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Biomed Pharmacother. 2017 Mar;87:568-574. doi: 10.1016/j.biopha.2016.12.128. Epub 2017 Jan 9.

Role of miR-15a in intervertebral disc degeneration through targeting MAP3K9.

Author information

1
Department of Orthopedics and Traumatology, Jiangsu Province Hospital of TCM, Nanjing 210029, PR China.
2
First Clinical College of Nanjing University of Chinese Medicine, Nanjing 210046, PR China.
3
Department of Orthopedics, The Affiliated Hospital of Nantong University, Nantong 226001, PR China.
4
Department of Spine Surgery, Nanfang Hospital, Southern Medical University, Guangzhou 510515, PR China.
5
Department of Orthopedics, The First People's Hospital of Lianyungang, Lianyungang 222002, PR China.
6
First Clinical College of Nanjing University of Chinese Medicine, Nanjing 210046, PR China. Electronic address: xiajianlong2016@163.com.

Abstract

BACKGROUND:

Accumulating evidence indicates that microRNAs are involved in various cellular processes, including cell proliferation, differentiation, apoptosis and metastasis. miR-15a is an important regulator of immune responses and angiogenesis, endogenous controls as well as potential targets and hallmarks of cancer. However, the role of miR-15a in intervertebral disc degeneration (IDD) has not been elucidated.

METHODS:

Total RNA was extracted from degenerative nucleus pulposus (NP) tissues of 20 patients with IDD and NP cells, respectively. The expression levels of miR-15a were examined by quantitative real-time PCR. The stable overexpress or silence miR-15a expression cell lines and control cell lines were constructed by lentivirus infection. Subsequently, 3-(4,5-dimethylthia zol-2-yl)-2,5-diphenylte trazolium bromide (MTT) assay, flow cytometry test, TdT-mediated dUTP Nick-End Labeling (TUNEL) experiment, colony formation assay and western blot analysis were performed to detect the biological functions of miR-15a. Moreover, a luciferase reporter assay was conducted to confirm its target associations.

RESULTS:

Herein, the results found that miR-15a was dramatically up-regulated in degenerative NP tissues and NP cells compared with the controls. Overexpression of miR-15a promoted NP cells proliferation and induced apoptosis. Moreover, apoptosis-related protein caspase-3 was significantly up-regulated and bcl-2 was observably down-regulated when NP cells were transfected with miR-15a mimics, while bax and caspase-3 were significantly down-regulated as well as bcl-2 was observably up-regulated when NP cells were transfected with miR-15a inhibitors. Further, luciferase reporter assay showed that MAP3K9, an upstream activator of MAPK kinase, was putative target of miR-15a. There was a negatively relationship between miR-15a and MAP3K9 expression in NP cells. In addition, knockdown MAP3K9 inhibited NP cells proliferation and promoted apoptosis, which further inhibited the activation of p38 and ERK MAPK pathway.

CONCLUSION:

This present study revealed that miR-15a might be considered as a novel therapeutic target for IDD treatment.

KEYWORDS:

Apoptosis; IDD; MAP3K9; MAPK pathway; miR-15a

PMID:
28081468
DOI:
10.1016/j.biopha.2016.12.128
[Indexed for MEDLINE]

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