Table_6_The RNA N6-Methyladenosine Methyltransferase METTL3 Promotes the Progression of Kidney Cancer via N6-Methyladenosine-Dependent Translational Enhancement of ABCD1.XLSX

The role of N6-methyladenosine (m6A)-modifying proteins in cancer progression depends on the cell type and mRNA affected. However, the biological role and underlying mechanism of m6A in kidney cancer is limited. Here, we discovered the variability in m6A methyltransferase METTL3 expression was significantly increased in clear cell renal cell carcinoma (ccRCC) the most common subtype of renal cell carcinoma (RCC), and high METTL3 expression predicts poor prognosis in ccRCC patients using a dataset from The Cancer Genome Atlas (TCGA). Importantly, knockdown of METTL3 in ccRCC cell line impaired both cell migration capacity and tumor spheroid formation in soft fibrin gel, a mechanical method for selecting stem-cell-like tumorigenic cells. Consistently, overexpression of METTL3 but not methyltransferase activity mutant METTL3 can promote cell migration, spheroid formation in cell line and tumor growth in xenograft model. Transcriptional profiling of m6A in ccRCC tissues identified the aberrant m6A transcripts were enriched in cancer-related pathways. Further m6A-sequencing of METTL3 knockdown cells and functional studies confirmed that translation of ABCD1, an ATP-binding cassette (ABC) transporter of fatty acids, was inhibited by METTL3 in m6A-dependent manner. Moreover, knockdown of ABCD1 in ccRCC cells decreased cancer cell migration and spheroid formation, and upregulation of ABCD1 acts as an adverse prognosis factor of kidney cancer patients. In summary, our study identifies that METTL3 promotes ccRCC progression through m6A modification-mediated translation of ABCD1, providing an epitranscriptional insight into the molecular mechanism in kidney cancer.

N6-甲基腺苷酸（N6-methyladenosine, m6A）修饰蛋白在癌症进展中的作用依赖于细胞类型与受影响的信使RNA（mRNA）。然而，目前关于m6A在肾癌中的生物学功能及潜在调控机制的研究仍较为有限。本研究利用癌症基因组图谱（The Cancer Genome Atlas, TCGA）数据集进行分析，发现作为肾细胞癌（renal cell carcinoma, RCC）最常见亚型的透明细胞肾细胞癌（clear cell renal cell carcinoma, ccRCC）中，m6A甲基转移酶METTL3的表达异质性显著升高，且METTL3高表达预示ccRCC患者预后不良。值得注意的是，在ccRCC细胞系中敲低METTL3，可同时削弱细胞迁移能力与软纤维蛋白凝胶中的肿瘤球体形成能力——该方法是筛选干细胞样致瘤细胞的力学分选手段。一致性实验结果显示，过表达野生型METTL3而非丧失甲基转移酶活性的METTL3突变体，能够促进细胞系中的细胞迁移与肿瘤球体形成，以及异种移植模型中的肿瘤生长。对ccRCC组织开展的m6A转录组分析表明，携带异常m6A修饰的转录本显著富集于癌症相关通路。后续通过METTL3敲低细胞的m6A测序及功能实验证实，脂肪酸ATP结合盒（ATP-binding cassette, ABC）转运蛋白ABCD1的翻译过程以m6A依赖的方式受到METTL3的抑制。此外，在ccRCC细胞中敲低ABCD1可降低癌细胞的迁移能力与肿瘤球体形成能力，而ABCD1的高表达则是肾癌患者的不良预后危险因素。综上，本研究证实METTL3通过m6A修饰介导的ABCD1翻译促进ccRCC进展，为肾癌的分子机制研究提供了表观转录组层面的全新视角。