【作者】 李鹏；

【导师】 李新营；

【作者基本信息】 中南大学 ， 临床医学（八年制）（专业学位）， 2023， 博士

【摘要】 研究背景:甲状腺癌是最常见的内分泌恶性肿瘤,近几十年来在世界范围内发病率迅速上升。与分化型甲状腺癌形成鲜明对比,甲状腺未分化癌（anaplastic thyroid cancer,ATC）是一种罕见的侵袭性恶性肿瘤,预后极差,平均生存时间短。迄今为止,尚无有效的治疗方案来延长甲状腺未分化癌患者的生存期。因此,探索ATC发生和发展的分子机制,进而识别新的候选靶点,改进治疗策略具有重要意义。翻译失调对肿瘤发生和癌症进展至关重要,这种增强的促癌翻译程序需要tRNA的稳定性和可用性来维持快速蛋白质合成,是可能的癌症治疗靶点。翻译过程中的核心分子之一,tRNA及其m⁵C甲基化修饰近年来逐渐成为研究热点,关于甲状腺未分化癌tRNA m⁵C修饰的研究尚未见报道。本研究以ATC为背景,目的在于探究m⁵C甲基转移酶NSUN2及其所介导的tRNA m⁵C修饰在肿瘤发生发展、恶性转化及耐药性中的作用。材料和方法:1)利用公共数据库确定感兴趣的目标分子tRNA m⁵C甲基转移酶NSUN2;通过公共数据库数据和收集标本免疫组化、免疫荧光对NSUN2表达水平进行相对定量;2)利用公共数据库对NSUN2进行泛癌表达谱分析,并进行甲状腺癌NSUN2单基因共表达分析、基因通路富集分析和免疫浸润分析,推测NSUN2在癌细胞中可能的生物学功能;3)利用增殖活性实验、Transwell侵袭及迁移、克隆形成和细胞周期实验探究NSUN2在癌细胞表型中的作用。通过测定顺铂或盐酸阿霉素对ATC细胞的半抑制浓度(half-maximal inhibitory concentration,IC₅₀)来分析NSUN2对ATC耐药性的影响;4)裸鼠皮下成瘤和心内注射肺转移模型探究活体内NSUN2对肿瘤增长或转移的意义。腹腔内注射顺铂或盐酸阿霉素探究药物治疗联合NSUN2敲除对荷瘤裸鼠的疗效;5)借助tRNA亚硫酸氢盐测序、高效液相色谱-质谱联用、核糖体印迹测序和mRNA测序探究NSUN2下游标靶分子和通路,并通过嘌呤霉素掺入实验、高频密码子报告实验、实时荧光定量PCR（qRT-PCR）和蛋白免疫印迹（Western blotting）进行验证;6)利用qRT-PCR和高碘酸钠氧化法对tRNA表达量及氨基酰化水平进行测定;tRNA测序和tRNA m⁵C甲基化水平联合分析探究两者相关性;实验推测相关tRNA衍生片段（tRFs）;7)利用si RNA敲低c-Myc来探究其对下游NSUN2的调控作用,在此基础上,过表达NSUN2对ATC细胞表型进行挽救。结果:1)在ATC中,tRNA m⁵C体系中的组分出现不同程度的上调,尤其是近来逐渐成为表观遗传甲基化修饰研究热点的m⁵C writer——NSUN2,在ATC中高表达且与甲状腺癌的分化程度相关;2)根据生物信息学分析,NSUN2表达上调引起了包括mRNA,mi RNA,lnc RNA在内的多种转录本的表达变化,主要与细胞增殖、代谢、蛋白合成与代谢及mi RNA处理等多个生物学过程相关;NSUN2与免疫细胞浸润,特别是巨噬细胞的浸润密切相关;3)靶向敲低NSUN2表现出广泛的抗癌作用,体外条件下,抑制NSUN2可阻遏ATC的形成、增殖、侵袭和迁移并且引起明星致癌信号通路中关键蛋白下调。敲低NSUN2后,顺铂或盐酸阿霉素对ATC细胞的半抑制浓度(IC₅₀)降低,ATC对基因毒性药物的敏感性增强;反之,过表达NSUN2则进一步增强ATC细胞的恶性表型;4)敲低NSUN2的ATC细胞在体内生长受限、增殖活性减低,显示出较弱的肺泡和血管侵袭性;腹腔注射顺铂或盐酸阿霉素对荷瘤裸鼠进行治疗,NSUN2缺失将进一步增强药物的疗效,有效抑制肿瘤生长;5)NSUN2广泛催化tRNA m⁵C修饰,与tRNA二级结构相关,支持包括c-Myc,BCL2,RAB31,JUNB和TRAF2在内的促癌翻译程序;6)以具有代表性的tRNA Leu-CAA及tRNA Leu-CAG（作为对照）为主要研究对象,发现可变环m⁵C修饰实质上维持某类tRNA的稳定性,tRNA表达量和m⁵C水平呈正相关,进而促进高效率翻译程序;7)缺乏m⁵C修饰的tRNA由于其不稳定性,其中一条去路是生成tRNA衍生片段（tRNA-derived fragments,tRFs）,通过实验确定了由tRNA Leu-CAA生成的至少3种3’tRF;8)靶向NSUN2成功打断了ATC中由c-Myc到NSUN2恶性循环。结论:在甲状腺未分化癌中,tRNA m⁵C甲基转移酶NSUN2促进肿瘤发生发展、维持基因毒性药物抗性;机制上,NSUN2通过在tRNA可变环位置的m⁵C甲基化修饰维持tRNA稳定性,维持下游致癌基因（包括c-Myc等）的快速翻译,从而实现下游密码子依赖性致癌基因翻译网络反应的调控;值得注意的是,ATC细胞中存在由c-Myc到NSUN2的恶性循环。NSUN2及m⁵C甲基化修饰的tRNA可能成为ATC治疗的潜在靶点。图36幅,表7个,参考文献168篇更多还原

【Abstract】 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.更多还原