INTRODUCTION: Low back pain is a global health concern, and intervertebral disc degeneration is recognized as one of its independent causes. Autophagy, a cellular survival mechanism under stress including nutrient deprivation, is negatively controlled by the mammalian target of rapamycin (mTOR) signaling pathway which also regulates cell proliferation and protein synthesis. We hypothesized that mTOR signaling would be influential in the disc, the largest avascular, low-nutrient organ in the body. The mTOR is an intracellular serine/threonine kinase, existing in the mTOR complexes 1 (mTORC1) containing the regulatory-associated protein of mTOR (RAPTOR) and 2 (mTORC2) containing the rapamycin-insensitive companion of mTOR (RICTOR). Our objective was to elucidate roles of mTOR signaling in disc cells under nutritional and inflammatory stress from the comparison between gene-silencing strategies using RNA interference (RNAi) and CRISPR–Cas9.
METHODS: (1) Selective gene knockdown of mTOR-signaling components using small interfering RNA (siRNA)-mediated RNAi was applied to human disc nucleus pulposus (NP) cells surgically obtained (total n=12). Monolayer cells were cultured in 10% serum-supplemented Dulbecco’s modified Eagle’s medium (DMEM) under hypoxic 2% oxygen. Then, siRNA against mTOR, RAPTOR, or RICTOR was 36-h reverse transfected through lipofection. Transfected cells were additionally 24-h cultured in 10% serum-supplemented DMEM. Western blotting for mTOR-signaling components and autophagic flux was performed to assess successful transfection with RNAi knockdown efficiency (n=6). Transfected cells were alternatively 24-h cultured in serum-free DMEM with pro-inflammatory 10-ng/ml IL-1β. Western blotting was conducted to examine the incidence of apoptosis, senescence, pyroptosis, and matrix metabolism (n=6). (2) Selective gene knockout of mTOR-signaling components using CRISPR–Cas9 was applied to human disc NP cells (total n=6). Western blotting analysis was performed similarly to RNAi experiments (n=6), time-course changes of which were further compared.
RESULTS: Both RNAi and CRISPR–Cas9 successfully achieved the specific suppression of mTOR, RAPTOR, and RICTOR protein expression. However, the transfection efficiency significantly differed: 53.8%–55.6% in RNAi and 88.1%–89.3% in CRISPR–Cas9 (p<0.001). In both treatments, mTOR-signaling suppression-mediated induction of autophagy (increased LC3 and decreased p62/SQSTM1) and inhibition of apoptosis (increased PARP and decreased cleaved PARP, cleaved caspase-9), senescence (increased p16/INK4A), pyroptosis (decreased GASDMD, cleaved caspase-1), and matrix catabolism (increased Aggrecan, Col2a1 and decreased MMP-3, MMP-13) were consistently observed; moreover, maintained expression of matrix components was the most prominent by CRISPR–Cas9-mediated RAPTOR knockout. In time profiles of RAPTOR gene expression, cells 7 d after transfection maintained the suppression to 81.8% by CRISPR-Cas9 but only 9.5% by RNAi (p<0.001), indicating a significantly longer-term, higher impacts of CRISPR-Cas9 compared to RNAi.
DISCUSSION: We confirmed the involvement of mTOR signaling in human disc NP cells by using RNAi and CRISPR–Cas9 gene-silencing methods. Silencing RAPTOR through both the techniques suppressed pyroptosis as well as apoptosis and senescence. While CRISPR–Cas9 provides extensive gene silencing and also unsolved safety issues, RNAi offers temporary and safer gene silencing for chronic disc degeneration. Selective interference of RAPTOR/mTORC1 is thus a potential biological therapeutic strategy for degenerative disc disease, which is relatively robust based on consistent findings from the two gene-silencing interventions