Gene-Silencing Therapeutic Approaches Targeting PI3K/Akt/mTOR Signaling in Degenerative Intervertebral Disk Cells: An
Masao Ryu1, Takashi Yurube1, Yoshiki Takeoka1
1Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan.
Abstract:
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.
Insights
Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 gene editing effectively targets the PI3K/Akt/mTOR pathway in degenerative disk cells, inducing autophagy and inhibiting cell death. Targeting RAPTOR/mTORC1 shows significant therapeutic potential for degenerative disk disease.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- The mammalian target of rapamycin (mTOR) pathway is crucial for cell growth and survival.
- Dysregulation of the PI3K/Akt/mTOR pathway contributes to degenerative disk disease.
- Intervertebral disc cells are avascular, hypoxic, and nutrient-deprived, making them susceptible to degeneration.
Purpose of the Study:
- To compare the efficacy of RNA interference (RNAi) and CRISPR-Cas9 gene editing for targeting the PI3K/Akt/mTOR pathway in human degenerative disk cells.
- To investigate the therapeutic potential of targeting mTOR, RAPTOR, or RICTOR in degenerative disk disease models.
Main Methods:
- Human nucleus pulposus cells were transfected with siRNA or CRISPR-Cas9 targeting mTOR, RAPTOR, or RICTOR.
- Transfection efficiency and protein suppression were assessed.
- Cellular responses including autophagy, apoptosis, senescence, pyroptosis, and matrix catabolism were evaluated.
Main Results:
- CRISPR-Cas9 demonstrated significantly higher transfection efficiency (88.1-89.3%) compared to RNAi (53.8-60.3%).
- Targeting mTOR, RAPTOR, and RICTOR induced autophagy and inhibited apoptosis, senescence, pyroptosis, and matrix catabolism.
- CRISPR-Cas9 mediated more sustained gene suppression (83.2% RAPTOR suppression at 168h) than RNAi (8.8%).
- Targeting RAPTOR with CRISPR-Cas9 yielded the most prominent therapeutic effects.
Conclusions:
- CRISPR-Cas9 gene editing is a highly efficient method for targeting the PI3K/Akt/mTOR pathway in degenerative disk cells.
- The RAPTOR/mTORC1 complex represents a promising therapeutic target for degenerative disk disease.
- CRISPR-Cas9 offers a more robust and sustained gene modification approach compared to RNAi for this application.
Related Concept Videos
02:15Experimental RNAi
01:59CRISPR
02:30siRNA - Small Interfering RNAs
In the cytoplasm, siRNA is processed from a double-stranded RNA, which comes from either endogenous DNA transcription or exogenous sources like a virus. This double-stranded RNA is then cleaved by the...
01:23RNA Interference
This process occurs naturally in cells, often through the activity of genomically-encoded microRNAs. Researchers can take advantage of this mechanism by introducing synthetic RNAs to deactivate specific genes for research or therapeutic purposes. For example, RNAi could be used...