The novel angiogenesis regulator circFAM169A promotes the metastasis of colorectal cancer through the angiopoietin-2 signaling axis

Figures

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  Figure 1. Screening of angiogenesis-related circular RNAs (circRNAs) in colorectal cancer (CRC). (A) Heatmap showing the up-regulated hallmarks in CRC patients with liver metastasis. (B) Spearman’s correlation analysis showing the correlation between circRNA expression and angiogenesis scores assessed by gene set variation analysis (GSVA). (C) Volcano diagram of circRNAs differentially expressed between CRC patients with and without liver metastasis. (D) Venn diagram of circRNAs highly associated with angiogenesis and upregulated circRNAs. (E) Correlation analysis between circFAM169A and angiogenesis scores assessed by GSVA. (F) Expression level of circFAM169A in CRC samples and non-tumoral tissues from GSE13572. (G) Expression level of circFAM169A in CRC patients with and without liver metastasis from GSE147711. (H) Receiver operating characteristic (ROC) curve showing the diagnostic value of circFAM169A compared with that of SNAI1, SNAI2, MACC1, and MMP1.

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  Figure 2. Identification of the potential biological functions of circFAM169A in CRC. (A) Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that many metastasis-related pathways were activated. (B) KEGG analysis showed that some synthesis-related pathways were inhibited. (C) Gene Ontology (GO) analysis showing the biological processes that were activated. (D) GO analysis showing the biological processes that were inhibited. (E) Protein–protein interaction network showing the possible protein networks that might be regulated by circFAM169A.

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  Figure 3. Characteristics of circFAM169A in CRC cells. (A) The diagram represents the structure of circFAM169A and the arrows denote the different primers. (B) The reverse transcription–PCR (RT-PCR) products from different primers were detected by agarose gel electrophoresis. (C) Samples were treated with RNase (+) or buffer (−), and the expression of circFAM169A was determined by quantitative RT–PCR (qRT–PCR). (D) SW480 cells were treated with (+) or without (−) actinomycin D, and the stability of circFAM169A was assessed by qRT–PCR. (E) Cells were analyzed by immunofluorescence cytochemistry. Scale bar = 20 μm. (F) The location of circFAM169A, GAPDH, and Malat1 in CRC cells. (G) Relative expression of circFAM169A in 20 paired CRC tumor and adjacent normal tissues determined using qRT–PCR. (H) ROC curves illustrating the capacity of circFAM169A to distinguish between tumors and adjacent tissues from patients with CRC. All data are presented as means ± standard deviation (SD) (n = 3 independent experiments). *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001.

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  Figure 4. circFAM169A promotes CRC angiogenesis and metastasis in vitro and in vivo. (A) The expression of circFAM169A in FHC, HT29, SW480, HCT116, HCT8, LOVO, and RKO cells determined using qRT–PCR. All data are presented as means ± SD (n = 3 independent experiments). *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001. (B, C) Construction of stable inducible circFAM169A-knockdown or -overexpressed CRC cell lines. All data are presented as means ± SD (n = 3 independent experiments). ***p ≤ 0.001 and ****p ≤ 0.0001. (D, E) Transwell migration assay and Transwell invasion assay were performed using HCT116 and RKO cells. (F, G) An in vitro Matrigel tube formation assay was performed to evaluate the angiogenic ability of human umbilical vein endothelial cells (HUVECs). The representative micrographs are shown at 200× magnification. (H, I) Representative images and bar graphs of liver and lung metastases with circFAM169A-knocked down HCT116 cells (H) and circFAM169A-overexpressing SW480 cells (I) in a nude mouse metastatic tumor model.

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  Figure 5. circFAM169A promotes CRC by sponging miR-518a-5p. (A) Venn diagram of miRNA response elements in circBank, CircInteractome, and TargetScan that may interact with circFAM169A. (B) The luciferase activities of different plasmids were measured by a dual-luciferase assay. (C) RNA antisense purification (RAP) assay showed that miR-518a-5p was significantly enriched when circFAM169A was used as a probe. (D) Construction of a luciferase plasmid encoding mut-circFAM169A. (E) The luciferase activities of different groups were measured by a dual-luciferase assay. (F, G) Transwell migration assay and Transwell invasion assay were performed using HCT116 and RKO cells. (H, I) An in vitro Matrigel tube formation assay was performed to evaluate the angiogenic ability of HUVECs. The representative micrographs are shown at 200× magnification.

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  Figure 6. circFAM169A enhances CRC angiogenesis and metastasis by targeting angiopoietin-2 (ANGPT2). (A) Venn diagram of mRNA that was highly correlated with circFAM169A and the downstream target genes of miR-518a-5p as predicted by TargetScan. (B) Survival analysis of ANGPT2. (C) ANGPT2 expression in normal and tumor tissues. The luciferase activities were measured by a dual-luciferase assay, and the Renilla/firefly luciferase light-unit ratio was measured. (D) ROC curve showed the diagnostic value of ANGPT2. (E) Construction of a luciferase plasmid encoding mut-ANGPT2. (F) The luciferase activities of different groups were measured by a dual-luciferase assay. (G, H) Transwell migration assay and Transwell invasion assay were performed using HCT116 and RKO cells. All data are presented as means ± SD (n = 3 independent experiments). ***p ≤ 0.001 and ****p ≤ 0.0001. (I) Western blotting analysis of ANGPT2, total TIE2, and p-TIE2 expression in circFAM169A-knocked down HCT116 cells treated with miR-518a-5p mimics. (J) Western blotting analysis of ANGPT2, total TIE2, and p-TIE2 expression in circFAM169A-overexpressing RKO cells treated with miR-518a-5p inhibitors. (K) Western blotting analyses…