m⁶A甲基化修饰在甲状腺癌发生发展中的作用研究进展

Role of m⁶A methylation modification in the occurrence and development of thyroid cancer: a review of research progress

摘要

甲状腺癌（TC）是最常见的内分泌恶性肿瘤，尽管大多数患者经规范化治疗后预后良好，但部分患者病情进展迅速且预后不良。近年来研究发现，N6-甲基腺苷（m⁶A）甲基化是RNA最常见的修饰，参与调节RNA的转录、成熟、降解和稳定，贯穿于肿瘤的全过程，因此在肿瘤的治疗和预防中被广泛研究。m⁶A水平变化可引起TC中相关癌基因或抑癌基因的异常激活或抑制，从而参与TC的发生发展。笔者就m⁶A甲基化修饰的概念、调控因子的组成成分和功能、在TC发生发展中的作用及治疗和预后作用进行综述。

Abstract

Thyroid cancer (TC) is the most common endocrine malignancy. Although most patients have a favorable prognosis following standardized treatment, a subset experiences rapid disease progression and poor outcomes. Recent studies have identified N6-methyladenosine (m⁶A) methylation as the most prevalent RNA modification, which regulates RNA transcription, maturation, degradation, and stability, playing a role throughout the tumorigenesis process. Consequently, m⁶A methylation has been extensively studied in tumor treatment and prevention. Changes in m⁶A levels can lead to abnormal activation or inhibition of oncogenes or tumor suppressor genes in TC, thereby contributing to its initiation and progression. This review summarizes the concept of m⁶A methylation, the components and functions of its regulatory factors, its role in the development and progression of TC, and its implications for treatment and prognosis.

关键词

甲状腺肿瘤 / N6-甲基腺嘌呤 / 预后 / 综述

Key words

Thyroid Neoplasms / N6-Methyladenosine / Prognosis / Review

甲状腺癌（thyroid cancer，TC）是最常见的内分泌恶性肿瘤，约占美国新发癌症总数的2.2%^[1]，且发病率以每年4.5%的速度持续增长^[2]。根据TC的病理特点可分为分化型、髓样型、低分化型和未分化型^[3]。低危分化型TC的死亡风险<1%且大多数复发可治愈，但高危患者常发生死亡且肿瘤频繁复发^[4]；甲状腺髓样癌的侵袭性较分化型TC强，其Ⅳ期的5年生存率仅有28%^[5]；晚期低分化型TC和甲状腺未分化癌（anaplastic thyroid cancer，ATC）的恶性程度极高，尚缺乏有效的治疗方法，占TC相关死亡总数的40%~50%^[6-7]。基于目前部分TC对常规治疗反应不佳和预后不良，表观遗传修饰可为该疾病的治疗和预后评估提供新的研究方向。N6-甲基腺苷（N6-methyladenosine，m⁶A）甲基化修饰作为一种动态可逆的表观遗传修饰，通过修饰信使RNA（messenger RNA，mRNA）和非编码RNA进而影响其分子功能来调控靶RNA的表达，在肿瘤的发生、进展和耐药中具有关键作用^[8]。因此，本文就m⁶A甲基化修饰在TC的分子机制、治疗和预后进行综述，旨在加深其在TC中表观转录组学的理解，从而指导基础向临床转化的方向。

1 m⁶A甲基化修饰的组成单位及功能

m⁶A甲基化是腺嘌呤N6位的甲基化，亦是真核细胞发生基因转录后最常见的mRNA内源性修饰，主要沉积在3'非编码区或终止密码子区附近的DRACH（D=A，G或U；R=A或G；H=A，C或U）共有序列内^[9]。m⁶A甲基化修饰发挥生物学作用主要受m⁶A三类调控因子介导，包括m⁶A甲基转移酶（编码器，writers）、m⁶A去甲基化酶（擦除器，erasers）和m⁶A结合蛋白（阅读器，readers），三者共同构成m⁶A甲基化修饰的复杂调控网络，参与调节mRNA的转录、成熟、降解和稳定等代谢过程^[10]。

