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...
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2024-12-01
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| author | 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 |
| author_facet | 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 |
| author_sort | Masao Ryu |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-2c2946945e674ebba5f9a96aa48e8276 |
| institution | OA Journals |
| issn | 2073-4409 |
| language | English |
| publishDate | 2024-12-01 |
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| spelling | doaj-art-2c2946945e674ebba5f9a96aa48e82762025-08-20T02:38:36ZengMDPI AGCells2073-44092024-12-011323203010.3390/cells13232030Gene-Silencing Therapeutic Approaches Targeting PI3K/Akt/mTOR Signaling in Degenerative Intervertebral Disk Cells: An In Vitro Comparative Study Between RNA Interference and CRISPR–Cas9Masao Ryu0Takashi Yurube1Yoshiki Takeoka2Yutaro Kanda3Takeru Tsujimoto4Kunihiko Miyazaki5Hiroki Ohnishi6Tomoya Matsuo7Naotoshi Kumagai8Kohei Kuroshima9Yoshiaki Hiranaka10Ryosuke Kuroda11Kenichiro Kakutani12Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, JapanDepartment of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, JapanDepartment of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, JapanDepartment of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, JapanDepartment of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, JapanDepartment of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, JapanDepartment of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, JapanDepartment of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, JapanDepartment of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, JapanDepartment of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, JapanDepartment of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, JapanDepartment of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, JapanDepartment of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, JapanThe 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.https://www.mdpi.com/2073-4409/13/23/2030intervertebral diskdisk degenerationnucleus pulposus (NP) cellsphosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signalingautophagyRNA interference (RNAi) |
| spellingShingle | 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 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 Cells intervertebral disk disk degeneration nucleus pulposus (NP) cells phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling autophagy RNA interference (RNAi) |
| 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 |
| title_full | 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 |
| title_fullStr | 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 |
| title_full_unstemmed | 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 |
| title_short | 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 |
| title_sort | 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 |
| topic | intervertebral disk disk degeneration nucleus pulposus (NP) cells phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling autophagy RNA interference (RNAi) |
| url | https://www.mdpi.com/2073-4409/13/23/2030 |
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