甲基化酶 METTL7B 对肺腺癌的发生转移作用机制研究

背景 肺腺癌是临床上常见恶性肿瘤之一，随着科技的进步，患者的生存率大 大提高，但是仍有相当一部分患者临床效果不佳，且肿瘤发生转移和复发。 近些年来，随着针对肺腺癌的治疗靶点研究的逐渐深入，肺腺癌的发生和转 移机制得到较大突破，尤其是在表观修饰方面近些年受到广泛关注。m6A甲 基化作为翻译后常见的一种修饰，与肿瘤发生发展密切相关。METTL7B作 为 m6A 甲基转移酶家族中的一员，对肺腺癌的发生发展具有重要影响。对 METTL7B 在肺癌发生发展中的作用进行深入研究，有助于揭示肺腺癌发展 的新机制，也有望发现新的调控分子网络和药物治疗靶标。 方法 使用RNA高通量测序检测30例肺腺癌临床样本组织中METTL7B的表 达情况，将METTL7B按照高低表达分为两组，通过GO富集和KEGG富集 分析肺腺癌组织中上调及下调的基因涉及的生物学功能和信号通路，分析 METTL7B 在肺癌发生和转移中的作用。同时通过联合 MeRIP-RNA 高通量 测序及RNA高通量测序结合分析METTL7B所介导的m6A修饰以及在RNA 转录及蛋白合成中的作用。运用GO，KEGG和 GSEA富集分析 METTL7B 影响的下游信号，分析METTL7B在肺腺癌发生发展中扮演中的角色。利用 Crispr Cas9 对肺腺癌细胞进行敲除 METTL7B以及过表达METTL7B的蛋白 模型构建，进而检测METTL7B对肺腺癌细胞的生长转移等生物学功能的影 响。最后通过RIP验证METTL7B对下游基因的甲基化作用。 结果 通过对临床肺腺癌样本组织的分析，我们发现与癌旁正常组织相比，肺 腺癌组织的METTL7B的表达量明显升高，与RNA-seq数据联合分析发现， 406 个表达上调以及28个表达下调。在这其中，有52个基因与METTL7B 存在强相关性，其中有37个基因呈正相关，有15个基因呈负相关。利用GO 对差异基因进行富集，提示METTL7B的功能与DNA复制、RNA甲基转移 酶活性有关。同时用KEGG进行富集，提示相关分子通路主要在碱基切除修 复、DNA 复制和 Hippo 信号通路中。结合 TIMER 数据库、TISIDB 数据库 以及临床样本，METTL7B与多种免疫分析相关，能够抑制肺腺癌免疫浸润， I 摘 要 促进免疫抑制。在细胞实验上，我们分别利用构建敲除和过表达METTL7B 稳转细胞系，证实METTL7B能够调控人肺腺癌细胞增殖和转移。在分子水 平上在分子水平上可以通过CDK2、CDK4等细胞周期蛋白促进细胞周期以 及调控N-cadherin、E-cadherin、Vimentin 等促进上皮间质转化。我们还发现， METTL7B 能够调控细胞的mRNA甲基化水平，在构建METTL7B敲除细胞 系后，联合METTL7B-KO MeRIP 与 RNA-seq 数据分析，METTL7B 缺失与 癌症的中心碳代谢、MAPK 途径和 ATP结合等代谢途径相关。通过测序我 们发现，METTL7B 能够调控 NTRK3 的甲基化修饰，qPCR 结果显示 METTL7B 下调 NTRK3 的表达，RIP 实验证实 METTL7B 参与 NTRK3 的 m6a 修饰从而影响NTRK3的基因表达水平。 结论 甲基转移酶METTL7B在肺腺癌中高表达，且高表达的METTL7B导致 肺腺癌患者预后不良。METTL7B不仅可调控肺腺癌细胞的细胞周期以及促 进肺腺癌细胞的迁移和侵袭，同时具有调控细胞 mRNA 甲基化的功能。结 合MeRIP 和 RNA-seq 分析以及体外实验，METTL7B可以调控NTRK3的甲 基化修饰，从而影响 NTRK3 的 mRNA的稳定性进而降低了其 mRNA水平 的表达。以上研究揭示METTL7B可能是肺腺癌转移的一个重要的分子标志 物，这些发现有助于更好地理解肺腺癌的生长转移机制，提供潜在的治疗靶 点。

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