OBJECTIVE: We aim to develop a multisite dCasRx- m6A editor for assessing the effects of the multisite dCasRx-m6A editor targeted m6A demethylation of ITGA6 mRNA in BC growth and progression.
METHODS: The multisite dCasRx- m6A editor was generated by cloning. m6A-methylated RNA immunoprecipitation (meRIP), luciferase reporter, a single-base T3 ligase-based qPCR-amplification, Polysome profiling and meRIP-seq experiments were performed to determine the targeting specificity of the multisite dCasRx-m6A editor. We performed cell phenotype analysis and used in vivo mouse xenograft models to assess the effects of the multisite dCasRx-m6A editor in BC growth and progression.
RESULTS: We designed a targeted ITGA6 multi-locus guide (g)RNA and established a bidirectional deactivated RfxCas13d (dCasRx)-based m6A-editing platform, comprising a nucleus-localized dCasRx fused with the catalytic domains of methyltransferase-like 3 (METTL3-CD) or α-ketoglutarate-dependent dioxygenase alkB homolog 5 (ALKBH5-CD), to simultaneously manipulate the methylation of ITGA6 mRNA at four m6A sites. The results confirmed the dCasRx-m6A editor modified m6A at multiple sites in ITGA6 mRNA, with low off-target effects. Moreover, targeted m6A demethylation of ITGA6 mRNA by the multisite dCasRx-m6A editor significantly reduced BCa cell proliferation and migration in vitro and in vivo. Furthermore, the dCasRx-ALKBH5-CD and ITGA6 multi-site gRNA delivered to 5-week-old BALB/cJNju-Foxn1nu/Nju nude mice via adeno-associated viral vectors significantly inhibited BCa cell growth.
CONCLUSIONS: Our study proposes a novel therapeutic tool for the treatment of BC by applying the multisite dCasRx-m6A editor while highlighting its potential efficacy for treating other diseases associated with abnormal m6A modifications.
目的:我们旨在开发一种多位点dCasRx-m6A编辑器,用于评估多位点dCasRx-m6A编辑器靶向m6A去甲基化ITGA6mRNA在BC生长和进展中的作用。
方法:通过克隆产生多位点dCasRx-m6A编辑器。m6A甲基化RNA免疫沉淀(meRIP),荧光素酶报告基因,基于单碱基T3连接酶的qPCR扩增,进行多聚体谱分析和meRIP-seq实验以确定多位点dCasRx-m6A编辑器的靶向特异性。我们进行了细胞表型分析,并在体内使用小鼠异种移植模型来评估多位点dCasRx-m6A编辑器在BC生长和进展中的作用。
结果:我们设计了一种靶向的ITGA6多位点指导(g)RNA,并建立了基于双向失活RfxCas13d(dCasRx)的m6A编辑平台,包含与甲基转移酶样3(METTL3-CD)或α-酮戊二酸依赖性双加氧酶alkB同源物5(ALKBH5-CD)的催化结构域融合的细胞核定位的dCasRx,同时操纵ITGA6mRNA在四个m6A位点的甲基化。结果证实dCasRx-m6A编辑器在ITGA6mRNA的多个位点修饰了m6A,低目标效应。此外,多位点dCasRx-m6A编辑器对ITGA6mRNA的靶向m6A去甲基化在体外和体内显着降低了BCa细胞的增殖和迁移。此外,dCasRx-ALKBH5-CD和ITGA6多位点gRNA通过腺相关病毒载体递送至5周龄BALB/cJNju-Foxn1nu/Nju裸鼠显著抑制BCa细胞生长。
结论:我们的研究提出了一种新的治疗工具,通过应用多位点dCasRx-m6A编辑器来治疗BC,同时强调其治疗与m6A异常修饰相关的其他疾病的潜在功效。