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. 2024 Feb 23;29(1):28.
doi: 10.1186/s11658-024-00543-3.

N6-methyladenosine-modified circ_104797 sustains cisplatin resistance in bladder cancer through acting as RNA sponges

Congjie Xu #  1 , Jiaquan Zhou #  1 , Xiaoting Zhang #  2 , Xinli Kang  1 , Shuan Liu  1 , Mi Song  1 , Cheng Chang  1 , Youtu Lin  3 , Yang Wang  4
Affiliations

N6-methyladenosine-modified circ_104797 sustains cisplatin resistance in bladder cancer through acting as RNA sponges

Congjie Xu et al. Cell Mol Biol Lett. .

Abstract

Background: Bladder cancer (BCa) ranks among the predominant malignancies affecting the urinary system. Cisplatin (CDDP) remains a cornerstone therapeutic agent for BCa management. Recent insights suggest pivotal roles of circular RNA (circRNA) and N6-methyladenosine (m6A) in modulating CDDP resistance in BCa, emphasizing the importance of elucidating these pathways to optimize cisplatin-based treatments.

Methods: Comprehensive bioinformatics assessments were undertaken to discern circ_104797 expression patterns, its specific interaction domains, and m6A motifs. These findings were subsequently corroborated through experimental validations. To ascertain the functional implications of circ_104797 in BCa metastasis, in vivo assays employing CRISPR/dCas13b-ALKBH5 were conducted. Techniques, such as RNA immunoprecipitation, biotin pull-down, RNA pull-down, luciferase reporter assays, and western blotting, were employed to delineate the underlying molecular intricacies.

Results: Our investigations revealed an elevated expression of circ_104797 in CDDP-resistant BCa cells, underscoring its pivotal role in sustaining cisplatin resistance. Remarkably, demethylation of circ_104797 markedly augmented the potency of cisplatin-mediated apoptosis. The amplification of circ_104797 in CDDP-resistant cells was attributed to enhanced RNA stability, stemming from an augmented m6A level at a distinct adenosine within circ_104797. Delving deeper, we discerned that circ_104797 functioned as a microRNA reservoir, specifically sequestering miR-103a and miR-660-3p, thereby potentiating cisplatin resistance.

Conclusions: Our findings unveil a previously uncharted mechanism underpinning cisplatin resistance and advocate the potential therapeutic targeting of circ_104797 in cisplatin-administered patients with BCa, offering a promising avenue for advanced BCa management.