1.1 m⁶A甲基转移酶

编码器复合体（m⁶A methyltransferase complex，MTC）由甲基转移酶样3（methyltransferase like 3，METTL3）、METTL14、Wilms肿瘤1相关蛋白（Wilms' tumor 1-associating protein，WTAP）、RNA结合基序蛋白15（RNA-binding motif protein 15，RBM15）、锌指CCCH结构域蛋白13（zinc finger CCCH domain-containing protein 13，ZC3H13）和病毒样m⁶A甲基转移酶相关蛋白（vir-Like m⁶A methyltransferase associated，VIRMA，亦称KIAA1429）等组成，具有特异性识别mRNA中DRACH序列并启动m⁶A甲基化的作用^[11]。METTL3是MTC的唯一催化亚基，通过与S-腺苷甲硫氨酸结合后可介导m⁶A甲基基团的催化加成反应^[12]。METTL14作为MTC的第二支持酶，虽然没有催化活性作用，但在特异性识别mRNA中DRACH序列和激活METTL3并提高其催化能力方面具有关键作用^[13]。WTAP与细胞核中METTL3-METTL14共同构成MTC的核心组分并将其定位于核斑点^[14]。MTC的其他辅助因子生物学功能如下，ZC3H13可将WTAP-Virilizer-Hakai复合物锚定于细胞核以提升MTC的催化效率^[15]。RBM15可结合MTC并将其招募到特殊的RNA位点进行m⁶A修饰^[16]。KIAA1429作为MTC中已知最大的支架蛋白，可优先招募核心组分METTL3-METTL14-WTAP来指导m⁶A区域的选择性甲基化^[17]。目前研究主要围绕在METTL3增强靶mRNA中m⁶A甲基化位点的丰富度，其修饰的促癌基因主要包括MYC、Bcl2和SOX2。MYC位于人类染色体8q24基因位点，在白血病、肺癌、口腔癌、食管癌、胃癌、结直肠癌、膀胱癌和前列腺癌中发生m⁶A甲基化修饰后表达增加，进而增强肿瘤细胞的增殖、转移和侵袭性等恶性表型；Bcl2在白血病、肺癌和乳腺癌以及SOX2在结直肠癌、胶质瘤和乳腺癌中亦发生m⁶A甲基化修饰而发挥致癌作用，表明上述促癌基因可作为不同类型癌症的潜在治疗靶点^[10,12,18]。

1.2 m⁶A去甲基化酶

擦除器需在辅因子α-酮戊二酸和Fe²⁺的介导下，可将m⁶A甲基化位点从靶mRNA中清除，主要包括脂肪量和肥胖相关蛋白（fat mass and obesity-associated protein，FTO）和ALKB同系物5（ALKB homolog 5，ALKBH5），两者都属于α-酮戊二酸依赖性双加氧酶家族成员，但两者诱导去甲基化的作用机制存在差异，ALKBH5可特异性直接清除m⁶A修饰，而FTO需将m⁶A依次氧化为N6-羟甲基腺苷和N6-甲酰基腺苷，最后水解为腺嘌呤^[19]。互为相反作用的编码器和擦除器都可促进致癌基因的表达，但两者的作用机制略有不同，前者通过增强mRNA m⁶A的水平来提升其翻译水平，而后者需增强mRNA的稳定性来促进其表达。FOXM1（forkhead box M1）是一种关键的增殖相关转录因子，ALKBH5诱导的FOXM1 m⁶A去甲基化可不改变FOXM1 mRNA的数量，但可增强FOXM1 mRNA稳定性，进而参与胶质瘤、肺癌和葡萄膜黑色素瘤的发生和进展^[10,20]。MYC亦是FTO在宫颈癌中的关键靶基因，虽然FTO可增强MYC的m⁶A去甲基化来抑制其表达，但FTO也可增加MYC mRNA的稳定性和MYC的异位过表达来促进宫颈癌细胞的恶性表型^[21]。

