Circular RNA circ-FIRRE interacts with HNRNPC to promote esophageal squamous cell carcinoma progression by stabilizing GLI2 mRNA

Abstract

Increasing evidence has shown that circular RNAs (circRNAs) interact with RNA-binding proteins (RBPs) and promote cancer progression. However, the function and mechanism of the circRNA/RBP complex in esophageal squamous cell carcinoma (ESCC) are still largely unknown. Herein, we first characterized a novel oncogenic circRNA, circ-FIRRE, by RNA sequencing (Ribo-free) profiling of ESCC samples. Furthermore, we observed marked circ-FIRRE overexpression in ESCC patients with high TNM stage and poor overall survival. Mechanistic studies indicated that circ-FIRRE, as a platform, interacts with the heterogeneous nuclear ribonucleoprotein C (HNRNPC) protein to stabilize GLI2 mRNA by directly binding to its 3'-UTR in the cytoplasm, thereby resulting in elevated GLI2 protein expression and subsequent transcription of its target genes MYC, CCNE1, and CCNE2, ultimately contributing to ESCC progression. Moreover, HNRNPC overexpression in circ-FIRRE knockdown cells notably abolished circ-FIRRE knockdown-mediated Hedgehog pathway inhibition and ESCC progression impairment in vitro and in vivo. Clinical specimen results showed that circ-FIRRE and HNRNPC expression was positively correlated with GLI2 expression, which reveals the clear significance of the circ-FIRRE/HNRNPC-GLI2 axis in ESCC. In summary, our results indicate that circ-FIRRE could serve as a valuable biomarker and potential therapeutic target for ESCC and highlight a novel mechanism of the circ-FIRRE/HNRNPC complex in ESCC progression regulation.

Keywords: GLI2; HNRNPC; circRNA; esophageal squamous cell carcinoma; hedgehog signaling.

FIGURE 1

Circular RNA circ‐FIRRE is upregulated and correlated with malignant clinicopathologic characteristics in esophageal squamous cell carcinoma (ESCC). (A) Volcano plot of circRNA profiles. (B) Genomic loci of circ‐FIRRE in the FIRRE gene are shown. Sanger sequencing results of the PCR product are shown. (C) Expression of circ‐FIRRE and FIRRE was measured by real‐time quantitative PCR (RT‐qPCR) analysis after RNase R treatment in KYSE‐410 and KYSE‐510 cells. (D) Stabilities of circ‐FIRRE and FIRRE in KYSE‐410 and KYSE‐510 cells after actinomycin D treatment are shown. (E) Expression of circ‐FIRRE in the cytoplasm and nucleus of KYSE‐410 and KYSE‐510 cells. (F) Cy3 probes targeting circ‐FIRRE showed their location in KYSE‐410 and KYSE‐510 cells. Scale bar, 10 μm. (G) Representative FISH fluorescence images of ESCC tissues and matched adjacent normal tissues are shown. Scale bar, 10 μm. (H) Expression of circ‐FIRRE in ESCC tissues and normal tissues was analyzed by RT‐qPCR. (I–K) RT‐qPCR analysis of circ‐FIRRE expression in tissues of ESCC patients; (I) clinical I + II stage vs. III + IV, (J) no lymphatic metastasis vs. lymphatic metastasis, (K) T1 + T2 stage vs. T3 + T4 stage are shown. (L) Area under the curve (AUC) values of circ‐FIRRE for distinguishing ESCC patients from healthy controls are shown. (M) Kaplan–Meier analysis of circ‐FIRRE in ESCC patients. LM, lymphatic metastasis; ns, no significance. *p < 0.05, ***p < 0.001.

FIGURE 2

Circular RNA circ‐FIRRE promotes proliferation, migration, and invasion of esophageal squamous cell carcinoma (ESCC) in vitro. (A) circ‐FIRRE expression levels were detected in an esophageal epithelial cell line and five ESCC cell lines. (B) Efficacy of circ‐FIRRE knockdown in KYSE‐410 and KYSE‐510 cells. (C–E) Proliferation abilities of KYSE‐410 and KYSE‐510 cells transfected with circ‐FIRRE shRNAs were tested by CCK‐8, EdU (scale bar, 50 μm), and colony formation assays (scale bar, 50 μm). (F) Flow cytometry shows the percentages of cells in the G₁, S, or G₂ phase in KYSE‐410 and KYSE‐510 cells. (G) Migration and invasion abilities of KYSE‐410 and KYSE‐510 cells transfected with circ‐FIRRE shRNAs are shown by Transwell assays. Scale bar, 100 μm. **p < 0.01, ***p < 0.001, and all the above experiments were repeated three times. ns, no significance.

