piRNA调节m6A RNA甲基化参与MM发生发展的机制研究

Our previous study supported by National Natural Science Foundation of China demonstrated piRNA is an important pathogenesis of multiple myeloma. Recently we found piRNA can exert other epigenetic regulation function besides regulating DNA methylation, and this new function might be related to driving the formation of myeloma stem cell, but its detailed mechanism remained elusive. m6A is a newly discovered modification of RNA methylation which is involved in regulating the stability, splice and translation of mRNA at post-transcriptional level. Recent study revealed that m6A RNA methylation might play critical role in the pathogenesis of malignances, especially in driving cancer stem cells. Our preliminary data showed RNA demethylase ALKBH5 is highly expressed in multiple myeloma cells and up-regulated by piRNA. Therefore, combining with others’ research, we speculated that m6A RNA methylation regulated by piRNA is involved in the tumorigenesis of multiple myeloma through affecting the function of myeloma cells and myeloma stem cells. Therefore, in this study, we endeavour to systematically answer the following questions by clinical, cell and mouse model experiments: ①Does m6A RNA methylation play important role in the tumorigenesis of multiple myeloma？②Does piRNA exert its function through regulating m6A RNA methylation？③Is myeloma stem cell the main target cell population affected by piRNA-regulated m6A RNA methylation？This will help find a completely new pathogenesis and therapeutic target for multiple myeloma.

我们前一个国基金研究表明piRNA是多发性骨髓瘤（MM）的重要发病机制，近期我们发现 piRNA除调控DNA甲基化外，还存在新的表观遗传学调控作用，且可能与其调控MM干细胞的形成相关，但机制不详。m6A是新发现的RNA甲基化修饰，在转录后水平调控mRNA的稳定性、剪切和翻译，最近研究发现它可能在肿瘤发病、特别是肿瘤干细胞的驱动中发挥关键作用。我们预试验发现RNA去甲基化酶ALKBH5在MM细胞中高表达，且被piRNA上调，结合他人研究可推测piRNA通过影响m6A RNA甲基化来调节MM和MM干细胞的功能参与MM的发病。因此本课题拟通过体内外实验及临床研究系统回答下述问题：①m6A RNA甲基化是否在MM发病中起重要作用？②piRNA是否通过调控m6A RNA甲基化发挥作用？③MM干细胞是否是piRNA调控m6A RNA甲基化影响的主要靶细胞？这将有助于发现一个新的MM发病机制和治疗靶点。

RNA m6A动态、可逆的修饰在真核生物中广泛存在，被发现与肿瘤发生、预后不良关系密切，备受科学家们关注。我们通过前期预实验已经发现RNA m6A去甲基化酶ALKBH5在多发性骨髓瘤患者及细胞系中异常高表达并与MM患者分期呈正相关，提示ALKBH5可能是促进MM进展的新机制。在本课题研究过程中，我们证实了ALKBH5正向调控MM细胞的增殖、迁移，反向调控MM细胞凋亡。动物实验证实：ALKBH5在小鼠体内有促进多发性骨髓瘤疾病进展的作用。MeRIP-seq测序提示ALKBH5靶向SAV1 mRNA，推测ALKBH5通过SAV1/HIPPO通路影响多发性骨髓瘤的增殖和干细胞水平。动物实验证实：ALKBH5在小鼠体内通过SAV1促进多发性骨髓瘤疾病进展。另一方面，MeRIP-seq测序也发现ALKBH5靶向长链非编码RNA SNHG15，进一步补偿实验证实ALKBH5通过lncRNA SNHG15影响多发性骨髓瘤细胞的增殖，凋亡和迁移。这些结果表明：ALKBH5可望做完靶点来开发新的MM治疗策略。