Effect And Mechanism Of M~6A Reader YTHDF3 On Biological Behavior Of Thyroid Papillary Carcinoma

background:Papillary thyroid cancer(PTC)is the most common histological subtype of thyroid cancer,and its incidence has steadily increased in recent decades.The main driving factor is the advancement of imaging techniques.But PTC has a good prognosis after standardized treatment and is considered to be one of the least deadly cancers.In recent years,the problem of overdiagnosis of PTC has attracted more and more attention.The benefits of thyroid cancer screening may be far less than the associated risks of subsequent treatment.Therefore,how to balance the relationship between diagnosis and treatment as well as postoperative follow-up has become a difficult problem at present.m~6A,as the most common methylation modification in eukaryotic RNA,is dynamically regulated by“writers”and“erasers”.“Readers”participate in all stages of RNA regulation.The abnormal expression of m~6A regulatory factor is closely related to the occurrence and development of many diseases and cancers.However,the role of m~6A regulatory factor in the occurrence and development of thyroid papillary carcinoma is not clear.Therefore,this study makes a systematic and comprehensive research on the role of m~6A regulatory factor in PTC.Methods:firstly,the expression,mutation and interaction of m~6A regulatory factors were analyzed in TCGA-THCA data by bioinformatics.Univariate Cox and Lasso Cox regression analysis was used to screen the prognostic m~6A regulators to build a risk model,and combined with clinicopathological information to build a nomogram.Then explore the effect of the key“reader”YTHDF3 on the biological behavior of PTC,and clarify its effect on the proliferation,migration,cell cycle and apoptosis of PTC cells through cell function experiment(CCK-8,Ed U staining,plate clone,transwell migration assay,cell scratch assay,cell cycle and apoptosis assay).Finally,m RNA-seq technology was used to explore the downstream signal pathway regulated by YTHDF3,and Me RIP-q PCR confirmed that the regulation of YTHDF3 on target genes was m~6A dependent.Results:we found that most m~6A regulatory factors were abnormally expressed in thyroid cancer,and the relationship between them was rich,which played a vital role in the occurrence and development of thyroid cancer.The survival analysis of tumor samples screened out 4 m~6A regulatory factors related to prognosis.The prognostic risk model of thyroid cancer constructed based on 4-m~6A regulatory factors(YTHDF3、YTHDF1、RBM15 and IGF2BP2)can well stratify the risk of patients,and high risk means poor prognosis.The nomogram drawn by combining traditional clinical information can combine gene research with clinic.Compared with traditional clinical information,nomogram has excellent prediction ability and better clinical utility for the long-term survival probability of patients.The key“reader”YTHDF3 was selected for the subsequent study on the biological behavior of PTC.From the results of cell function experiment,we found that YTHDF3 could significantly inhibit the proliferation and migration of PTC cells,block the cell cycle in G1 phase and promote apoptosis.m RNA-seq technology was used to screen the downstream signal pathway regulated by YTHDF3.Hippo signal pathway was involved in regulating a variety of biological processes,including cell proliferation,cell fate determination and EMT.We determined by Me RIP-q PCR that YAP1,the core effector of Hippo signaling pathway,was regulated by YTHDF3 by m~6A dependent,and the expression of YAP1 increased after down regulating YTHDF3.Western blotting showed that down-regulation of YTHDF3 increased the expression of anti-apoptosis related proteins and decreased the expression of apoptosis related proteins.EMT characteristic proteins were also affected by the down-regulation of YTHDF3,and the expression of epithelial markers decreased,while the expression of interstitial markers increased.Conclusion:m~6A regulatory factor is abnormally expressed in thyroid cancer,and the risk model based on 4-m~6a regulatory factor can be used as a new tool for risk stratification in patients with thyroid cancer.YTHDF3is an inhibitor of malignant biological behavior of PTC cells.It regulates YAP1 to activate Hippo signal pathway in a m~6A dependent manner,inhibits the proliferation and migration of PTC cells,and promotes apoptosis.