FIGURE. 3

GLI2 is a downstream target gene of circ‐FIRRE. (A, B) Western blot analysis and real‐time quantitative PCR showed the expression of GLI2, C‐MYC, CCNE1, and CCNE2 in vector and circ‐FIRRE knockdown KYSE‐410 cells. (C) Western blot showing the expression of GLI2, C‐MYC, CCNE1, and CCNE2 in cells transfected with vector or sh circ‐FIRRE or cotransfected with sh circ‐FIRRE and GLI2 overexpression plasmid. (D–F) CCK‐8, colony formation, and Transwell assays (scale bar, 100 μm) were carried out in KYSE‐410 cells transfected with vector or sh circ‐FIRRE or cotransfected with sh circ‐FIRRE and the GLI2 overexpression plasmid. (G) Representative images and quantitative analysis of GLI2 expression in esophageal squamous cell carcinoma (ESCC) patients. Scale bars: up, 500 μm; down, 50 μm. (H) Kaplan–Meier analysis of GLI2 in ESCC patients. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 4

Circular RNA circ‐FIRRE–heterogeneous nuclear ribonucleoprotein C (HNRNPC) complex facilitates the malignant phenotype of esophageal squamous cell carcinoma (ESCC) cells by regulating GLI2 expression. (A) RNA pull‐down efficacy of sense and antisense probes in KYSE‐410 cells. (B) Results of the RNA pull‐down assay analyzed by western blotting. (C) Enrichment of circ‐FIRRE pulled down by the HNRNPC‐FLAG protein is shown. (D, E) Western blotting and quantitative PCR showed the expression of GLI2, C‐MYC, CCNE1, and CCNE2 in cells transfected with vector or sh circ‐FIRRE or cotransfected with sh circ‐FIRRE and the HNRNPC overexpression plasmid. (F–H) Proliferation, migration, and invasion abilities of KYSE‐410 cells transfected with sh‐circ‐FIRRE or cotransfected with the HNRNPC overexpression plasmid by CCK‐8, colony formation, and Transwell assays. Scale bar, 100 μm. (I) Representative images of immunohistochemistry (IHC) and quantitative analysis of HNRNPC expression in ESCC patients. Scale bars: up, 500 μm; down, 50 μm. (J) Correlation between HNRNPC and GLI2 expression in IHC data of ESCC patients. **p < 0.01, ***p < 0.001.

FIGURE 5

Circular RNA circ‐FIRRE–heterogeneous nuclear ribonucleoprotein C (HNRNPC) complex promotes esophageal squamous cell carcinoma (ESCC) tumor growth in vivo. (A) FISH and immunofluorescence analyses showed circ‐FIRRE and HNRNPC colocalization in KYSE‐410 cells. Scale bar, 10 μm. (B) RNA binding protein immunoprecipitation showed the combination of HNRNPC and GLI2 mRNA in KYSE‐410 cells. (C) Enrichment of GLI2 mRNA pulled down by the circ‐FIRRE probe is shown. (D) mRNA stability of GLI2 under treatment with actinomycin D in KYSE‐410 and KYSE‐510 cells with circ‐FIRRE knockdown or co‐overexpression of HNRNPC measured by real‐time quantitative PCR. (E) Schematic illustration of the GLI2 3′‐UTR full‐length or mutant luciferase reporter. (F, G) The relationship between the circ‐FIRRE/HNRNPC complex and the 3′‐UTR of GLI2 mRNA was verified by the dual‐luciferase reporter assay. (H) GLI2 Δ 2 is the binding site of circ‐FIRRE/HNRNPC and GLI2 verified by the dual‐luciferase reporter assay. (I) Schematic illustration of the GLI2‐dependent luciferase reporter. (J) Hedgehog signaling activity in circ‐FIRRE knockdown or co‐overexpressing HNRNPC KYSE‐410 cells tested by the dual‐luciferase reporter assay. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 6

Circular RNA circ‐FIRRE–heterogeneous nuclear ribonucleoprotein C (HNRNPC) complex promotes esophageal squamous cell carcinoma (ESCC) tumor growth in vivo. (A) Representative images of the inoculated tumor tissues of each group. (B) Time‐course evaluation of the tumor volumes in the indicated groups. (C) Tumor weight in each group. (D) Representative immunohistochemical staining images of Ki‐67, HNRNPC, and GLI2 in different groups. Scale bar, 50 μm. (E) Average optical density of GLI2 and Ki‐67 in the indicated groups. (F) Correlation between the average optical density of HNRNPC and GLI2 in tumor tissues. (G) Relative RNA levels extracted from tumor tissues of each group are shown. (H) Protein expression levels of tumor tissues from each group with five mixed samples. (I) Schematic diagram elucidating the mechanism of the circ‐FIRRE/HNRNPC‐GLI2 axis, which mediates the promotion of ESCC. *p < 0.05, **p < 0.01, ***p < 0.001.