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Abstract：
BACKGROUND: N6-methyladenosine (m6A) modification contributes to the pathogenesis and development of various cancers, including bladder cancer (BCa). In particular, integrin α6 (ITGA6) promotes BCa progression by cooperatively regulating multisite m6A modification. However, the therapeutic effect of targeting ITGA6 multisite m6A modifications in BCa remains unknown.
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.
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.
摘要：
背景：N6-甲基腺苷（m6A）修饰有助于各种癌症的发病机理和发展，包括膀胱癌（BCa）。特别是,整合素α6(ITGA6)通过协同调节多位点m6A修饰促进BCa进展。然而,在BCa中靶向ITGA6多位点m6A修饰的治疗效果仍然未知.
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。
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。
