Regenerating Urethral Striated Muscle by CRISPRi/dCas9-KRAB-Mediated Myostatin Silencing for Obesity-Associated Stress Urinary Incontinence.
Regenerating Urethral Striated Muscle by CRISPRi/dCas9-KRAB-Mediated Myostatin Silencing for Obesity-Associated Stress Urinary Incontinence.
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DOI：
10.1089/crispr.2020.0077
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发表时间：
2020-12
期刊：
影响因子：
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通讯作者：
Huixing Yuan;Y. Ruan;Yan Tan;A. Reed-Maldonado;Yinwei Chen;Dehua Zhao;Zhao Wang;Feng Zhou
中科院分区：
文献类型：
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作者：
Huixing Yuan;Y. Ruan;Yan Tan;A. Reed-Maldonado;Yinwei Chen;Dehua Zhao;Zhao Wang;Feng Zhou
超重女性容易出现与肥胖相关的压力性尿失禁（OA-SUI），并且对于这种常见的泌尿系统疾病没有明确的药物治疗方法。本研究旨在检验以下假设：恢复尿道横纹肌 (stM) 和盆底肌肉的再生疗法可能是一种有价值的治疗方法。在体外实验中，使用靶向肌生长抑制素 (MSTN) 的单引导 RNA 进行 CRISPRi/dCas9-Kruppel 相关盒 (KRAB) 介导的基因沉默。对于体内实验，总共 14 只雌性瘦 ZUC-Leprfa 186 和 14 只肥胖 ZUC-Leprfa 185 大鼠分别用作对照组和 CRISPRi-MSTN 治疗组。结果表明，慢病毒介导的 MSTN CRISPRi/dCas9-KRAB 表达导致大鼠 L6 成肌细胞中 MSTN 持续下调，并显着增强体外肌生成。在体内，尿道括约肌注射慢病毒-MSTN sgRNA和慢病毒-dCas9-KRAB显着增加了渗漏点压力、stM层厚度、stM与平滑肌的比率以及神经肌肉接头的数量。通过 CRISPRi/dCas9-KRAB 介导的基因沉默下调 MSTN 可显着增强体外和体内的肌生成。它还改善了 OA-SUI 大鼠模型的尿道失禁。
Overweight females are prone to obesity-associated stress urinary incontinence (OA-SUI), and there are no definitive medical therapies for this common urologic condition. This study was designed to test the hypothesis that regenerative therapy to restore urethral striated muscle (stM) and pelvic floor muscles might represent a valuable therapeutic approach. For the in vitro experiment, single-guide RNAs targeting myostatin (MSTN) were used for CRISPRi/dCas9-Kruppel associated box (KRAB)-mediated gene silencing. For the in vivo experiment, a total of 14 female lean ZUC-Leprfa 186 and 14 fatty ZUC-Leprfa 185 rats were used as control and CRISPRi-MSTN treated groups, respectively. The results indicated that lentivirus-mediated expression of MSTN CRISPRi/dCas9-KRAB caused sustained downregulation of MSTN in rat L6 myoblast cells and significantly enhanced myogenesis in vitro. In vivo, the urethral sphincter injection of lentiviral-MSTN sgRNA and lentiviral-dCas9-KRAB significantly increased the leak point pressure, the thickness of the stM layer, the ratio of stM to smooth muscle, and the number of neuromuscular junctions. Downregulation of MSTN with CRISPRi/dCas9-KRAB-mediated gene silencing significantly enhanced myogenesis in vitro and in vivo. It also improved urethral continence in the OA-SUI rat model.