IGF2BP2-induced circRUNX1 facilitates the growth and metastasis of esophageal squamous cell carcinoma through miR-449b-5p/FOXP3 axis

Abstract

Background: Esophageal squamous cell carcinoma (ESCC) is one of the most common digestive malignancies with relatively high morbidity and mortality. Emerging evidence suggests circular RNAs (circRNAs) play critical roles in tumor cell malignancy. However, the biological function and clinical significance of many circRNAs in ESCC remain elusive.

Methods: The expression level and clinical implication of circRUNX1 in ESCC tissues were evaluated using qRT-PCR. In vitro and in vivo functional studies were conducted to investigate the underlying biological effects of circRUNX1 on ESCC cell growth and metastasis. Moreover, bioinformatics analysis, RNA sequencing (RNA-seq), RNA immunoprecipitation (RIP) assays, dual-luciferase reporter assays, and rescue experiments were performed to explore the relationships between circRUNX1, miR-449b-5p, Forkhead box protein P3 (FOXP3), and insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2).

Results: CircRUNX1 was found to be significantly up-regulated in ESCC tissues and associated with TNM stage and differentiation grade. Functionally, circRUNX1 promoted ESCC cell proliferation and metastasis in vitro and in vivo. CircRUNX1 enhanced FOXP3 expression by competitively sponging miR-449b-5p. Notably, both miR-449b-5p mimics and FOXP3 knockdown restored the effects of circRUNX1 overexpression on cell proliferation and metastasis. Furthermore, IGF2BP2 binding to circRUNX1 prevented its degradation.

Conclusions: IGF2BP2 mediated circRUNX1 functions as an oncogenic factor to facilitate ESCC progression through the miR-449b-5p/FOXP3 axis, implying that circRUNX1 has the potential to be a promising diagnostic marker and therapeutic target for ESCC patients.

Keywords: Biomarker; CircRUNX1; Esophageal squamous cell carcinoma (ESCC); FOXP3; IGF2BP2.

Fig. 1

Expression and validation of circRUNX1 in ESCC tissues and cells. A Expression of top eight circRNAs in 25 pairs of ESCC tissues was detected by qRT-PCR. B qRT-PCR analysis of circRUNX1 expression in 54 pairs of ESCC tissues (25 pairs in panel A and another 29 pairs of ESCC tissues). C Diagnosed value of circRUNX1 was displayed in ROC curve. D Relative expression of circRUNX1 in a panel of ESCC cell lines and normal esophageal epithelial cells (HEEC). E Schematic illustration of circRUNX1 formation via the circularization from exon 5 to exon 6 in the RUNX1 gene located on chromosome 21. F The expression of circRUNX1 and RUNX1 mRNAs after treatment with RNase R in KYSE150 cells. G Convergent and divergent primers were used to verify closed loop structure of circRUNX1. H The expression changes of circRUNX1 (cRUNX1) and RUNX1 (mRUNX1) were detected by qRT-PCR in KYSE150 and TE1 cells treated with actinomycin D at the indicated time points. I The abundance of circRUNX1 in the nuclear and cytoplasmic of KESE150 and TE1 cells was measured by qRT-PCR. J FISH assays for circRUNX1 in KESE150 and TE1 cells. Nuclei were stained with DAPI and cytoplasm was staind with 18S. (Magnification × 400. Scale bar: 20 μm and Magnification × 1000. Scale bar: 10 μm). The data are presented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 2

CircRUNX1 knockdown suppresses ESCC progression. A and B Expression levels of circRUNX1 (A) and RUNX1 (B) post transfection of three siRNAs targeting circRUNX1. C Colony formation assays were performed in ESCC cell lines with circRUNX1 knockdown. D The cell viability of KYSE150 and TE1 cells were evaluated by CCK-8 assays. E Changes of cell proliferation after knocking down circRUNX1 were detected by EDU assay (Magnification × 100. Scale bar: 100 μm). F Migration and invasion abilities of KYSE150 and TE1 cells after circRUNX1 inhibition were evaluated by transwell assays. G Migration abilities of KYSE150 and TE1 cells after depletion of circRUNX1 were evaluated by wound healing assay. **P < 0.01, ***P < 0.001

Fig. 3

CircRUNX1 promotes cell proliferation and metastasis by upregulating FOXP3 expression. A Volcano plot showing differentially expressed genes in TE1 cells transfected with si-circRUNX1 and si-NC. B Differential expression of FOXP3 in 41 pairs of ESCC tissues and matched esophageal normal tissues. C and D Relative mRNA levels of FOXP3 were detected in ESCC cells with circRUNX1 knockdown or overexpression using qRT-PCR. E and F Relative protein levels of FOXP3 after inhibition or ectopic expression of circRUNX1 were determined using western blotting. G The correlation between circRUNX1 and FOXP3 was detected in 41 clinical ESCC samples. H-J CCK-8 (H), colony formation (I) and EdU assays (J) were performed to examine the proliferative effects of circRUNX1 and FOXP3 on TE1 cells. K-M Wound healing and transwell assays were adopted to evaluate the migration and invasion capabilities of TE1 cells after overexpression of circRUNX1 or disruption of  FOXP3. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 4

