Overexpressed ZC3H13 Suppresses Papillary Thyroid Carcinoma Growth Through M6A Modification-mediated IQGAP1 Degradation

Background and Objective:Thyroid cancer is the most common type of endocrine tumor,and its incidence has been increasing for years.Although thyroid cancer has a good prognosis in terms of long-term survival,PTC still exist a considerable recurrence rate,and the diagnosis and treatment of metastatic recurrence in advanced PTC remains a challenge.Studies based on molecular mechanisms are expected to help diagnose and treat PTC.Therefore,exploring the molecular pathological mechanisms of PTC has become a very important issue in the field of thyroid cancer diagnosis and treatment.m~6A methylation is a common eukaryotic RNA epigenetic modification that affects m RNA stability,splicing,export,and translation to regulate m RNA dynamics,and is involved in regulating not only coding RNAs but also long-stranded non-coding RNAs.The modification process involves the methylation of the N element at position 6 of the adenine(A)base in the RNA with the m~6A methylation site that can be recognized on the RNA.This modification process is related to the catalysis of the methyltransferase complex formed by the"methylation recognition enzymes"YTH family protein 1/2/3,insulin-like growth factor 2 m RNA-binding protein 1/2/3,and common"methyltransferases"such as methyltransferase-like factor3 and RNA-binding motif protein 15.ZC3H13(zinc finger CCCH domain-containing protein 13)is the m~6A methyltransferase responsible for m~6A installation and is involved in the regulation of RNA metabolism and embryonic stem cell renewal.and diagnosis,but its role in thyroid cancer is not yet clear.Initially,we determined the expression level of ZC3H13 in thyroid cancer cells by WB and explored the effect of ZC3H13 on the proliferation and invasion ability of thyroid cancer cells by in vitro experiments.Secondly,through bioinformatics analysis,we identified the presence of multiple m~6A methylation sites IQGAP1m RNA,and further validated the relationship between ZC3H13 and IQGAP1 m RNA and the effect of ZC3H13 on thyroid cancer proliferation through IQGAP1 m RNA by in vitro experiments.Finally,the specific mechanism of ZC3H13 regulation of IQGAP1 m RNA was clarified.This study provides noval research approach for the diagnosis and drug target treatment of thyroid cancer.Methods:1.Clinical levelThe expression of ZC3H13,IQGAP1 and other related proteins were determined by protein blotting;the m RNA levels of ZC3H13 and IQGAP1 were determined by q RT-PCR.2.Cell levelsChanges in proliferation,migration,and invasion capacity of thyroid cancer cells were determined using CCK-8 assay,cross-well migration,and invasion assay,respectively.3.Animal levelsAn in vivo nude mouse xenograft tumor model was established.In vivo validation of ZC3H13-overexpression,by LV-NC,LV-ZC3H13,LV-ZC3H13,+si-YTHDF2 by reducing IQGAP1 on tumor-growth.4.Molecular levelThe m6A level of IQGAP1 m RNA was assessed first using Me RIP q PCR,then the effect of ZC3H13 on IQGAP1 m RNA stability was observed in the presence of actinomycin D(an RNA polymerase inhibitor),and the interaction between YTHDF2and IQGAP1 m RNA was explored by RIP analysis.5.BioinformaticsUALCAN and GEPIA network resources analyzed ZC3H13 expression online,and UALCAN was used to investigate the relationship between ZC3H13 expression and clinicopathology.Results:1.ZC3H13 expression was decreased in PTC cell lines BCPAP,KTC-1,k1,HTH83 and TPC-1.2.Overexpression of ZC3H13 inhibited the proliferation,invasion and migration of PTC cells,and knockdown of ZC3H13 increased the proliferation,invasion and migration of PTC cells.3.ZC3H13 overexpression inhibited IQGAP1 expression,but ZC3H13knockdown enhanced IQGAP expression.In ZC3H13 overexpressed PTC cells,the m~6A level of IQGAP1 m RNA was elevated,and IQGAP1 m RNA expression decreased with increasing time of actinomycin D treatment.YTHDF2 enriched more IQGAP1 m RNA than Ig G,and the knockdown of YTHDF2 reversed the effect of ZC3H13 overexpression on IQGAP1 m RNA stability.4.Overexpression of ZC3H13 inhibited tumor growth,while overexpression of IQGAP1 reversed the inhibitory effect of ZC3H13 overexpression on tumor growth.Conclusion:1.ZC3H13 was downexpressed in PTC2.ZC3H13 inhibits the proliferation,invasion,and migration of KTC-1 and TPC-1 cells,and conversely ZC3H13 knockdown promotes the proliferation,invasion and migration of BCPAP and TPC-1 cells3.ZC3H13 regulates the stability of IQGAP1 m RNA through m~6A modification4.In vivo validation of the inhibitory effect of ZC3H13 overexpression on tumor growth by reducing IQGAP1.