MiR-146 regulates the repair and regeneration of intervertebral nucleus pulposus cells via Notch1 pathway

R.-S. Yang, Y.-H. Wang, C. Ding, X.-H. Su, X.-B. Gong

Department of Nuclear Medicine, Zhongshan Hospital Xiamen University, Xiamen, China. xuetume@126.com

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• Orthopedics

OBJECTIVE: The aim of this study was to investigate the effects of regulation of micro-ribonucleic acid (miR)-146 expression targeting the Notch1 pathway on the repair and regeneration of intervertebral nucleus pulposus cells, and to explore the possible underlying mechanism.

MATERIALS AND METHODS: Intervertebral nucleus pulposus cells were harvested from rats and cultured in vitro. All cells were randomly divided into 4 groups, including the normal group, the miR-146 inhibitor group, the miR-146 over-expression group and the negative control group. The cells in the normal group were cultured normally. Meanwhile, the cells in the miR-146 inhibitor group and miR-146 over-expression group were transfected with inhibitor-containing plasmid and miR-146-containing plasmid, respectively. However, the cells in the negative control group were transfected with empty plasmid. 24 h after transfection, the cells were collected for subsequent experiments. The differentiation of nucleus pulposus cells was detected via toluidine blue staining. The relative protein expression levels of Notch1, aggrecan (ACAN) and COL II were detected via Western blotting. Meanwhile, the mRNA expressions of miR-146 and Notch1 were detected via quantitative Polymerase Chain Reaction (qPCR). Furthermore, the apoptosis and proliferation of cells were detected via terminal deoxynucleotidyl transferase-mediated dUTP Nick End Labeling (TUNEL) and Cell Counting Kit-8 (CCK-8; Dojindo, Kumamoto, Japan) assay, respectively.

RESULTS: Compared with the normal group, toluidine blue positive staining was significantly increased in miR-146 overexpression group (p<0.05), whereas was significantly decreased in the miR-146 inhibitor group (p<0.05). The results of Western blotting revealed that compared with normal group, the protein expression of Notch1 was markedly decreased in miR-146 over-expression group (p<0.05), whereas the expression levels of ACAN and COL II were notably increased (p<0.05). However, the miR-146 inhibitor group exhibited significantly increased the protein expression level of Notch1 (p<0.05), as well as markedly decreased the expressions of ACAN and COL II (p<0.05). The results of qPCR showed that compared with the normal group, the expression level of miR-146 was significantly increased in the miR-146 over-expression group. However, the mRNA expression level of Notch1 was remarkably decreased (p<0.05). Similarly, the miR-146 inhibitor group exhibited significantly decreased expression level of miR-146, as well as markedly increased mRNA expression level of Notch1 (p<0.05). Compared with those in the normal group, the cell proliferation rate was markedly increased, whereas cell apoptosis was remarkably decreased (p<0.05) in the miR-146 over-expression group. Furthermore, the cell proliferation rate was significantly decreased, while the cell apoptosis was remarkably increased (p<0.05) in the miR-146 inhibitor group.

CONCLUSIONS: Regulating miR-146 expression can target the Notch1 signaling pathway, thereby exerting important influences on the repair and regeneration of intervertebral nucleus pulposus cells.

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