CircRUNX1 serves as miR-449b-5p sponge to promote ESCC progression. A Venn diagram showing the candidate miRNAs predicted to be the binding targets of circRUNX1 and FOXP3 by circBank (http://www.circbank.cn) and Targetscan (http://www.targetscan.org). B Relative expression of 14 candidate miRNAs after knocking down circRUNX1. C Relative expression of miR-449b-5p in KYSE150 and TE1 cells stably expressing circRUNX1. D The luciferase activity was determined in 293 T cells co-transfected with miR-449b-5p mimics/miR-NC and circRUNX1-WT/circRUNX1-MUT. E Anti-Ago2 RIP assays were performed in KYSE150 and TE1 cells to detect the enrichment ability of Ago2 to circRUNX1 and miR-449b-5p. F The correlation between miR-449b-5p and circRUNX1 expression in 41 primary ESCC tissues. G Relative expression of miR-449b-5p in a panel of ESCC cell lines and normal esophageal epithelial cells (HEEC). H-J CCK-8 (H), colony formation (I) and EdU assays (J) were performed to measure the proliferative abilities of TE1 cells transfected with circRUNX1 or circRUNX1 plus miR-449b-5p mimics. K-M Wound healing and transwell assays were utilized to evaluate the migration and invasion capacities of TE1 cells transfected with indicated vectors. Data are represented as the mean ± SD from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 5

CircRUNX1/miR-449b-5p/FOXP3 axis facilitates ESCC proliferation and metastasis. A Relative expression of FOXP3 in KYSE150 cells and TE1 cells transfected with miR-449b-5p mimics or miR-NC. B Western blot analysis of FOXP3 level in ESCC cells after transfecting miR-449b-5p mimics or miR-NC. C The luciferase activity was determined in 293 T cells co-transfected with miR-449b-5p mimics/miR-NC and FOXP3-WT/FOXP3-MUT. D-F CCK-8 (D), colony formation (E) and EdU assays (F) were performed to measure the proliferative effects of miR-449b-5p and FOXP3 on KYSE150 cells. G-I The migratory or invasion properties of the miR-449b-5p inhibition with or without FOXP3 depletion in KYSE150 cells and control cells were analyzed by wound healing and transwell experiments. Data are represented as the mean ± SD from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 6

IGF2BP2 silencing retards ESCC progression via accelerating circRUNX1 degradation. A Venn diagram showing the candidate RNA binding proteins predicted to combine with circRUNX1by circAtlas (http://circatlas.biols.ac.cn) and RBPmap (http://rbpmap.technion.ac.il). B IGF2BP2 mRNA levels were detected by qRT-PCR in 41 pairs of ESCC tissues and their corresponding normal tissues. C Expression and scoring of IGF2BP2 protein were detected by IHC in 30 pairs of ESCC tissues. D IGF2BP2 immunostaining scores were detected by IHC in ESCC tissues with lymph node metastasis (n = 13) and non-metastasis (n = 17). E and F Changes of circRUNX1 and IGF2BP2 levels were determined in KYSE150 cells upon silencing or overexpression of IGF2BP2. G The correction between circRUNX1 and IGF2BP2 were examined in 41 ESCC tissues. H IHC results showing that higher circRUNX1 expression was related to higher abundance of IGF2BP2 protein in ESCC tissues. I qRT-PCR analysis showing circRUNX1 expression in KYSE150 cells treated with actinomycin D for specific time points. J RIP assay using anti-IGF2BP2 antibody and IgG antibody was executed in KYSE150 cells with or without overexpressing IGF2BP2. K-M CCK-8 (K), colony formation (L) and EdU assays (M) were conducted to investigate the proliferative abilities of KYSE150 cells with IGF2BP2 overexpression or circRUNX1 inhibition. N-P Wound healing and transwell assays showing the migration and invasion capabilities of KYSE150 cells with IGF2BP2 overexpression or circRUNX1 inhibition. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 7

CircRUNX1 targets FOXP3 to accelerate tumorigenesis and metastasis in vivo. A and B The process of constructing subcutaneous tumor models (A) and pulmonary metastasis models (B) in nude mice. C Tumor volumes of indicated groups were measured every 5 days. D Image of subcutaneous tumors from three groups were displayed. E The weights of subcutaneous tumors were quantified. F Representative IHC staining images and quantification of FOXP3 and Ki-67 positive cells in the indicated xenograft tumors. Magnification × 100. G FOXP3 mRNA levels of subcutaneous tumor tissues were detected by qRT-PCR. H The images of lung metastases and statistical analysis of metastatic lung foci. I Representative images from H&E staining of the lung metastasis nodules. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 8

Schematic illustration of the biological role of IGF2BP2/circRUNX1/miR-449b-5p/FOXP3 axis in modulating ESCC malignant progression