.

Figure 7

Effects of miR-524-5p on tumor growth in vivo. (a) An image of tumor tissue. (b) Tumor volume. (c) Tumor weight. (d) The relative miR-524-5p expression. (e) Tumor tissue sections were harvested for immunohistochemical staining of CXCR7, Ki67, VEGF, AKT, p-AKT, ERK, p-ERK, and CD34. (f) The count of CD34 microvessels reflected the MVD values. (g) The relative mRNA expression of CXCR7. (h) Protein levels of CXCR7, AKT, p-AKT, ERK, p-ERK, VEGF, and PDGF.