1.3 m⁶A结合蛋白

阅读器可识别并结合经m⁶A修饰的转录本，进而调节mRNA的剪接、输出、降解、翻译和miRNA的生物发生等多种代谢过程来调控基因表达，核阅读器包括含YT521-B homology结构域的1（YTH domain-containing 1，YTHDC1），异质核糖核蛋白（heterogeneous nuclear ribonucleoprotein，HNRNP）A2B1（HNRNPA2B1）、HNRNPC和HNRNPG；胞质阅读器包括YTH结构域家族蛋白1-3（YTH N6-methyladenosine RNA-binding protein 1-3，YTHDF1-3）、YTHDC2和胰岛素样生长因子-2 mRNA结合蛋白1-3（insulin-like growth factor 2 mRNA-binding protein1-3，IGF2BP1-3），不同的阅读器具有不同的m⁶A定位功能和生物学功能^[16]。含YTH结构域的蛋白是最重要的阅读器，主要参与mRNA的剪接、降解和提高翻译效率^[22]。与YTH家族的功能略有差异，IGF2BP家族可通过招募其辅助因子人类抗原R和基质蛋白3来增强mRNA的稳定性和翻译效率^[23]。HNRNP家族可充当m⁶A开关，其通过m⁶A甲基化修饰引起前mRNA 的结构改变并增强其剪接和加工效能^[24]。CTNNB1是一种关键的Wnt信号调节因子，YTHDF3能够识别并结合CTNNB1 mRNA上的m⁶A峰来增强CTNNB1的翻译，促进眼黑色素瘤细胞的繁殖和迁移^[25]。IGF2BP1是IGF2BP家族中最保守的致癌分子，可通过m⁶A修饰增加转录因子SRF的表达，从而增强肺癌、肝癌和卵巢癌细胞的生长和侵袭性^[23]。Toll样受体4作为一种模式识别受体且与癌症进展相关，HNRNPA2B1可介导其mRNA m⁶A修饰来促进多发性骨髓瘤的发生和进展^[26]。

2 TC m⁶A甲基化修饰的调控因子及相关信号通路

2.1 METTL3

Lin等^[27]研究发现，METTL3在TC细胞和组织中高表达，可通过介导前miR-222-3p的m⁶A甲基化修饰来促进其表达，后者靶向负调控丝氨酸/苏氨酸蛋白激酶4，从而加速TC细胞的恶性表型。另一项研究^[28]发现，METTL3诱导TCF1 mRNA的m⁶A甲基化可正向调控TCF1蛋白表达水平，进一步激活Wnt信号通路来促进TC细胞的恶性进展。然而，与上述两项研究不同的是，He等^[29]表明，METTL3在甲状腺乳头状癌（papillary thyroid cancer，PTC）细胞和组织中表达下调，沉默METTL3可降低转录因子c-Rel和家族亚基RelA的m⁶A甲基化水平，其与YTHDF2结合后可抑制c-Rel和RelA mRNA的衰变，从而触发NF-κB信号通路来促进PTC细胞的增殖和迁移；此外，METTL3激活的NF-κB途径还可增加IL-8的产生，进而招募肿瘤相关中性粒细胞并促进PTC的进展。Zhu等^[30]发现，前列腺跨膜上皮抗原2具有抑癌作用，METTL3启动的m⁶A甲基化以YTHDF1依赖性方式可增强其mRNA的稳定性和翻译水平，随后导致Hedgehog及上皮—间充质转化信号通路失活，从而抑制PTC的侵袭性表型。此外，单核苷酸多态性变异可以影响mRNA m⁶A修饰，Ruan等^[31]鉴定了m⁶A相关的最优功能性单核苷酸多态性变异基因：酰基辅酶A合成酶中链家族成员5，其在PTC中低表达且与预后不良相关，初步机制探讨发现METTL3可正向调控该基因的甲基化和表达水平来抑制PTC的进展，其详细的信号通路有待深入研究。

2.2 METTL14

Zhang等^[32]研究发现，敲除METTL14可减弱与长链非编码RNA（long non-coding RNA，lncRNA）OIP5-AS1结合而导致OIP5-AS1过表达，后者通过海绵吸附miR-98并上调ADAMTS8表达，进而激活EGFR和MEK/ERK信号通路来促进PTC细胞的恶性表型。

2.3 ZC3H13

Xie等^[33]研究发现，低表达ZC3H13与YTHDF2可维持IQGAP1mRNA m⁶A修饰来发挥促癌作用；然而，过表达ZC3H13可增强IQGAP1 mRNA m⁶A的修饰水平，但YTHDF2同时加强与IQGAP1的修饰位点结合并加速其mRNA衰变，最终抑制PTC细胞的恶性表型。

