Cargando…
Split‐Cas9‐based targeted gene editing and nanobody‐mediated proteolysis‐targeting chimeras optogenetically coordinated regulation of Survivin to control the fate of cancer cells
BACKGROUND: 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‐...
Autores principales: | , , , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
John Wiley and Sons Inc.
2023
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10449816/ https://www.ncbi.nlm.nih.gov/pubmed/37620295 http://dx.doi.org/10.1002/ctm2.1382 |
_version_ | 1785095043530358784 |
---|---|
author | Deng, Changping Li, Shihui Liu, Yuping Bao, Wen Xu, Chengnan Zheng, Wenyun Wang, Meiyan Ma, Xingyuan |
author_facet | Deng, Changping Li, Shihui Liu, Yuping Bao, Wen Xu, Chengnan Zheng, Wenyun Wang, Meiyan Ma, Xingyuan |
author_sort | Deng, Changping |
collection | PubMed |
description | BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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. |
format | Online Article Text |
id | pubmed-10449816 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | John Wiley and Sons Inc. |
record_format | MEDLINE/PubMed |
spelling | pubmed-104498162023-08-26 Split‐Cas9‐based targeted gene editing and nanobody‐mediated proteolysis‐targeting chimeras optogenetically coordinated regulation of Survivin to control the fate of cancer cells Deng, Changping Li, Shihui Liu, Yuping Bao, Wen Xu, Chengnan Zheng, Wenyun Wang, Meiyan Ma, Xingyuan Clin Transl Med Research Articles BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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. John Wiley and Sons Inc. 2023-08-24 /pmc/articles/PMC10449816/ /pubmed/37620295 http://dx.doi.org/10.1002/ctm2.1382 Text en © 2023 The Authors. Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics. https://creativecommons.org/licenses/by/4.0/This is an open access article under the terms of the http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Research Articles Deng, Changping Li, Shihui Liu, Yuping Bao, Wen Xu, Chengnan Zheng, Wenyun Wang, Meiyan Ma, Xingyuan Split‐Cas9‐based targeted gene editing and nanobody‐mediated proteolysis‐targeting chimeras optogenetically coordinated regulation of Survivin to control the fate of cancer cells |
title | Split‐Cas9‐based targeted gene editing and nanobody‐mediated proteolysis‐targeting chimeras optogenetically coordinated regulation of Survivin to control the fate of cancer cells |
title_full | Split‐Cas9‐based targeted gene editing and nanobody‐mediated proteolysis‐targeting chimeras optogenetically coordinated regulation of Survivin to control the fate of cancer cells |
title_fullStr | Split‐Cas9‐based targeted gene editing and nanobody‐mediated proteolysis‐targeting chimeras optogenetically coordinated regulation of Survivin to control the fate of cancer cells |
title_full_unstemmed | Split‐Cas9‐based targeted gene editing and nanobody‐mediated proteolysis‐targeting chimeras optogenetically coordinated regulation of Survivin to control the fate of cancer cells |
title_short | Split‐Cas9‐based targeted gene editing and nanobody‐mediated proteolysis‐targeting chimeras optogenetically coordinated regulation of Survivin to control the fate of cancer cells |
title_sort | split‐cas9‐based targeted gene editing and nanobody‐mediated proteolysis‐targeting chimeras optogenetically coordinated regulation of survivin to control the fate of cancer cells |
topic | Research Articles |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10449816/ https://www.ncbi.nlm.nih.gov/pubmed/37620295 http://dx.doi.org/10.1002/ctm2.1382 |
work_keys_str_mv | AT dengchangping splitcas9basedtargetedgeneeditingandnanobodymediatedproteolysistargetingchimerasoptogeneticallycoordinatedregulationofsurvivintocontrolthefateofcancercells AT lishihui splitcas9basedtargetedgeneeditingandnanobodymediatedproteolysistargetingchimerasoptogeneticallycoordinatedregulationofsurvivintocontrolthefateofcancercells AT liuyuping splitcas9basedtargetedgeneeditingandnanobodymediatedproteolysistargetingchimerasoptogeneticallycoordinatedregulationofsurvivintocontrolthefateofcancercells AT baowen splitcas9basedtargetedgeneeditingandnanobodymediatedproteolysistargetingchimerasoptogeneticallycoordinatedregulationofsurvivintocontrolthefateofcancercells AT xuchengnan splitcas9basedtargetedgeneeditingandnanobodymediatedproteolysistargetingchimerasoptogeneticallycoordinatedregulationofsurvivintocontrolthefateofcancercells AT zhengwenyun splitcas9basedtargetedgeneeditingandnanobodymediatedproteolysistargetingchimerasoptogeneticallycoordinatedregulationofsurvivintocontrolthefateofcancercells AT wangmeiyan splitcas9basedtargetedgeneeditingandnanobodymediatedproteolysistargetingchimerasoptogeneticallycoordinatedregulationofsurvivintocontrolthefateofcancercells AT maxingyuan splitcas9basedtargetedgeneeditingandnanobodymediatedproteolysistargetingchimerasoptogeneticallycoordinatedregulationofsurvivintocontrolthefateofcancercells |