CRISPR/dCas9-Mediated Parkin Inhibition Impairs Mitophagy and Aggravates Apoptosis of Rat Nucleus Pulposus Cells Under Oxidative Stress

Tao Lan; Yu-Chen Zheng; Ning-Dao Li; Xiao-Sheng Chen; Zhe Shen; Bin Yan

doi:10.3389/fmolb.2021.674632

CRISPR/dCas9-Mediated Parkin Inhibition Impairs Mitophagy and Aggravates Apoptosis of Rat Nucleus Pulposus Cells Under Oxidative Stress

Front Mol Biosci. 2021 Apr 15:8:674632. doi: 10.3389/fmolb.2021.674632. eCollection 2021.

Authors

Tao Lan¹, Yu-Chen Zheng¹, Ning-Dao Li², Xiao-Sheng Chen¹, Zhe Shen¹, Bin Yan¹

Affiliations

¹ Department of Spine Surgery, Shenzhen Second People's Hospital, The First Affiliated Hospital of Shenzhen University, Shenzhen, China.
² Department of Orthopedic Surgery, Shenzhen Luohu People's Hospital, The Third Affiliated Hospital of Shenzhen University, Shenzhen, China.

Abstract

Objective: The aim of this study is to explore the role of Parkin in intervertebral disk degeneration (IDD) and its mitophagy regulation mechanism.

Study design and methods: Rat nucleus pulposus (NP) cells were stimulated with hydrogen peroxide (H₂O₂) to a mimic pathological condition. Apoptosis and mitophagy were assessed by Western blot, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, and immunofluorescence staining. The CRISPR-dCas9-KRAB system was used to silence the expression of Parkin.

Result: In this study, we found that Parkin was downregulated in rat NP cells under oxidative stress. In addition, treatment with H₂O₂ resulted in mitochondrial dysfunction, autophagy inhibition, and a significant increase in the rate of apoptosis of NP cells. Meanwhile, mitophagy inhibition enhanced H₂O₂-induced apoptosis. Furthermore, repression of Parkin significantly attenuated mitophagy and exacerbated apoptosis.

Conclusion: These results suggested that Parkin may play a protective role in alleviating the apoptosis of NP cells via mitophagy, and that targeting Parkin may provide a promising therapeutic strategy for the prevention of IDD.

Keywords: CRISPR/dCas9; IDD; Parkin; apoptosis; mitophagy.