2.4 RBM15和KIAA1429

在PTC中，敲低RBM15和KIAA1429可抑制抑癌细胞的增殖，前者具有阻止PI3K/Akt/mTOR信号通路作用，但两者m⁶A修饰的具体分子机制仍需探究^[34]。

2.5 FTO

FTO是真核细胞中首次报道的RNA擦除器，在PTC组织中表达下调，进一步发现高FTO表达与腺体外侵犯和淋巴结转移呈负相关^[35-36]。Ji等^[35]指出，FTO过表达可直接介导溶质载体家族7成员11的mRNA m⁶A甲基化水平和表达降低，从而激活PTC的铁死亡机制并阻止肿瘤进展。另有研究发现，FTO通过IGF2BP2介导的m⁶A修饰可降低载脂蛋白E mRNA m⁶A甲基化水平和稳定性，继而遏制IL-6/JAK2/STAT3信号通路来阻碍PTC的糖酵解代谢和恶性表型；相反，沉默FTO后，载脂蛋白E表达上调，促进PTC的发生和发展^[36]。

2.6 ALKBH5

ALKBH5是第二个被鉴定的RNA擦除器，在TC组织中低表达，与FTO介导铁死亡的分子机制不同，过表达ALKBH5通过m⁶A去甲基化修饰靶向负调控T淋巴瘤侵袭转移诱导因子1的表达，随后导致核因子E2相关因子2/血红素加氧酶1轴失活，进而诱导TC细胞发生铁死亡^[37]。此外，ALKBH5还可调控PTC的糖酵解途径，如敲低ALKBH5可显著增加circNRIP1的m⁶A甲基化水平并上调其表达，后者通过直接海绵吸附致癌分子miR-541-5p和miR-3064-5p来上调靶基因丙酮酸激酶M2的表达，进而促进PTC细胞的增殖和糖酵解^[38]。

2.7 YTHDC2

Zhou等^[39]研究发现，YTHDC2在TC的组织和细胞中低表达，而YTHDC2过表达可正向调控下游靶点圆柱瘤基因的表达，后者可介导Akt信号通路失活来阻止PTC细胞的增殖并诱导细胞凋亡。

2.8 IGF2BP1

IGF2BP1可稳定mRNA，过表达LINC00641可与GLI1mRNA竞争性结合IGF2BP1来促进GLI1mRNA降解，进而抑制Akt通路并阻止PTC的恶性进展^[40]。此外，IGF2BP1作为癌基因且仅在ATC中从头表达，主要是由于表观遗传和（或）转录失调引起，具体机制尚待研究^[41]。

2.9 IGF2BP2

IGF2BP2是阅读器的核心成分且在TC组织中高表达，研究^[42]发现，IGF2BP2可通过m⁶A依赖的方式增加与lncRNA HALGR结合并增强转录本的稳定性来正向调控HAGLR的表达，从而发挥致癌作用。另有研究^[43]显示，MALAT1在TC中高表达，机制上lncRNA MALAT1通过海绵吸附miR-204导致IGF2BP2表达增加，继而识别TC细胞中MYC的m⁶A修饰并提高其表达水平，最终加剧TC细胞的恶性表型。Sa等^[44]研究发现，IGF2BP2亦参与PTC的分化调节，IGF2BP2与RUNT相关转录因子2的m⁶A修饰位点结合可增强其mRNA的稳定性，后者可结合钠－碘同向转运体（sodium/iodide symporter，NIS）基因的启动子区域来下调NIS表达水平，阻止PTC的分化。IGF2BP2还可直接识别V-Erb-B2禽红细胞白血病病毒癌基因同源物2 mRNA CDS的m⁶A甲基化位点来正向调节该分子的蛋白表达，继而加剧ERK/MAPK、PI3K/Akt和JNK/STAT3信号通路的组成性激活，共同参与PTC的去分化^[45]。此外，CircSH2B3通过海绵吸附hsa-miR-4640-5p可上调IGF2BP2的表达来提高细胞核芳基烃受体蛋白水平，上调的IGF2BP2和芳基烃受体蛋白可协同抑制NIS蛋白的表达而诱导PTC去分化^[46]。

