Bibliographic Details
| Title: |
Gene-Silencing Therapeutic Approaches Targeting PI3K/Akt/mTOR Signaling in Degenerative Intervertebral Disk Cells: An In Vitro Comparative Study Between RNA Interference and CRISPR–Cas9 |
| Authors: |
Masao Ryu, Takashi Yurube, Yoshiki Takeoka, Yutaro Kanda, Takeru Tsujimoto, Kunihiko Miyazaki, Hiroki Ohnishi, Tomoya Matsuo, Naotoshi Kumagai, Kohei Kuroshima, Yoshiaki Hiranaka, Ryosuke Kuroda, Kenichiro Kakutani |
| Source: |
Cells, Vol 13, Iss 23, p 2030 (2024) |
| Publisher Information: |
MDPI AG, 2024. |
| Publication Year: |
2024 |
| Collection: |
LCC:Cytology |
| Subject Terms: |
intervertebral disk, disk degeneration, nucleus pulposus (NP) cells, phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling, autophagy, RNA interference (RNAi), Cytology, QH573-671 |
| More Details: |
The mammalian target of rapamycin (mTOR), a serine/threonine kinase, promotes cell growth and inhibits autophagy. The following two complexes contain mTOR: mTORC1 with the regulatory associated protein of mTOR (RAPTOR) and mTORC2 with the rapamycin-insensitive companion of mTOR (RICTOR). The phosphatidylinositol 3-kinase (PI3K)/Akt/mTOR signaling pathway is important in the intervertebral disk, which is the largest avascular, hypoxic, low-nutrient organ in the body. To examine gene-silencing therapeutic approaches targeting PI3K/Akt/mTOR signaling in degenerative disk cells, an in vitro comparative study was designed between small interfering RNA (siRNA)-mediated RNA interference (RNAi) and clustered regularly interspaced short palindromic repeat (CRISPR)–CRISPR-associated protein 9 (Cas9) gene editing. Surgically obtained human disk nucleus pulposus cells were transfected with a siRNA or CRISPR–Cas9 plasmid targeting mTOR, RAPTOR, or RICTOR. Both of the approaches specifically suppressed target protein expression; however, the 24-h transfection efficiency differed by 53.8–60.3% for RNAi and 88.1–89.3% for CRISPR–Cas9 (p < 0.0001). Targeting mTOR, RAPTOR, and RICTOR all induced autophagy and inhibited apoptosis, senescence, pyroptosis, and matrix catabolism, with the most prominent effects observed with RAPTOR CRISPR–Cas9. In the time-course analysis, the 168-h suppression ratio of RAPTOR protein expression was 83.2% by CRISPR–Cas9 but only 8.8% by RNAi. While RNAi facilitates transient gene knockdown, CRISPR–Cas9 provides extensive gene knockout. Our findings suggest that RAPTOR/mTORC1 is a potential therapeutic target for degenerative disk disease. |
| Document Type: |
article |
| File Description: |
electronic resource |
| Language: |
English |
| ISSN: |
2073-4409 |
| Relation: |
https://www.mdpi.com/2073-4409/13/23/2030; https://doaj.org/toc/2073-4409 |
| DOI: |
10.3390/cells13232030 |
| Access URL: |
https://doaj.org/article/2c2946945e674ebba5f9a96aa48e8276 |
| Accession Number: |
edsdoj.2c2946945e674ebba5f9a96aa48e8276 |
| Database: |
Directory of Open Access Journals |
|
Full text is not displayed to guests. |
Login for full access.
|
