CircRNF13 enhances IGF2BP1 phase separation-mediated ITGB1 mRNA stabilization in an m6A-dependent manner to promote oral cancer cisplatin chemoresistance

Abstract

Oral cancer ranks among the most common malignancies within the head and neck region; however, its etiology remains inadequately understood despite substantial research advances in recent years. Many studies highlight the regulatory role of circular RNAs (circRNAs) in human cancers, suggesting their potential as cancer biomarkers. However, their specific mechanisms in oral cancer are not well understood. This study analyzed circRNAs expression in oral cancer, identifying circRNF13 (circbaseID: has_circ_0006801) as having elevated expression in oral cancer cells and tissues. Our study demonstrated that circRNF13 is correlated with increased tumor grade and stage in oral cancer. Results from both in vitro and in vivo experiments indicated that circRNF13 enhances cancer cell proliferation and tumor growth, while concurrently diminishing tumor sensitivity to cisplatin. Mechanistically, circRNF13 interacts with the m6A "reader" protein IGF2BP1, inhibiting its ubiquitin-mediated degradation and promoting its phase separation formation. Subsequently, circRNF13 augments the stability of ITGB1 mRNA via IGF2BP1 in a manner dependent on m6A modification. The m6A modification of ITGB1 mRNA is modulated by the phase separation of IGF2BP1, thereby promoting the malignant progression of oral cancer cells. This evidence positions circRNF13 as a crucial regulatory molecule in the pathogenesis of oral cancer and suggests its potential as a therapeutic target. This discovery enriches our understanding of the mechanistic role of circRNAs.

Keywords: IGF2BP1; Liquid-liquid phase separation; Oral cancer; circRNF13; m6A.

Fig. 1

Characteristics of circRNF13 in oral cancer cells and tissues. (a) Volcano plot of circRNAs expression in datasets GSE131182 and GSE131568 (significant at Log 2 FC = 2.0, P < 0.05). (b) Venn diagram of differentially expressed circRNAs in these datasets. (c) Diagram of circRNF13’s genomic location and splicing, confirmed by Sanger sequencing. (d) RNA digestion assay showing circRNF13’s stability in Cal-27 cells. (e) Assess circRNF13 and RNF13 mRNA stability via RT-qPCR post-actinomycin D treatment. f-g. Determine circRNF13 localization in Cal-27 and Scc-9 cells using RNA nucleocytoplasmic separation. h. Use FISH assay to detect circRNF13 localization in Cal-27 and Scc-9 cells. i. Display IHC results of circRNF13 expression in oral cancer versus adjacent normal tissues. j. Compare circRNF13 expression levels and scores in oral cancer and adjacent normal tissues (n = 96). k-m. Correlation of circRNF13 expression with N, T, and clinical stages in oral cancer patients. Data are mean ± SD from three independent experiments. **P < 0.01

Fig. 2

Effects of circRNF13 on oral cancer cell proliferation and migration. (a) RT-qPCR analysis of circRNF13 levels after knockdown. (b) CCK-8 assay on circRNF13 knockdown’s effect on Cal-27 and Scc-9 proliferation. (c) EdU assay with statistics on circRNF13’s influence on Cal-27 and Scc-9 proliferation. (d) Plate cloning assay with statistics on circRNF13’s impact on Cal-27 and Scc-9 growth. (e) Transwell assay with statistics on circRNF13’s effect on Cal-27 and Scc-9 migration. (f) Tumor formation images in nude mice post-cell injection. (g) Tumor weight analysis in nude mice. (h) Tumor growth curve in nude mice. i-l. IHC staining for Ki-67 expression and TUNEL staining for apoptosis in tumors, with statistical analyses. Data are mean ± SD. Significance levels: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001

Fig. 3

The effect of circRNF13 on oral cancer biological functions by regulating IGF2BP1 ubiquitination. (a) Bioinformatics analysis predicts the interaction between circRNF13 and IGF2BP1. (b) RNA pull-down assay confirms the binding of circRNF13 to IGF2BP1. (c) The RNA immunoprecipitation (RIP) assay is employed to detect the interaction between circRNF13 and IGF2BP1. (d) The co-localization of circRNF13 with IGF2BP1 in oral cancer cells is detected using fluorescence in situ hybridization combined with immunofluorescence assays. e-f. The impact of circRNF13 on IGF2BP1 mRNA and protein expression levels is assessed using RT-qPCR and western blot analysis. g. Silencing circRNF13 reduces IGF2BP1 translation activity with CHX treatment (left), and IGF2BP1 degradation rate is quantified by gray scale analysis (right). h. Western blot shows IGF2BP1 protein levels after circRNF13 knockdown and 12 h MG132 treatment. i. Immunoprecipitation measures ubiquitinated IGF2BP1 following circRNF13 knockdown. j. IHC assay results display IGF2BP1 expression in oral cancer tissues versus adjacent normal tissues

