m6A modified circPTK2 mediates TNBC chemotherapy resistance

Circular RNAs (circRNAs) act as novel biomarkers associated with drug resistance in triple-negative breast cancer (TNBC). Our previous study has demonstrated that the Wnt/β-catenin pathway mediates TNBC chemoresistance, and the present study further investigates its upstream regulatory mechanisms.

Gene chip analysis was used to screen for circRNAs in drug-resistant cells. The expression levels of circPTK2 in cells and tissues were detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Cell Counting Kit-8 (CCK-8), Transwell, flow cytometry, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL), three-dimensional (3D) multicellular tumor spheroid culture model, 5-ethynyl-2'-deoxyuridine (EdU) proliferation staining, western blot and immunofluorescence were used to detect the influence of circPTK2 on the drug sensitivity, cell invasion, proliferation, apoptosis and DNA damage repair of TNBC cells. The pull-down and dual-luciferase reporter gene assays were used to determine the interaction between circPTK2 and miR-495. Dual luciferase reporter gene assay and RNA immunoprecipitation (RIP) experiment verified the binding of miR-495 to β-catenin. A transplanted tumor model was established in nude mice to determine circPTK2 effects on the chemosensitivity of drug-resistant cells to cisplatin (DDP). Methylated RNA immunoprecipitation (MeRIP) and RNA electrophoretic mobility shift assay (RNA-EMSA) verified the enrichment of the N6-methyladenosine (m6A) methylated region of circPTK2. The m6A quantitative kit was used to detect the level of m6A modification. MeRIP-PCR was used to detect the enrichment amount of m6A-modified circPTK2 (m6A⁺ circPTK2).

qRT-PCR results indicated that the expression level of circPTK2 in drug-resistant cells was higher than that in parental cells. The expression level of circPTK2 in DDP-resistant tissues was significantly higher than that in DDP-sensitive tissues. circPTK2 has the characteristics of resisting Rnase R digestion and having stronger stability than linear RNA. Knocking down of circPTK2 can reduce the viability and invasion ability of TNBC DDP-resistant cells, promote the DNA damage induced by DDP, and enhance the sensitivity of TNBC cells to DDP. RNA fluorescence in situ hybridization (RNA FISH) results showed that circPTK2 and miR-495 were co-localized in the cytoplasm. The dual luciferase reporter gene activity assay showed the targeted binding of miR-495 to the 3'UTR region of β-catenin. The RNA immunoprecipitation-polymerase chain reaction (RIP-PCR) experiment confirmed that YTH domain-containing protein 1 (YTHDC1) could bind to circPTK2. m6A quantitative kit showed that the m6A modification in the resistant cells was higher than that in the parental cells. qRT-PCR and Western blot results showed that the mRNA and protein expression level of YTHDC1 in the DDP resistant cells was higher than that in the parental cells.

The m6A methylation reader YTHDC1 mediates the regulation of DDP resistance by circPTK2 in TNBC, and circPTK2 may serve as a non-invasive biomarker for clinical detection of DDP resistance in TNBC.