Keywords: Bladder cancer; Cisplatin resistance; N6-methyladenosine; circ_104797; miR-103a.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
circRNA_104797 is upregulated in BCa. A Heatmap illustrating the differential expression profiles of circRNAs between four matched sets of bladder cancer and adjacent normal tissues; B volcano plot representing the results of circRNA microarray analysis; C schematic diagram outlining the selection criteria for identifying candidate regulatory circRNAs enriched in bladder cancer; D comparative expression levels of circRNA_104797 across SV-HVC-1, T24, T24/CDDP, 5637, and 5637/CDDP cell lines; E Kaplan–Meier survival analysis depicting overall survival rates in bladder cancer patients stratified by low or high expression levels of circRNA_104797 (n = 40)
Fig. 2
Fig. 2
The characteristics of circ_104797. A Diagrammatic representation depicting the formation of circ_104797; B gel electrophoresis demonstrates amplification of circ_104797 using divergent primers from total cDNA, but not from genomic DNA (gDNA); C, D RT-qPCR analysis indicating the relative abundance of GAPDH mRNA and circ_104797 in T24/CDDP (C) and 5637/CDDP (D) cells post actinomycin D (Act-D) treatment at specified time intervals; E, F RT-qPCR results confirm the resistance of circ_104797 to RNase R digestion in T24/CDDP (E) and 5637/CDDP (F) cells; G subcellular localization of circ_104797 in BCa cells as determined by FISH assay; H, I RT-qPCR assessment of circ_104797, actin, and U6 distribution in the cytoplasmic and nuclear fractions of T24/CDDP (H) and 5637/CDDP (I) cells
Fig. 3
Fig. 3
circ_104797 is modulated by m6A RNA methylation. A Identification of the potential m6A site in circ_104797, as determined by the convergence of results from the sequence-based N6-methyladenosine (m6A) site prediction tools SRAMP and RMBase v2.0; B MeRIP-qPCR analysis illustrating the relative enrichment of circ_104797 when immunoprecipitated with m6A antibody (m6A-IP) compared with IgG in BCa cells; C bioinformatics analysis using online platforms (https://circinteractome.nia.nih.gov/, http://rbpdb.ccbr.utoronto.ca/) suggested potential interactions between circ_104797 and specific RNA binding proteins (RBP); D RNA pulldown assays complemented by western blotting revealed interactions of circ_104797 with METTL3, ALKBH5, and IGF2BP1/2; E, F RBP immunoprecipitation (RIP) followed by qPCR analysis indicated significant enrichment of circ_104797 in the anti-ALKBH5 and anti-IGF2BP2 antibody groups compared to the IgG control in T24/CDDP (E) and 5637/CDDP (F) cells
Fig. 4
Fig. 4
ALKBH5 inhibited the m6A modification process of circ_104797. A, B MeRIP-qPCR analyses depicted the relative enrichment of circ_104797 when immunoprecipitated with m6A antibody (m6A-IP) versus immunoglobulin G (IgG) in T24/CDDP (A) and 5637/CDDP (B) cells in both the presence and absence of ALKBH5 overexpression; C expression levels of circ_104797 were assessed using RT-qPCR following ALKBH5 overexpression; D, E examination of circ_104797 expression in T24/CDDP (D) and 5637/CDDP (E) cells post actinomycin D treatment considering the presence or absence of ALKBH5 overexpression; F a schematic representation highlights the AGACU m6A motif situated at the junction of exon 6 and exon 7 in circ_104797; G dual-luciferase assays revealed the expression efficiency differences between m6A-WT and m6A-Mut in BCa cells overexpressing ALKBH5; H, I RT-qPCR analyses determined the relative abundance of GAPDH mRNA and circ_104797 in T24/CDDP (H) and 5637/CDDP (I) cells post Act-D treatment, considering the overexpression status of ALKBH5
Fig. 5
Fig. 5
Targeting m6A methylation of circ_104797 by CRISPR/dCas13b-ALKBH5 to regulate cisplatin-resistant BCa cells proliferation and apoptosis. A A diagrammatic representation delineated the m6A site locations within circ_104797 and the regions targeted by specific guide RNA, B,C In both T24/CDDP and 5637/CDDP cells, m6A modifications (B) and expression patterns (C) of circ_104797 were assessed post-transfection with dCas13b-ALKBH5, either in conjunction with control gRNA or circ_104797-specific gRNA; D,E the proliferative capacity of T24/CDDP (D) and 5637/CDDP (E) cells, post-transfection with dCas13b-ALKBH5 and either control gRNA or circ_104797-specific gRNA, was gauged using the CCK-8 assay; F,G wound healing assays were conducted on T24/CDDP and 5637/CDDP cells post-transfection with dCas13b-ALKBH5 and the respective gRNAs, with representative images (F) and subsequent quantitative evaluations (G) presented; H apoptotic rates in T24/CDDP and 5637/CDDP cells, post-transfection with dCas13b-ALKBH5 and the respective gRNAs, were determined through ELISA; I, J the tumor volume and weights of BCa cells stably transfected with dCas13b-ALKBH5 combined with gRNA control or gRNA for circ_104797
Fig. 6
Fig. 6
circ_104797 acts as an efficient miRNA sponge for miR-103a and miR-660-3p. A A diagrammatic representation highlights the potential binding regions of miRNAs in relation to circ_104797; B qRT-PCR results depict the expression levels of the selected miRNAs following the overexpression of circ_104797 in BCa cells; C a schematic overview of the Ago2-RIP procedure is provided; D, E post-Ago2 RIP assay, qRT-PCR was conducted to assess the expression levels of circ_104797, miR-103a, and miR-660-3p in T24/CDDP (D) and 5637/CDDP (E) cells; F CCK-8 assays were performed on cisplatin-resistant BCa cells post-transfection with respective miRNA mimics and control sequences
Fig. 7
Fig. 7
circRNA_104797 sustains cisplatin resistance by acting as a miRNA sponge for miR-103a and miR-660-3p. A Diagrammatic representation of the luciferase reporter vectors for wild-type (WT) and mutant (MUT) circ_104797; B, C luciferase activity measurements for both WT and MUT linear circ_104797 post-transfection with miR-103a (B) and miR-660-3p (C) mimics in cisplatin-resistant BCa cells; D, E post-demethylation of circ_104797 via CRISPR/dCas13b-ALKBH5, RT-qPCR was conducted to determine the expression levels of miR-103a and miR-660-3p in T24/CDDP (D) and 5637/CDDP (E) cells
Fig. 8
Fig. 8
circ_104797 demethylation inhibited CDDP resistance in BCa cells through targeting miR-103a and miR-660-3p. A, B Proliferation analysis of T24/CDDP (A) and 5637/CDDP (B) cells following demethylation of circ_104797 and concurrent knockdown of miR-103a and miR-660-3p; C, D migration evaluation of T24/CDDP (C) and 5637/CDDP (D) cells post-demethylation of circ_104797 and simultaneous suppression of miR-103a and miR-660-3p; E, F apoptosis assessment in T24/CDDP (E) and 5637/CDDP (F) cells after circ_104797 demethylation and concurrent downregulation of miR-103a and miR-660-3p
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