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
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 phospha...
Saved in:
| Main Authors: | , , , , , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2024-12-01
|
| Series: | Cells |
| Subjects: | |
| Online Access: | https://www.mdpi.com/2073-4409/13/23/2030 |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| Summary: | 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 <i>mTOR</i>, <i>RAPTOR</i>, or <i>RICTOR</i>. 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 (<i>p</i> < 0.0001). Targeting <i>mTOR</i>, <i>RAPTOR</i>, and <i>RICTOR</i> all induced autophagy and inhibited apoptosis, senescence, pyroptosis, and matrix catabolism, with the most prominent effects observed with <i>RAPTOR</i> 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. |
|---|---|
| ISSN: | 2073-4409 |