title: Role of miR-15a in intervertebral disc degeneration through targeting MAP3K9.
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.
  
  
  CONCLUSIONS
  This present study revealed that miR-15a might be considered as a novel therapeutic target for IDD treatment.
authors.affiliation: Department of Orthopedics and Traumatology, Jiangsu Province Hospital of TCM, Nanjing 210029, PR China.
authors.forename: Ping
authors.initials: P
authors.lastname: Cai
authors.affiliation: First Clinical College of Nanjing University of Chinese Medicine, Nanjing 210046, PR China.
authors.forename: Ting
authors.initials: T
authors.lastname: Yang
authors.affiliation: Department of Orthopedics, The Affiliated Hospital of Nantong University, Nantong 226001, PR China.
authors.forename: Xingjie
authors.initials: X
authors.lastname: Jiang
authors.affiliation: Department of Spine Surgery, Nanfang Hospital, Southern Medical University, Guangzhou 510515, PR China.
authors.forename: Minghui
authors.initials: M
authors.lastname: Zheng
authors.affiliation: Department of Orthopedics, The First People's Hospital of Lianyungang, Lianyungang 222002, PR China.
authors.forename: Gang
authors.initials: G
authors.lastname: Xu
authors.affiliation: First Clinical College of Nanjing University of Chinese Medicine, Nanjing 210046, PR China. Electronic address: xiajianlong2016@163.com.
authors.forename: Jianlong
authors.initials: J
authors.lastname: Xia
mesh_terms: D017209:Apoptosis / Q000235:genetics; D051017:Apoptosis Regulatory Proteins / Q000235:genetics; D053148:Caspase 3 / Q000235:genetics; D002454:Cell Differentiation / Q000235:genetics; D049109:Cell Proliferation / Q000235:genetics; D015536:Down-Regulation / Q000235:genetics; D006801:Humans; D055959:Intervertebral Disc Degeneration / Q000235:genetics*; D020930:MAP Kinase Kinase Kinases / Q000235:genetics*; D035683:MicroRNAs / Q000235:genetics*; D015854:Up-Regulation / Q000235:genetics
chemical_list: D051017:Apoptosis Regulatory Proteins; C505361:MIRN15 microRNA, human; D035683:MicroRNAs; D020930:MAP Kinase Kinase Kinases; C433243:MAP3K9 protein, human; D053148:Caspase 3
keywords: Apoptosis; IDD; MAP3K9; MAPK pathway; miR-15a