State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, P. R. China.
Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, Shanghai, P. R. China.
Clin Transl Med. 2023 Aug;13(8):e1382. doi: 10.1002/ctm2.1382.
Precise regulation of partial critical proteins in cancer cells, such as anti-apoptotic proteins, is one of the crucial strategies for treating cancer and discovering related molecular mechanisms. Still, it is also challenging in actual research and practice. The widely used CRISPR/Cas9-based gene editing technology and proteolysis-targeting chimeras (PROTACs) have played an essential role in regulating gene expression and protein function in cells. However, the accuracy and controllability of their targeting remain necessary.
Construction of UMUC-3-EGFP stable transgenic cell lines using the Sleeping Beauty system, Flow cytometry, quantitative real-time PCR, western blot, fluorescence microplate reader and fluorescence inverted microscope analysis of EGFP intensity. Characterization of Survivin inhibition was done by using Annexin V-FITC/PI apoptosis, calcein/PI/DAPI cell viability/cytotoxicity assay, cloning formation assay and scratch assay. The cell-derived xenograft (CDX) model was constructed to assess the in vivo effects of reducing Survivin expression.
Herein, we established a synergistic control platform that coordinated photoactivatable split-Cas9 targeted gene editing and light-induced protein degradation, on which the Survivin gene in the nucleus was controllably edited by blue light irradiation (named paCas9-Survivin) and simultaneously the Survivin protein in the cytoplasm was degraded precisely by a nanobody-mediated target (named paProtacL-Survivin). Meanwhile, in vitro experiments demonstrated that reducing Survivin expression could effectively promote apoptosis and decrease the proliferation and migration of bladder cancerous cells. Furthermore, the CDX model was constructed using UMUC-3 cell lines, results from animal studies indicated that both the paCas9-Survivin system and paProtacL-Survivin significantly inhibited tumour growth, with higher inhibition rates when combined.
In short, the coordinated regulatory strategies and combinable technology platforms offer clear advantages in controllability and targeting, as well as an excellent reference value and universal applicability in controlling the fate of cancer cells through multi-level regulation of key intracellular factors.
精确调控癌细胞中的部分关键蛋白，如抗凋亡蛋白，是治疗癌症和发现相关分子机制的关键策略之一。然而，在实际研究和实践中，这也是具有挑战性的。广泛使用的基于 CRISPR/Cas9 的基因编辑技术和蛋白水解靶向嵌合体（PROTACs）在调节细胞中的基因表达和蛋白功能方面发挥了重要作用。然而，它们的靶向准确性和可控性仍然是必要的。
利用 Sleeping Beauty 系统构建 UMUC-3-EGFP 稳定转染细胞系，流式细胞术、实时定量 PCR、western blot、荧光微孔板读数仪和荧光倒置显微镜分析 EGFP 强度。通过 Annexin V-FITC/PI 凋亡、钙黄绿素/PI/DAPI 细胞活力/细胞毒性测定、克隆形成测定和划痕试验来研究 Survivin 抑制作用。构建细胞衍生的异种移植（CDX）模型来评估降低 Survivin 表达的体内效果。
本文建立了一个协同调控平台，该平台协调了光激活的分裂 Cas9 靶向基因编辑和光诱导的蛋白降解，在该平台上，细胞核中的 Survivin 基因可通过蓝光照射进行可控编辑（命名为 paCas9-Survivin），同时细胞质中的 Survivin 蛋白可通过纳米体介导的靶标进行精确降解（命名为 paProtacL-Survivin）。同时，体外实验表明，降低 Survivin 表达可有效促进膀胱癌的细胞凋亡，并降低其增殖和迁移。此外，使用 UMUC-3 细胞系构建了 CDX 模型，动物研究结果表明，paCas9-Survivin 系统和 paProtacL-Survivin 均显著抑制肿瘤生长，联合使用时抑制率更高。
总之，协同调控策略和可组合的技术平台在可控性和靶向性方面具有明显优势，通过对关键细胞内因子进行多层次调控来控制癌细胞命运，为其提供了良好的参考价值和普遍适用性。
