The circular RNA 001971/miR-29c-3p axis modulates colorectal cancer growth, metastasis, and angiogenesis through VEGFA

Abstract

Background: Colorectal cancer (CRC) is one of the most common malignant tumors globally. Angiogenesis is a key event maintaining tumor cell survival and aggressiveness. The expression of vascular endothelial growth factor A (VEGFA), one of the most significant tumor cell-secreted proangiogenic factors, is frequently upregulated in CRC.

Methods: The MTT assay was used to detect the viability of CRC cells. Transwell assays were performed to detect the invasion capacity of target cells. Relative protein levels were determined by immunoblotting. Pathological characteristics of tissues were detected by H&E staining and immunohistochemical (IHC) staining. A RIP assay was conducted to validate the predicted binding between genes.

Results: We observed that circ-001971 expression was dramatically increased in CRC tissue samples and cells. Circ-001971 knockdown suppressed the capacity of CRC cells to proliferate and invade and HUVEC tube formation in vitro, as well as tumor growth in mice bearing SW620 cell-derived tumors in vivo. The expression of circ-001971 and VEGFA was dramatically increased whereas the expression of miR-29c-3p was reduced in tumor tissue samples. Circ-001971 relieved miR-29c-3p-induced inhibition of VEGFA by acting as a ceRNA, thereby aggravating the proliferation, invasion and angiogenesis of CRC. Consistent with the above findings, the expression of VEGFA was increased, whereas the expression of miR-29c-3p was decreased in tumor tissue samples. miR-29c-3p had a negative correlation with both circ-001971 and VEGFA, while circ-001971 was positively correlated with VEGFA.

Conclusions: In conclusion, the circ-001971/miR-29c-3p axis modulated CRC cell proliferation, invasion, and angiogenesis by targeting VEGFA.

Keywords: Angiogenesis; Circ-001971; Colorectal cancer (CRC); Metastasis; miR-29c-3p.

Fig. 1

Selection of circ-001971 (a) Pathological characteristics of the tumor and non-cancerous tissues revealed by H&E staining. b The expression of the top 11 upregulated circRNAs reported by the previous study was verified in 13 paired tumor and noncancerous tissues using real-time PCR. The data are presented as the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01. c The expression of circ-001971 was examined in 70 paired tumor and noncancerous tissues using real-time PCR. d Circ-001971 expression was analyzed according to the TNM stage. e ROC curve of circ-001971 to distinguish colorectal cancer (CRC) from controls. f Colorectal cancer (CRC) cases were divided into two groups according to circ-001971 expression. Overall survival was analyzed by Kaplan-Meier overall survival analysis using the log-rank test. g ROC curve of circ-001971 to assess the diagnostic sensitivity and specificity of circ-001971 for CRC

Fig. 2

The effect of circ-001971 on CRC cell invasion and HUVEC tube formation (a) The expression of circ-001971 in five CRC cell lines, HT29, HCT116, LoVo, SW480, and SW620, and a normal cell line was examined using real-time PCR. b The knockdown of circ-001971 was achieved in HCT116 and SW620 cells by transfection with si-circ-001971#1 or si-circ-001971#2, as confirmed using real-time PCR. (C-E) HCT116 and SW620 cells were transfected with si-circ-001971 and examined for DNA synthesis (c) and cell proliferation (d-e). f-g HCT116 and SW620 cells were transfected with si-circ-001971 and examined for cell invasion using Transwell assays. h-i We collected conditioned medium from circ-001971-knockdown or control SW620 cells (si-circ-001971 CM and si-NC CM), cultured HUVECs in these two CM samples, and examined for the tube formation capacity. The data are presented as the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, compared to FHC cells or the si-NC group

Fig. 3

Effects of circ-001971 on CRC growth in vivo (a) Lentivirus containing shRNA targeting circ-001971 was constructed and the knockdown efficiency was verified by real-time PCR. b Six animals were randomly assigned into two groups (n = 3). SW620 cells infected with Lv-sh-NC or Lv-sh-circ-001971 were hypodermically injected into the left axillaries of mice in different groups. Twenty-five days after the injection, when the diameters of the resulting tumors measured approximately 5 mm, the rats were sacrificed under anesthesia. c The length (L) and width (W) of the tumors were measured with calipers every 5 days for 25 days and the tumor volumes were calculated. d At the end of the animal experiments, all the animals were sacrificed under anesthesia, and the tumor weight was determined. **P < 0.01, compared to Lv-sh-NC group