3 m⁶A甲基化调控因子作为TC的预后标志物

m⁶A调控因子的表达与TC的发生发展相关，构建其风险预后模型可有效地评估患者的生存期。鉴于各调控因子对TC的致癌风险存在差异，应根据LASSO回归算法构建风险评分模型，即风险评分=∑e各调控因子的表达水平×相应的回归系数，并且以风险评分的中位数可将患者分为高危组和低危组。与低危组患者相比，高危组患者的生存期或无进展生存期明显缩短。根据目前m⁶A风险基因构建的生存预测模型：RBM15、KIAA1429和FTO模型^[34,47-48]，IGF2BP2、RBM15、YTHDF1和YTHDF3模型^[49]，FTO、YTHDF3、IGF2BP1模型^[50]，HNRNPC、ZC3H13、ALKBH5和WTAP模型^[51]，IGF2BP2、YTHDF1和YTHDF3模型^[52]，以上研究模型的AUC值均大于0.716。此外，糖代谢和脂质代谢对TC的发生和发展具有重要作用，脂质代谢相关基因PDZK1IP1、TMC3、LRP2和KCNJ13构建预后模型的AUC值为0.7^[53]，糖酵解相关基因HSPA5、KIF20A和SDC2构建预后模型的AUC值为0.681^[54]。因此，相较于糖脂代谢基因预后模型的预测价值，m⁶A调控因子预后模型的预测效能更优。

4 m⁶A调控因子抑制剂与TC的治疗现状

目前m⁶A调控因子抑制剂治疗TC的证据仅有少数基础研究，主要通过影响m⁶A调节因子的水平来降低致癌基因的甲基化水平。METTL3低表达可增加免疫检查点阻断的不良反应、免疫抑制调节性T细胞和终末耗竭T细胞的丰度，继而驱动TC患者产生抗PD-1治疗的耐药性，库沙珠单抗可阻断该途径抗PD-1治疗的耐药性，为晚期PTC和ATC的疗法提供新思路^[55]。人参皂苷Rh2不仅在乳腺癌、结肠癌、肺癌和前列腺癌中具有抑癌作用，还可通过降低ZC3H13的核定位来抑制m⁶A RNA甲基化，从而抑制TC细胞的生长^[56]。IGF2BP2可诱导PTC去分化，而拉帕替尼和吉非替尼可导致IGF2BP2表达降低来逆转PTC去分化作用^[45]。IGF2BP3过表达可诱导TC细胞的Akt激活并促进癌细胞的恶性表型，但IGF1R抑制剂（林西替尼）在体外能够阻断Akt的激活和抑制IGF1R，后者可导致IGF2BP3以剂量依赖性方式来抑制癌细胞的生长^[57]。虽然上述抑制剂对m⁶A调控因子的作用具有抗癌作用，但特异性较差，尚需利用人工智能和化学合成技术进一步探索特异性更强的m⁶A抑制剂。此外，FTO抑制剂是目前m⁶A调控的研究热点，甲氯芬酸、羟基戊二酸和FTO-43可加强去甲基化修饰发挥抑制白血病细胞增殖和增强化疗药物敏感性；传统医药中的天然产物亦可参与m⁶A修饰发挥抗癌作用，如METTL3抑制剂（槲皮素和黄芩苷）可介导去甲基化调控^[58]，其治疗作用仍有待临床试验论证。

5 总结与展望

综上所述，m⁶A甲基化修饰在TC中的研究已取得初步进展，其三大调控因子修饰的癌基因或抑癌基因在TC的发生发展、预后和治疗中具有关键作用，并且构建m⁶A调控因子预后模型为TC提供新的预测生物标志物。然而，一些m⁶A修饰分子的上游调控因子和下游靶点以及它们本身的致癌或抑癌机制尚不清楚，今后需要进一步研究，进而为预后不良的TC提供新的治疗策略。因此，m⁶A甲基化修饰的深入研究对TC的治疗和预后都具有广阔的研究及临床应用前景。

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基金资助

甘肃省自然科学基金资助项目(20JR5RA342)

甘肃省自然科学基金资助项目(21JR11RA109)

甘肃省兰州市城关区科技计划基金资助项目(2022SHFZ0010)