Circular RNA ARHGAP5 inhibits cisplatin resistance in cervical squamous cell carcinoma by interacting with AUF1

Abstract

Cervical squamous cell carcinoma (CSCC) is one of the leading causes of cancer death in women worldwide. Patients with advanced cervical carcinoma always have a poor prognosis once resistant to cisplatin due to the lack of effective treatment. It is urgent to investigate the molecular mechanisms of cisplatin resistance. Circular RNAs (circRNAs) are known to exert their regulatory functions in a series of malignancies. However, their effects on CSCC remain to be elucidated. Here, we found that cytoplasmic circARHGAP5, derived from second and third exons of the ARHGAP5 gene, was downregulated in cisplatin-resistant tissues compared with normal cervix tissues and untreated cervical cancer tissues. In addition, experiments from overexpression/knockdown cell lines revealed that circARHGAP5 could inhibit cisplatin-mediated cell apoptosis in CSCC cells both in vitro and in vivo. Mechanistically, circARHGAP5 interacted with AU-rich element RNA-binding protein (AUF1) directly. Overexpression of AUF1 could also inhibit cell apoptosis mediated by cisplatin. Furthermore, we detected the potential targets of AUF1 related to the apoptotic pathway and found that bcl-2-like protein 11 (BIM) was not only negatively regulated by AUF1 but positively regulated by circARHGAP5, which indicated that BIM mRNA might be degraded by AUF1 and thereby inhibited tumor cell apoptosis. Collectively, our data indicated that circARHGAP5 directly bound to AUF1 and prevented AUF1 from interacting with BIM mRNA, thereby playing a pivotal role in cisplatin resistance in CSCC. Our study provides insights into overcoming cancer resistance to cisplatin treatment.

Keywords: AUF1; BIM; CSCC; cervical cancer; circARHGAP5.

FIGURE 1

Identification and characterization of circular RNA ARHGAP5 (circARHGAP5) in cervical squamous cell carcinoma (CSCC) cells and tissues. (A) Heatmap of circRNA expression profile in SiHa/Con with SiHa/ΔNp63α and ME‐180/NC with ME‐180/shΔNp63α stable cell lines. (B) Overlap of 193 differentially expressed circRNAs in these two groups of cells. (C) Verification of circARHGAP5 expression levels in the above two groups of cells. (D) Sanger sequencing of backsplice junction site for circARHGAP5. (E) PCR analysis for circARHGAP5 in cDNA and genomic DNA (gDNA) with divergent and convergent primers. (F) Relative RNA levels of circARHGAP5 and ARHGAP5 with or without RNase R digestion. (G) Expression level of circARHGAP5 in normal cervical tissues and untreated and cisplatin (CP)‐resistant CSCC tissues. (H) Nuclear (nuc) and cytoplasmic (cyto) levels of circARHGAP5 in ME‐180 and SiHa cells. GADPH served as cytoplasmic control, while U1 served as nuclear control. (I) FISH assay of circARHGAP5 (red) in ME‐180 and SiHa cells. The location of circARHGAP5 (red) in SiHa and ME‐180 cells was determined by FISH assay. DAPI‐stained nuclei are blue. Scale bar = 5 μm. **p < 0.01, ***p < 0.001. Con, control; NC, negative control; ns, not significant; OE, overexpression.

FIGURE 2

Knockdown of circular RNA ARHGAP5 (circARHGAP5) promoted cervical squamous cell carcinoma drug resistance in vivo and in vitro. (A) Knockdown efficiency of circARHGAP5 shRNAs in SiHa/NC with SiHa/shcircARHGAP5s and ME‐180/NC with ME‐180/shcircARHGAP5s stable cells. (B) Relative change levels of ARHGAP5 in the above two groups of cells. (C) CCK‐8 assays with gradient concentration of cisplatin (CP) in the above two groups of cells. (D, E) Colony formation and apoptosis assays with IC₅₀ concentration of drug cisplatin in the two groups of cells. IC₅₀ for SiHa is 8 μg/ml, and 9 μg/ml for ME‐180. (F) Representative pictures of tumors. (G, H) Weight and volume of the xenograft tumors in the ME‐180/Con and ME180/shcircARHGAP5s cells after 15 days of injection of cisplatin. *p < 0.05, **p < 0.01, ***p < 0.001. Con, control; NC, negative control; ns, not significant; PI, propidium iodide.

