Effect and mechanism of miR-640 in lumbar disc degeneration

English Keywords:Lumbar disc degeneration  miR-640  Nucleus pulposus  Extracellular matrix  Apoptosis  Rat

English Abstract:

  【Abstract】 Objectives: To investigate the effect and mechanism of miR-640 in lumbar disc degeneration(LDD). Methods: The nucleus pulposus cells isolated from normal rat nucleus pulposus tissue were allocated into four groups: control group, TNF-α group(50ng/mL TNF-α treated the cells for 48h), TNF-α+miR-640-NC group(50ng/mL TNF-α treated the cells transfected with miR-640-NC for 48h) and TNF-α+miR-640-inhibitor group(50ng/mL TNF-α treated the cells transfected with miR-640-inhibitor for 48h). The expression of miR-640 in nucleus pulposus cells was detected by RT-qPCR. The cell viability was detected by CCK-8 assay. Cell apoptosis was examined by flow cytometry. The expressions of Bcl-2, Bax, Cleaved caspase-3, Collagen Ⅱ and Aggrecan were detected by Western blot. The expression of MMP-9 was observed by immunofluorescence staining. The LDD rat model was established through the intradiscal injection of TNF-α. The 25 rats were randomly allocated into 5 groups(each consisting of 5 rats): sham group, LDD group, Sham+miR-640-NC group, LDD+miR-640-NC group, and LDD+miR-640-inhibitor group. Specifically, one week prior to sham surgery or LDD modeling, rats in the Sham+miR-640-NC, LDD+miR-640-NC, and LDD+miR-640-inhibitor groups were administered with the lentivirus vector carrying miR-640-inhibitor or miR-640-NC into the intervertebral disc space. The expression of miR-640 in nucleus pulposus tissue was detected by RT-qPCR. The morphology of lumbar intervertebral discs was observed by MRI and HE staining. The expression of MMP-9 in nucleus pulposus was observed by immunofluorescence. And the expressions of Cleaved caspase-3, Collagen Ⅱ and Aggrecan in nucleus pulposus was observed by immunohistochemistry. Results: The expression of miR-640 was increased in nucleus pulposus tissue of LDD model rats and nucleus pulposus cells treated with TNF-α(P<0.05). Compared with TNF-α+miR-640-NC group, cell viability and the expressions of Bcl-2, Collagen Ⅱ and Aggrecan were all increased in TNF-α+miR-640-inhibitor group(P<0.05), while cell apoptosis, and Bax, Cleaved caspase-3 and MMP-9 expressions were decreased(P<0.05). The LDD+miR-640-NC group showed obvious degenerative changes in lumbar intervertebral disc, the expressions of Collagen Ⅱ and Aggrecan in nucleus pulposus were decreased(P<0.05), and the expressions of Cleaved caspase-3 and MMP-9 were increased(P<0.05), however, the above changes were reversed to a certain extent in LDD+miR-640-inhibitor group(P<0.05). Conclusions: In the LDD rat model and in vitro cell model, miR-640 expression is upregulated. Inhibition of miR-640 can alleviate LDD by potentially suppressing nucleus pulposus cell apoptosis, reducing extracellular matrix degradation, and promoting extracellular matrix synthesis.