Mechanism Of TRNA M~5C Methyltransferase NSUN2 Involved In The Development Of Anaplastic Thyroid Cancer

Background:Thyroid cancer is the most common endocrine malignancy and its incidence has increased rapidly worldwide in recent decades.In stark contrast to differentiated thyroid carcinoma,anaplastic thyroid cancer（ATC）is a rare invasive malignant tumor with poor prognosis and short mean survival time.To date,there is no effective treatment to prolong the survival of patients with undifferentiated thyroid carcinoma.Therefore,it is of great significance to explore the molecular mechanism of the occurrence and development of ATC,so as to identify new candidate targets and improve therapeutic strategies.Dysregulated translation is critical to tumorigenesis and cancer progression,and this enhanced oncogenic translation program,which requires the stability and availability of tRNA to maintain rapid protein synthesis,is a possible cancer therapeutic target.As one of the core molecules in the translation process,tRNA and the m⁵C methylation modification has gradually become a focus of research in recent years.There have been no reports on the modification of tRNA m5C in anaplastic thyroid cancer.The purpose of this study was to investigate the role of m⁵C methyltransferase NSUN2 and its mediated tRNA m⁵C modification in tumor development,malignant transformation and drug resistance in the background of anaplastic thyroid cancer.Materials and methods:1)The target molecule of interest,tRNA m5C methyltransferase NSUN2,was identified using public databases;The expression level of NSUN2 was quantified by public database data analysis,immunohistochemistry and immunofluorescence of collected specimens.2)The public database was used to conduct pan-cancer expression profile analysis of NSUN2,and single gene co-expression analysis,gene pathway enrichment analysis and immune infiltration analysis in thyroid cancer were performed to speculate the possible biological function of NSUN2 in cancer cells.3)The role of NSUN2 in the phenotype of cancer cells was explored using proliferative activity assay,Transwell invasion and migration,clonal formation and cell cycle assay.The influence of NSUN2on ATC drug-resistance was analyzed by determining the half-inhibitory concentration(IC₅₀)of cisplatin or doxorubicin HCl for ATC cells.4)Subcutaneous tumor formation and pulmonary metastasis in nude mice were investigated to explore the significance of NSUN2 in vivo on tumor growth or metastasis.And we explored the effect of intraperitoneal injection of cisplatin or doxorubicin hydrochloride combined with NSUN2 knockout on nude mice with tumor;5)The downstream target molecules and pathways of NSUN2 were investigated by tRNA bisulfite sequencing,liquid chromatography-mass spectrometry（LC-MS）,ribosome sequencing and mRNA sequencing.The results were verified by puromycin intake assay,high-frequency codon reporter assay,real-time quantitative PCR（qRT-PCR）and western blotting.6)tRNA expression and charging levels were determined by qRT-PCR and sodium periodate oxidation.tRNA sequencing and methylation level of tRNA m⁵C were combined to explore the correlation between them.The tRNA-derived fragments（tRFs）were predicted.7)Knockdown of c-Myc by si RNA was used to explore its regulatory effect on downstream NSUN2.On this basis,overexpression of NSUN2 rescued the phenotype of ATC cells.Results:1)In ATC,the components of the tRNA m⁵C system are up-regulated to varying degrees.In particular,m⁵C writer--NSUN2,which has become a hot topic of epigenetic methylation modification,is highly expressed in ATC and is highly correlated with the differentiation degree of thyroid cancer.2)According to bioinformatics analysis,the up-regulation of NSUN2 expression caused the expression changes of various transcripts including mRNA,mi RNA and lnc RNA,which were mainly related to cell proliferation,metabolism,protein synthesis and metabolism,mi RNA processing and other biological processes.NSUN2 is closely related to the infiltration of immune cells,especially macrophages.3)Targeted knockdown of NSUN2 shows extensive anticancer effects.In vitro,inhibition of NSUN2 can inhibit the formation,proliferation,invasion and migration of ATC and cause downregulation of key proteins in the star oncogenic signaling pathway.After NSUN2knockdown,the semi-inhibitory concentration(IC₅₀)of cisplatin or doxorubicin hydrochloride on ATC cells decreased,and the sensitivity of ATC to genotoxic drugs increased.On the contrary,overexpression of NSUN2 further enhanced the malignant phenotype of ATC cells.4)NSUN2-knockdown ATC cells showed limited growth and decreased proliferative activity in vivo,showing weak alveolar and vascular invasion;Intraperitoneal injection of cisplatin or doxorubicin hydrochloride in nude mice with tumor could further enhance the efficacy of NSUN2 and effectively inhibit tumor growth.5)NSUN2 extensively catalyzed tRNA m⁵C modification, related to the secondary structure of tRNA,and supported cancer promoting translation programs including c-Myc,BCL2,RAB31,JUNB,and TRAF2;6)Taking the representative tRNA Leu-CAA and tRNA Leu-CAG（as the control）as the main research objects,it was found that m⁵C essentially maintained the stability of a type of tRNA,and the expression level of tRNA was positively correlated with the level of m⁵C,thus promoting efficient translation procedures;7)Due to the instability of Trnas lacking m⁵C modification,one of the ways is to generate Tr Na-derived fragments（tRFs）.At least three types of 3’tRFs derived from tRNA Leu-CAA were determined experimentally.8)Targeting NSUN2 successfully interrupted the vicious cycle from c-Myc to NSUN2 in ATC.Conclusion:In ATC,tRNA m⁵C methyltransferase NSUN2 promotes tumor development and maintains genotoxic drug resistance.Mechanistically,NSUN2 maintains the stability of tRNA and the rapid translation of downstream oncogenes（including c-Myc,etc.）through m⁵C methylation modification at the variable loop position of tRNA,thus achieving the regulation of downstream codon dependent oncogene translation network response.Notably,a vicious cycle from c-Myc to NSUN2 exists in ATC cells.NSUN2 and m⁵C methylated tRNAs may be potential targets for ATC treatment.