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Role of miR-15a in intervertebral disc degeneration through targeting MAP3K9
Ping Cai1, Ting Yang2, Xingjie Jiang3
1Department of Orthopedics and Traumatology, Jiangsu Province Hospital of TCM, Nanjing 210029, PR China.
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.
Insights
MicroRNA-15a (miR-15a) is upregulated in intervertebral disc degeneration (IDD), promoting cell proliferation and apoptosis. Targeting miR-15a offers a potential therapeutic strategy for IDD treatment.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- MicroRNAs (miRNAs) regulate crucial cellular functions like proliferation, differentiation, apoptosis, and metastasis.
- miR-15a plays a role in immune responses, angiogenesis, and cancer, but its function in intervertebral disc degeneration (IDD) remains unclear.
Purpose of the Study:
- To investigate the role and mechanism of miR-15a in intervertebral disc degeneration (IDD).
- To explore miR-15a as a potential therapeutic target for IDD.
Main Methods:
- Quantitative real-time PCR to measure miR-15a expression in degenerative nucleus pulposus (NP) tissues and cells.
- Cell lines with stable miR-15a overexpression or silencing were created using lentivirus infection.
- Functional assays including MTT, flow cytometry, TUNEL, colony formation, and Western blot were performed.
- Luciferase reporter assay was used to identify miR-15a targets.
Main Results:
- miR-15a expression was significantly upregulated in degenerative NP tissues and cells from IDD patients.
- Overexpression of miR-15a promoted NP cell proliferation and induced apoptosis, affecting caspase-3, bcl-2, and bax.
- MAP3K9 was identified as a direct target of miR-15a, with an inverse expression relationship.
- Knockdown of MAP3K9 inhibited NP cell proliferation, promoted apoptosis, and affected the p38 and ERK MAPK pathways.
Conclusions:
- miR-15a is upregulated in IDD and contributes to the degeneration process.
- miR-15a influences NP cell proliferation and apoptosis through the MAP3K9/MAPK signaling pathway.
- miR-15a represents a promising novel therapeutic target for treating intervertebral disc degeneration.
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