Methylation of microRNA-129-5P modulates nucleus pulposus cell autophagy by targeting Beclin-1 in intervertebral disc degeneration
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Kangcheng Zhao1,*, Yukun Zhang1,*, Liang Kang1, Yu Song1, Kun Wang1, Shuai Li1, Xinghuo Wu1, Wenbin Hua1, Zengwu Shao1, Shuhua Yang1 and Cao Yang1
1Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China
*These authors have contributed equally to this work
Cao Yang, email: [email protected]
Keywords: miR-129-5P, intervertebral disc degeneration, Beclin-1, autophagy, methylation
Received: January 13, 2017 Accepted: August 06, 2017 Published: September 21, 2017
MicroRNAs play an important role in the etiology and progression of many diseases, including intervertebral disc degeneration (IVDD). The miRNA miR-129-5P regulates autophagy in various cancers, but its role in human nucleus pulposus (NP) cells is unclear. The present study investigated whether miR-129-5p regulates the expression of Beclin-1 which is known to induce autophagy in NP cells by evaluating their levels in normal and degenerative disc tissues and human NP cells transfected with miR-129-5P mimic or inhibitor by quantitative real-time (qRT-)PCR, western blotting, flow cytometry, and immunofluorescence analysis. A bioinformatics analysis was used to predict the relationship between miR-129-5P and Beclin-1, which was confirmed by the dual luciferase assay. DNA methylation status was assessed by methylation-specific PCR, and the effect of demethylation on miR-129-5P level and autophagy was examined by qRT-PCR, western blotting, and flow cytometry. We found that miR-129-5P expression was downregulated while that of Beclin-1 and LC3-II was upregulated in degenerative disc NP cells. Meanwhile, autophagy was reduced in human NP cells transfected with miR-129-5P mimic, whereas the opposite result was observed upon treatment with miR-129-5P inhibitor. Bioinformatics analysis and the luciferase reporter assay revealed that Beclin-1 is a target of and is inhibited by miR-129-5P. We also found that CpG islands in the miR-129-5P promoter region were hypermethylated in degenerative as compared to normal disc tissue. Thus, miR-129-5P blocks NP cell autophagy by directly inhibiting Beclin-1, a process that is dependent on miR-129-5P promoter methylation.
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