Circ-MFN2 Positively Regulates the Proliferation, Metastasis, and Radioresistance of Colorectal Cancer by Regulating the miR-574-3p/IGF1R Signaling Axis

Abstract

Numerous studies have shown that the expression of circular RNA (circRNA) is closely related to the malignant progression of cancer. However, the role of circ-MFN2 in colorectal cancer (CRC) is unclear. Our study aims to explore the role and mechanism of circ-MFN2 in CRC progression. The relative expression levels of circ-MFN2, microRNA (miR)-574-3p and insulin-like growth factor 1 receptor (IGF1R) were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Cell viability was determined using 3-(4, 5-dimethyl-2 thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT) assay. The colony number and radioresistance of cells were assessed using colony formation assay. Moreover, the migration and invasion of cells were measured using transwell assay. Tumor xenograft model was constructed to evaluate the effect of circ-MFN2 knockdown on CRC tumor growth. Furthermore, dual-luciferase reporter assay was used to verify the interaction between miR-574-3p and circ-MFN2 or IGF1R. In addition, the protein level of IGF1R was evaluated by western blot (WB) analysis. Circ-MFN2 expression was elevated in CRC tissues and cells. Knockdown of circ-MFN2 restrained the proliferation, migration, invasion, and radioresistance of CRC cells in vitro. Furthermore, silenced circ-MFN2 also reduced the tumor volume and weight of CRC in vivo. MiR-574-3p could be sponged by circ-MFN2, and its inhibitor reversed the suppression effect of circ-MFN2 silencing on CRC progression. Moreover, IGF1R was a target of miR-574-3p, and its overexpression reversed the inhibition effect of miR-574-3p mimic on CRC progression. In addition, circ-MFN2 could positively regulate IGF1R expression by sponging miR-574-3p. Our results revealed that circ-MFN2 promoted the proliferation, metastasis and radioresistance of CRC through regulating the miR-574-3p/IGF1R axis, suggesting that circ-MFN2 might be a novel therapeutic biomarker for CRC.

Keywords: CRC; IGF1R; circ-MFN2; miR-574-3p; progression.

FIGURE 1

The expression of circ-MFN2 in CRC tissues and cells. (A) Heat map revealed that the differentially expressed circRNAs in 8 paired CRC tumor tissues and non-cancer tissues (GEO accession: GSE126094). (B) The expression of circ-MFN2 in CRC tumor tissues and non-cancer tissues was measured using qRT-PCR. (C) QRT-PCR was used to detect the circ-MFN2 expression in CRC cell lines (LOVO, HCT-116, SW620 and SW480) and NCM460 cells. (D) The expression of circ-MFN2 in different TNM stages (I-II and III-IV) of CRC patients was determined using qRT-PCR. (E) The expression of circ-MFN2 was detected by qRT-PCR in CRC patients with (Yes) or without (No) lymph node metastasis. (F) Kaplan-Meier analysis was performed to analyze the relationship between the circ-MFN2 expression and the overall survival rate of CRC patients. (G,H) RNase R assay was used to confirm the circular structure of circ-MFN2 compared to linear MFN2. *P < 0.05.

FIGURE 2

Circ-MFN2 knockdown inhibited CRC progression. SW620 and SW480 cells were transfected with si-NC or si-circ-MFN2, and non-transfected cells were used as control. (A) The expression of circ-MFN2 was detected by qRT-PCR to evaluate transfection efficiency. (B,C) MTT assay was used to measure the viability of cells. (D) The number of colonies was determined using colony formation assay. (E,F) Transwell assay was used to assess the migration and invasion of cells. (G,H) The survival fraction of cells was examined using colony formation assay. *P < 0.05.

FIGURE 3

Circ-MFN2 silencing reduced CRC tumor growth in vivo. SW480 cells transfected with sh-NC or sh-circ-MFN2 were injected into nude mice. 6 Gy radiation was given after 8 days, and then radiation was given every 4 days until 31 days. The tumor volume (A) and tumor weight (B) were measured in mice. (C) QRT-PCR was used to detect the expression of circ-MFN2 in tumors. *P < 0.05.

FIGURE 4

Circ-MFN2 directly interacted with miR-574-3p. (A) The predicted and mutated binding sites between circ-MFN2 and miR-574-3p were shown. (B,C) Dual-luciferase reporter assay was used to verify the interaction between circ-MFN2 and miR-574-3p. (D) The expression of miR-574-3p in CRC tumor tissues and non-cancer tissues was measured using qRT-PCR. (E) QRT-PCR was used to detect the miR-574-3p expression in CRC cell lines (SW620 and SW480) and NCM460 cells. (F) Pearson correlation analysis was used to assess the correlation between circ-MFN2 and miR-574-3p. (G) The transfection efficiencies of si-circ-MFN2 and pcDNA circ-MFN2 overexpression plasmid were confirmed using qRT-PCR. (H) QRT-PCR was employed to detect the expression of miR-574-3p to assess the effect of circ-MFN2 on miR-574-3p expression. *P < 0.05.

FIGURE 5

The regulation of circ-MFN2 silencing and miR-574-3p inhibitor on CRC progression. SW620 and SW480 cells were transfected with si-NC, si-circ-MFN2, si-circ-MFN2 + anti-miR-NC or si-circ-MFN2 + anti-miR-574-3p, and non-transfected cells were used as control. (A) The expression of miR-574-3p was measured by qRT-PCR to evaluate transfection efficiency. (B,C) The viability of cells was detected using MTT assay. (D) The number of colonies was assessed using colony formation assay. (E,F) The migration and invasion of cells were examined by transwell assay. (G,H) Colony formation assay was performed to detect the survival fraction of cells. *P < 0.05.

FIGURE 6

MiR-574-3p could target IGF1R. (A) The sequences of IGF1R 3′UTR-WT and IGF1R 3′UTR-MUT were shown. (B,C) The interaction between IGF1R 3′UTR and miR-574-3p was confirmed using dual-luciferase reporter assay. (D,E) The mRNA and protein levels of IGF1R in CRC tumor tissues and non-cancer tissues were measured using qRT-PCR and WB analysis. (F,G) QRT-PCR and WB analysis were used to detect the mRNA and protein levels of IGF1R in CRC cell lines (SW620 and SW480) and NCM460 cells. (H) The correlation between IGF1R and miR-574-3p was analyzed using Pearson correlation analysis. *P < 0.05.

FIGURE 7

The regulation of miR-574-3p mimic and IGF1R overexpression on CRC progression. SW620 and SW480 cells were transfected with miR-NC, miR-574-3p, miR-574-3p + pcDNA or miR-574-3p + IGF1R, and non-transfected cells were used as control. (A) The protein level of IGF1R was measured by WB analysis to evaluate transfection efficiency. (B,C) The viability of cells was assessed using MTT assay. (D) Colony formation assay was determined to evaluate the number of colonies. (E,F) The migration and invasion of cells were detected using transwell assay. (G,H) The survival fraction of cells was examined using colony formation assay. *P < 0.05.

FIGURE 8

Circ-MFN2 and miR-574-3p regulated IGF1R expression. SW620 and SW480 cells were transfected with si-NC, si-circ-MFN2, si-circ-MFN2 + anti-miR-NC or si-circ-MFN2 + anti-miR-574-3p, and non-transfected cells were used as control. The protein level of IGF1R in SW620 (A) and SW480 (B) cells was measured using WB analysis. *P < 0.05.