N6‐Methyladenosine reader HNRNPC‐mediated downregulation of circITCH prevents miR‐224‐3p sequestering and contributes to tumorigenesis in nasopharyngeal carcinoma

Abstract

AbstractBackgroundN6‐Methyladenosine (m6A) RNA methylation modulators are implicated in nasopharyngeal carcinoma (NPC). Circular RNAs (circRNAs) stimulate/inhibit the development of NPC by sponging microRNAs (miRNAs). Herein, m6A modifications affecting the circRNA/miRNA axis in NPC were explored.MethodsTwenty prognostic m6A RNA methylation regulators were identified from 504 head/neck squamous cell carcinoma and 44 normal samples from The Cancer Genome Atlas (TCGA). Differentially expressed miRNAs were screened from the TCGA and Gene Expression Omnibus (GEO) databases. RNA‐binding protein (RBP)–circRNA and circRNA–miRNA interactive pairs were verified using RBPmap and RNAhybrid, respectively. The RBP/circRNA/miRNA network was constructed using Cytoscape. Furthermore, CircITCH (hsa_circ_00059948), HNRNPC, and miR‐224‐3p expressions were detected by western blotting and quantitative polymerase chain reaction. The role of circITCH in NPC was examined using apoptosis, scratch wound healing, transwell invasion, and cell counting kit‐8 assays. Finally, CircITCH–miR‐224‐3p and circITCH–HNRNPC interactions were assessed by dual‐luciferase reporter and RNA‐immunoprecipitation (RIP) assays, respectively.ResultsBioinformatics analysis revealed that high pathological grade, late‐stage tumors, and low survival were associated with increased HNRNPC expression. MiR‐224‐3p was upregulated in NPC and sequestered by circITCH. Construction of the RBP/circRNA/miRNA network highlighted the HNRNPC/circITCH/miR‐224‐3p axis. In vitro experiments demonstrated decreased circITCH expression and increased HNRNPC and miR‐224‐3p expressions in NPC. In NPC cells overexpressing circITCH, HNRNPC and miR‐224‐3p expressions were significantly decreased. Dual‐luciferase assays demonstrated a targeting relationship between circITCH and miR‐224‐3p, and RIP assays demonstrated interaction of HNRNPC targets with circITCH.ConclusionCircITCH overexpression inhibited NPC progression by sequestering miR‐224‐3p, and HNRNPC reduced circITCH expression through direct interaction.