FIGURE 3

Overexpression (OE) of circular RNA ARHGAP5 (circARHGAP5) inhibited cervical squamous cell carcinoma drug resistance. (A) Overexpression efficiency of circARHGAP5 in ME‐180/NC and ME‐180/OE‐circARHGAP5 cells, and SiHa/NC and SiHa/OE‐circARHGAP5 cells. (B) Relative change in ARHGAP5 levels in NC/circ‐OE ME‐180 and SiHa cells. (C) CCK‐8 assay with gradient concentration of cisplatin (CP) in NC/circ‐OE ME‐180 cells and SiHa cells. (D) Apoptosis assays with IC₅₀ concentration of drug cisplatin in NC/circ‐OE ME‐180 cells and SiHa cells. IC₅₀ for SiHa is 8 μg/ml, and 9 μg/ml for ME‐180. *p < 0.05, **p < 0.01, ***p < 0.001. NC, negative control; ns, not significant; PI, propidium iodide.

FIGURE 4

Circular RNA ARHGAP5 (circARHGAP5) interacted with AUF1 in cervical squamous cell carcinoma. (A) Ago2 RIP for detection of circARHGAP5 in ME‐180 cells. PDP2 acted as a positive control, while U1 was a negative control. (B) Model of RNA pull‐down assay. (C) Enrichment of circARHGAP5 of RNA pull‐down in ME‐180 cells. (D) Silver staining of proteins binding with circARHGAP5. (E) Western blot of RNA pull‐down for detecting the binding protein of top enrichment proteins of mass spectrometry. (F–H) RNA pull‐down and RIP efficiency of AUF1 protein and enrichment of circARHGAP5 in ME‐180 and SiHa cells. Protein efficiency was validated through western blot, RNA enrichment was validated by quantitative RT‐PCR. (I) FISH of circARHGAP5 (red) along with immunofluorescence staining (IF) of AUF1 (green) in ME‐180 and SiHa cells. IF/FISH assay showed that circARHGAP5 was colocated with AUF1 in ME‐180 and SiHa cells. Scale bar, 5 μm. **p < 0.01, ***p < 0.001. IB, immunoblot; M, marker; ns, not significant; scr, scramble.

FIGURE 5

Circular RNA ARHGAP5 (circARHGAP5) bound to RNA recognition motif 1 (RRM1) and RRM2 of AUF1 in cervical squamous cell carcinoma. (A) Structure of AUF1 with four isoforms. (B) Model of truncations of AUF1. (C) Western blot of RNA pull‐down assay for detecting the truncations of AUF1 binding to circARHGAP5 in ME‐180 cells. (D) Model of circARHGAP5 truncations. (E) RIP efficiency of AUF1 protein and enrichment of circARHGAP5 (WTs and mutants [MTs]) in ME‐180 cells. Protein efficiency was validated through western blot, RNA enrichment was validated by quantitative RT‐PCR. *p < 0.05, ***p < 0.001. ns, not significant

FIGURE 6

AUF1 inhibited cervical squamous cell carcinoma drug resistance in vitro. (A, B) Overexpression (OE) efficiency of AUF1 upon mRNA and protein levels in ME‐180 and SiHa cells. (C, D) CCK‐8 assay with gradient concentration of cisplatin (CP) in AUF1/EV and AUF1/OE upon ME‐180 and SiHa cells. (E) Apoptosis assay with IC₅₀ concentration of drug cisplatin in the two groups of cells. IC₅₀ for SiHa is 8 μg/ml, and 9 μg/ml for ME‐180. *p < 0.05, **p < 0.01, ***p < 0.001. EV, empty vector; ns, not significant; PI, propidium iodide.

FIGURE 7

Circular RNA ARHGAP5 (circARHGAP5) compromised AUF1 degradation of BIM mRNA to promote cell apoptosis. (A) Relative AUF1 mRNA in NC and shcircARHGAP5 stable cells upon ME‐180 and SiHa cervical squamous cell carcinoma (CSCC) cells. (B) Relative circARHGAP5 levels in AUF1/EV and AUF1/overexpressed (OE) upon ME‐180 and SiHa cells.(C, D) Relative mRNA levels of several apoptosis‐related genes in AUF1/EV and AUF1/OE upon ME‐180 and SiHa cells. (E) Survival analysis of BIM in CSCC from Kaplan–Meier Plotter. (F) Relative BIM mRNA in the above NC and shcircARHGAP5 stable cells upon ME‐180 and SiHa cells. (G) Quantitative RT‐PCR showed relative BIM mRNA after circARHGAP5 overexpression in ME‐180 and SiHa cells. (H) Relative BIM mRNA in circARHGAP5‐mutated (MT) ME‐180 cells. (H) Proposed mechanism of circARHGAP5‐regulated cisplatin (CP) resistance. CircARHGAP5 derived from exons 2–3 of ARHGAP5, mainly located in cytoplasm, and then work as a sponge for AUF1 protein, which resulted in the decreased number of “free” AUF1 proteins that bind to BIM mRNAs to promote cell apoptosis in cisplatin resistance. *p < 0.05, **p < 0.01, ***p < 0.001. EV, empty vector; NC, negative control; ns, not significant.