Fig. 4

CircRNF13 drives the phase separation of IGF2BP1. (a) Bioinformatics identifies a major IDR in IGF2BP1’s amino acid sequence. (b) A droplet formation assay examines IGF2BP1’s cellular distribution and status. (c) FRAP experiments assess IGF2BP1 spot dynamics, with the right panel displaying the fluorescence recovery curve. (d) circRNF13’s impact on IGF2BP1 phase separation droplet formation. (e) circRNF13’s influence on the dynamic resilience of IGF2BP1 phase-separated droplets, based on three independent experiments. ****P < 0.0001

Fig. 5

CircRNF13 enhances ITGB1 mRNA stability by promoting phase separation of IGF2BP1. (a) SRAMP-predicted m6A sites on ITGB1 mRNA. (b) MeRIP-PCR with anti-m6A antibody to assess m6A levels in ITGB1 mRNA. (c) RIP-PCR with anti-IGF2BP1 antibody to examine IGF2BP1 and ITGB1 mRNA interaction. d-e. RT-qPCR and Western blot to evaluate circRNF13’s impact on ITGB1. f. RT-qPCR after ACTD treatment to analyze circRNF13’s effect on ITGB1 mRNA stability. g. RIP assay to study circRNF13’s influence on IGF2BP1 and ITGB1 mRNA interaction. h-j. After circRNF13 knockdown, IGF2BP1 overexpression was analyzed using RT-qPCR and western blot to assess ITGB1 mRNA and protein levels and mRNA stability. k. Examined how 1,6-hexanediol affects IGF2BP1 phase separation. l. RIP assay evaluated the impact of 1,6-hexanediol on IGF2BP1 and ITGB1 mRNA interaction. m. Assessed ITGB1 mRNA levels following 1,6-hexanediol treatment. n. HIC assay results showed ITGB1 expression in oral cancer versus adjacent normal tissues. o. Comparison of ITGB1 expression in oral cancer tissues versus adjacent normal tissues (n = 96), presented as mean ± SD from three independent experiments. Significance levels: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001

Fig. 6

CircRNF13 promotes oral cancer cells growth and migration through IGF2BP1. a-b.ITGB1 expression was measured post-overexpression using RT-qPCR and western blot. c. CCK-8 assay assessed ITGB1 overexpression’s impact on Cal-27 and Scc-9 cell growth after circRNF13 knockdown. d-e. EdU assay evaluated ITGB1 overexpression’s effect on Cal-27 and Scc-9 cellgrowth post-circRNF13 knockdown. f. Plate cloning assay examined the influence of ITGB1 overexpression on Cal-27 and Scc-9 cell growth after circRNF13 knockdown. g. Transwell assay results showing ITGB1 overexpression’s impact on Cal-27 and Scc-9 cell migration after circRNF13 knockdown. h. Images of tumors formed in nude mice via subcutaneous injection. i. Tumor weight analysis in nude mice. j. Tumor growth curve analysis in nude mice. k-m. IHC assay results for Ki-67 expression and TUNEL staining for apoptosis in tumor groups, with statistical analysis. Data are mean ± SD. Significance levels: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001

Fig. 7

CircRNF13 promotes oral cancer cells cisplatin resistance through ITGB1. a Representative and quantified results of colony formation assays for Cal-27 cells coexpressing si-circRNF13 (or siNC) and ITGB1 plasmid (or empty vector) after cisplatin treatment at specified concentrations. (b) Diagram of Cal-27 xenograft mice treated with cisplatin or saline in vivo. (c) Images of nude mice with tumors post cisplatin or saline treatment. (d) Tumor weight analysis in nude mice. (e) Statistical analysis of tumor growth in nude mice. (f) IHC assay and statistical analysis of Ki-67 expression in tumor groups. (g) Tunel staining and statistical analysis of apoptosis in tumor groups. Values are mean ± SD. Significance: *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 8

A proposed model suggests that circRNF13 promotes IGF2BP1 phase separation to stabilize ITGB1 mRNA, promoting oral cancer progression