RACK1 mediates the advanced glycation end product-induced degradation of HIF-1α in nucleus pulposus cells via competing with HSP90 for HIF-1α binding.

Hyperglycemia can drive advanced glycation end product (AGE) accumulation and associated nucleus pulposus cell (NPC) dysfunction, but the basis for this activity has not been elucidated. Hypoxia-inducible factor-1α (HIF-1α) is subject to cell-type-specific AGE-mediated regulation. In the current study, we assessed the mechanistic relationship between AGE accumulation and HIF-1α degradation in NPCs. Immunohistochemical staining of degenerated nucleus pulposus (NP) samples was used to assess AGE levels. AGE impact on NPC survival and glycolysis-related gene expression was assessed via 3-(4,5)-dimethylthiazol(-z-y1)-3,5-di-phenyltetrazolium bromide assay and quantitative reverse-transcription polymerase chain reaction (qRT-PCR), while HIF-1α expression in NPCs following AGE treatment was monitored via Western blot analysis and qRT-PCR. Additionally, a luciferase reporter assay was used to monitor HIF-1α transcriptional activity. The importance of the receptor for activated C-kinase 1 (RACK1) as a mediator of HIF-1α degradation was evaluated through gain- and loss-of-function experiments. Competitive binding of RACK1 and HSP90 to HIF-1α was evaluated via immunoprecipitation. Increased AGE accumulation was evident in NP samples from diabetic patients, and AGE treatment resulted in reduced HIF-1α protein levels in NPCs that coincided with reduced HIF-1α transcriptional activity. AGE treatment impaired the stability of HIF-1α, leading to its RACK1-mediated proteasomal degradation in a manner independent of the canonical PHD-mediated degradation pathway. Additionally, RACK1 competed with HSP90 for HIF-1α binding following AGE treatment. AGE treatment of NPCs leads to HIF-1α protein degradation. RACK1 competes with HSP90 for HIF-1α binding following AGE treatment, resulting in posttranslational HIF-1α degradation. These results suggest that AGE is an intervertebral disc degeneration risk factor, and highlight potential avenues for the treatment or prevention of this disease.

MiR-1224-5p acts as a tumor suppressor by targeting CREB1 in malignant gliomas.

The dysregulation of miR-1224-5p has been reported in several human cancers. However, the expression and function of miR-1224-5p in glioma remains unknown. The aim of our study was to investigate the effect of miR-1224-5p on glioma cells and to determine its functional signaling mediators. Using 198 glioma samples within the Chinese Glioma Genome Atlas expression dataset, we demonstrated that miR-1224-5p expression is decreased in high-grade gliomas when compared with low-grade gliomas. Differential miR-1224-5p expression in 50 randomly selected samples was verified by in situ hybridization. The expression of miR-1224-5p was shown to positively correlate with overall survival in 82 glioblastoma patients. Exogenous expression of miR-1224-5p in glioma cells suppressed proliferation and invasion and promoted apoptosis. Target prediction algorithms identified a consensus miR-1224-5p recognition site in the 3'UTR of the cAMP response element-binding protein (CREB1) gene, and this sequence was shown to directly confer miR-1224-5p repression in luciferase reporter assays. Furthermore, exogenous miR-1224-5p expression was shown to down-regulate CREB1, as well as its downstream target genes matrix metalloproteinase-9 and B-cell lymphoma-2. Conversely, over-expression of CREB1 reversed the effect of miR-1224-5p on the proliferation, invasion, and apoptosis of glioma cells. These data indicate that miR-1224-5p may inhibit tumor-associated activity in malignant gliomas by targeting CREB1.