Fig. 4

Selection and verification of miRNAs related to circ-001971 and CRC angiogenesis (a) Upregulated and downregulated miRNAs in CRC tissues compared to that in noncancerous tissues according to the online microarray expression profile GSE126093. b-c HCT116 and SW620 cells were transfected with si-circ-001971, and the protein levels of VEGFA were examined by immunoblotting. (d) TargetScan was used to predict miRNAs that might target VEGFA. Among the 627 predicted miRNAs, 16 miRNAs were downregulated miRNAs in CRC, as reported by GSE126093. Among the 16 miRNAs, 3 were predicted to target circ-001971, including miR-29c-3p, miR-497-5p, and miR-943. e Overexpression of the three candidate miRNAs was conducted in HCT116 and SW620 cells by the transfection with miRNA mimics. The transfection efficiency was confirmed by real-time PCR. Then, HCT116 and SW620 cells were transfected by miRNA mimics and examined for (f) the expression of circ-001971 by real-time PCR and (g) the expression of VEGFA by real-time PCR. miR-29c-3p was selected for further experiments. h miR-29c-3p inhibition was conducted in HCT116 and SW620 cells by the transfection of miR-29c-3p inhibitor. The transfection efficiency was verified by real-time PCR. i HCT116 and SW620 cells were transfected with miR-29c-3p inhibitor and the expression of circ-001971 was examined by real-time PCR. j HCT116 and SW620 cells were transfected by si-circ-001971 and examined for the expression of miR-29c-3p by real-time PCR. (K-L) HCT116 and SW620 cells were transfected with miR-29c-3p mimics or inhibitor, and the protein levels of VEGFA were by immunoblotting. *P < 0.05; **P < 0.01, compared to si-NC, mimics-NC or inhibitor NC group

Fig. 5

miR-29c-3p directly binds to circ-001971 and VEGFA (a-b) Two different types of luciferase reporter vectors were constructed: wild-type circ-001971 and VEGFA 3’UTR, and mutant-type circ-001971 and VEGFA 3’UTR; mutant-type vectors contained a 4 bp mutation in the predicted miR-29c-3p binding site. c-d The above vectors were cotransfected into HEK293 cells with miR-29c-3p mimics or miR-29c-3p inhibitor and examined for luciferase activity. e RIP assays were performed to confirm the binding of miR-29c-3p to circ-001971 and VEGFA using an AGO2 antibody. The levels of miR-29c-3p, circ-001971 and VEGFA in precipitated AGO2 proteins were examined using real-time PCR. f Endogenous circ-001971 and VEGFA 3’UTR pull-down by AGO2 upon overexpression of miR-29c-3p was determined using RIP assays. The data are presented as the mean ± SD of three independent experiments. **P < 0.01, compared to mimics-NC, inhibitor-NC, IgG group

Fig. 6

The circ-001971/miR-29c-3p axis modulates the tube formation capacity of HUVECs (a-b) HCT116 and SW620 cells were cotransfected with miR-29c-3p inhibitor and si-circ-001971, and the protein levels of VEGFA were determined. c-d HUVECs were cultured in miR-29c-3p inhibitor and si-circ-001971 transfected SW620 cell conditioned medium (CM) and assayed for tube formation capacity. *P < 0.05, **P < 0.01, compared to si-NC + inhibitor-NC group; ##P < 0.01, compared to si-circ-001971 + inhibitor-NC group

Fig. 7

Expression of miR-29c-3p, HIF1α and VEGFA in tissue specimens and their correlation with circ-001971 (a-b) Expression of VEGFA and miR-29c-3p in 70 paired tumor and noncancerous tissue samples was examined using real-time PCR. c VEGFA contents in tumor and noncancerous tissues were examined using IHC assays. d VEGFA protein levels in tumor and non-cancerous tissues were examined using immunoblotting. e-g The correlation of circ-001971, miR-29c-3p, and VEGFA was analyzed using Pearson’s correlation analysis. *P < 0.05, compared to noncancerous tissue samples