Center for Translational Medicine, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510080, China; Department of Urology, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510080, China.
Center for Translational Medicine, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510080, China.
J Adv Res. 2024 Feb;56:57-68. doi: 10.1016/j.jare.2023.03.010. Epub 2023 Mar 30.
N6-methyladenosine (mA) 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 mA modification. However, the therapeutic effect of targeting ITGA6 multisite mA modifications in BCa remains unknown.
We aim to develop a multisite dCasRx- mA editor for assessing the effects of the multisite dCasRx-mA editor targeted mA demethylation of ITGA6 mRNA in BC growth and progression.
The multisite dCasRx- mA editor was generated by cloning. mA-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-mA editor. We performed cell phenotype analysis and used in vivo mouse xenograft models to assess the effects of the multisite dCasRx-mA editor in BC growth and progression.
We designed a targeted ITGA6 multi-locus guide (g)RNA and established a bidirectional deactivated RfxCas13d (dCasRx)-based mA-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 mA sites. The results confirmed the dCasRx-mA editor modified mA at multiple sites in ITGA6 mRNA, with low off-target effects. Moreover, targeted mA demethylation of ITGA6 mRNA by the multisite dCasRx-mA 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.
Our study proposes a novel therapeutic tool for the treatment of BC by applying the multisite dCasRx-mA editor while highlighting its potential efficacy for treating other diseases associated with abnormal mA modifications.
N6-甲基腺苷（mA）修饰有助于各种癌症的发病和发展，包括膀胱癌（BCa）。特别是整合素α6（ITGA6）通过协同调节多部位 mA 修饰促进 BCa 的进展。然而，靶向 BCa 中 ITGA6 多部位 mA 修饰的治疗效果尚不清楚。
我们旨在开发一种多部位 dCasRx-mA 编辑器，用于评估靶向 ITGA6 mRNA 多部位 dCasRx-mA 编辑器脱甲基化对 BC 生长和进展的影响。
通过克隆生成多部位 dCasRx-mA 编辑器。进行 mA 甲基化 RNA 免疫沉淀（meRIP）、荧光素酶报告、单碱基 T3 连接酶 qPCR 扩增、多核糖体分析和 meRIP-seq 实验以确定多部位 dCasRx-mA 编辑器的靶向特异性。我们进行了细胞表型分析，并使用体内小鼠异种移植模型评估了多部位 dCasRx-mA 编辑器在 BC 生长和进展中的作用。
我们设计了靶向 ITGA6 多基因座指导（g）RNA，并建立了一个双向失活的 RfxCas13d（dCasRx）为基础的 mA 编辑平台，该平台由核定位的 dCasRx 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）的催化结构域融合而成，以同时操纵 ITGA6 mRNA 四个 mA 位点的甲基化。结果证实，dCasRx-mA 编辑器在 ITGA6 mRNA 中的多个位点修饰 mA，脱靶效应低。此外，多部位 dCasRx-mA 编辑器靶向 ITGA6 mRNA 的 mA 去甲基化显著降低了体外和体内 BCa 细胞的增殖和迁移。此外，通过腺相关病毒载体递送至 5 周龄 BALB/cJNju-Foxn1nu/Nju 裸鼠的 dCasRx-ALKBH5-CD 和 ITGA6 多部位 gRNA 显著抑制了 BCa 细胞的生长。
我们的研究提出了一种通过应用多部位 dCasRx-mA 编辑器治疗 BC 的新型治疗工具，同时强调了其治疗其他与异常 mA 修饰相关疾病的潜在疗效。
