DataSheet2_Clinical and molecular significance of the RNA m6A methyltransferase complex in prostate cancer.zip

N6-methyladenosine (m6A) is the most abundant internal mRNA modification and is dynamically regulated through distinct protein complexes that methylate, demethylate, and/or interpret the m6A modification. These proteins, and the m6A modification, are involved in the regulation of gene expression, RNA stability, splicing and translation. Given its role in these crucial processes, m6A has been implicated in many diseases, including in cancer development and progression. Prostate cancer (PCa) is the most commonly diagnosed non-cutaneous cancer in men and recent studies support a role for m6A in PCa. Despite this, the literature currently lacks an integrated analysis of the expression of key components of the m6A RNA methyltransferase complex, both in PCa patients and in well-established cell line models. For this reason, this study used immunohistochemistry and functional studies to investigate the mechanistic and clinical significance of the METTL3, METTL14, WTAP and CBLL1 components of the m6A methyltransferase complex in PCa specimens and cell lines. Expression of METTL3 and CBLL1, but not METTL14 and WTAP, was associated with poorer PCa patient outcomes. Expression of METTL3, METTL14, WTAP and CBLL1 was higher in PCa cells compared with non-malignant prostate cells, with the highest expression seen in castrate-sensitive, androgen-responsive PCa cells. Moreover, in PCa cell lines, expression of METTL3 and WTAP was found to be androgen-regulated. To investigate the mechanistic role(s) of the m6A methyltransferase complex in PCa cells, short hairpin RNA (shRNA)-mediated knockdown coupled with next generation sequencing was used to determine the transcriptome-wide roles of METTL3, the catalytic subunit of the m6A methyltransferase complex. Functional depletion of METTL3 resulted in upregulation of the androgen receptor (AR), together with 134 AR-regulated genes. METTL3 knockdown also resulted in altered splicing, and enrichment of cell cycle, DNA repair and metabolic pathways. Collectively, this study identified the functional and clinical significance of four essential m6A complex components in PCa patient specimens and cell lines for the first time. Further studies are now warranted to determine the potential therapeutic relevance of METTL3 inhibitors in development to treat leukaemia to benefit patients with PCa.

N6-甲基腺苷（m6A）是mRNA内部最丰富的修饰形式，其通过不同的蛋白质复合体动态调节，这些复合体负责甲基化、去甲基化以及/或解读m6A修饰。这些蛋白质及其m6A修饰与基因表达、RNA稳定性、剪接和翻译的调控密切相关。鉴于其在这些关键过程中的作用，m6A已被证实在多种疾病中发挥作用，包括癌症的发生和发展。前列腺癌（PCa）是男性最常见的非皮肤癌，近期研究支持m6A在PCa中的作用。尽管如此，目前文献中尚缺乏对m6A RNA甲基转移酶复合体关键组分在PCa患者及已建立的细胞系模型中的表达进行综合分析。因此，本研究利用免疫组化和功能研究，旨在探究m6A甲基转移酶复合体的METTL3、METTL14、WTAP和CBLL1组分在PCa标本和细胞系中的机制和临床意义。METTL3和CBLL1的表达与PCa患者的预后较差相关，而METTL14和WTAP则不然。与良性前列腺细胞相比，PCa细胞中METTL3、METTL14、WTAP和CBLL1的表达更高，且在去势敏感、雄激素反应性PCa细胞中表达最高。此外，在PCa细胞系中，METTL3和WTAP的表达被发现受雄激素调节。为了研究m6A甲基转移酶复合体在PCa细胞中的机制作用，本研究采用了短发夹RNA（shRNA）介导的敲低与下一代测序相结合的方法，以确定m6A甲基转移酶复合体催化亚基METTL3在转录组范围内的作用。METTL3功能耗竭导致雄激素受体（AR）及其134个AR调控基因的上调。METTL3敲低还导致剪接改变，以及细胞周期、DNA修复和代谢途径的富集。综上所述，本研究首次确定了四个m6A复合体关键组分在PCa患者标本和细胞系中的功能和临床意义。目前，有必要进行进一步研究，以确定METTL3抑制剂在治疗白血病中的潜在治疗相关性，从而惠及PCa患者。