IGF2BP3 Stabilizes CircHECTD2 In A M6A Modification-Dependent Manner To Promote Hypopharyngeal Carcinoma Progression

Background:Hypopharyngeal squamous cell carcinoma(HSCC)accounts for about 3%of head and neck malignancies,and is most common in the piriform fossa.Although the incidence rate is not high,HSCC has a high degree of malignancy and poor prognosis.The 5year survival rate is less than 40%,so it is urgent to explore new diagnostic and therapeutic targets.Increasing reports indicate m6A modification of circRNAs play important roles in regulating cancer progression.However,in hypopharyngeal squamous cell carcinoma(HSCC),the biological roles and mechanism of m6A modification of circRNAs remain unknown.Objectives:(1)To investigate the expression of IGF2BP3 in HSCC and its effect on the progression of HSCC;(2)To verify the structure and subcellular localization of circHECTD2;(3)To investigate the methylation regulation and stabilizing effects of IGF2BP3 on the downstream circHECTD2;(4)To investigate the signaling pathway of IGF2BP3/circHECTD on the progression of HSCC.Methods:First,MeRIP-Seq was used to detect the m6A modification profile of HSCC and adjacent normal tissue to determine whether the m6A modification of circRNA is involved in the progression of HSCC.Secondly,qRT-PCR technology was used to detect the upstream regulatory genes of m6A modification(common writers,readers and erasers involved in RNA m6A modification)to identify m6A modification-related regulators involved in HSCC progression.Thirdly,potential circRNAs involved in the progression of HSCC were identified by down-regulating this modification factor combined with circRNA-seq technology,and techniques such as RNA immunoprecipitation,RNA Pulldown,Knockdown,Knockout and Western Blot were performed to explore the molecular mechanism of circRNA m6A modification regulating HSCC progression.Results:The m6A modified profile of circRNA was significantly different between HSCC and adjacent normal tissue.While IGF2BP3 is a circRNA m6A modification related protein involved in the progression of hypopharyngeal cancer,high expression of IGF2BP3 is associated with poor clinical prognosis,and low expression of IGF2BP3 in FaDu cells decreased proliferation and migration ability in vitro and in vivo.CircHECTD2 was regulated by IGF2BP3 and was involved in HSCC progression,and this was because IGF2BP3 binded to circHECTD2 and promoted its stabilization.m6A modification of circHECTD2 enhanced the interaction between IGF2BP3 and circHECTD2,thus further enhancing the stability of circHECTD2 itself.Downregulation of IGF2BP3 or circHECTD2 induced downregulation of TGF-β signaling pathway.Conclusion:In this study,we found a key factor promoting the progression of HSCC.IGF2BP3,as a reader protein associated with RNA m6A modification,can interact with downstream circHECTD2 and promote its stability,and up-regulate the TGF-β signaling pathway to promote the progression of HSCC.The m6A modification of circHECTD2 can enhance the interaction between IGF2BP3 and circHECTD2.IGF2BP3 and circHECTD2 may be involved in clinical diagnosis and treatment as promising prognostic biomarkers and targets.