Effect and mechanism of miR-640 in lumbar disc degeneration
10.3969/j.issn.1004-406X.2024.09.07
- VernacularTitle:miR-640在腰椎间盘退变中的作用及相关机制研究
- Author:
Kun WANG
1
;
Dengyu LI
;
Wenqing LI
Author Information
1. 河南省洛阳正骨医院(河南省骨科医院)脊柱外科 471002洛阳市
- Keywords:
Lumbar disc degeneration;
miR-640;
Nucleus pulposus;
Extracellular matrix;
Apoptosis;
Rat
- From:
Chinese Journal of Spine and Spinal Cord
2024;34(9):950-959
- CountryChina
- Language:Chinese
-
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-in-hibitor 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 n and Aggrecan were detected by Western blot.The expression of MMP-9 was observed by im-munofluorescence 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 pri-or 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 expres-sion 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 pulpo-sus cells treated with TNF-α(P＜0.05).Compared with TNF-α+miR-640-NC group,cell viability and the ex-pressions 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 cas-pase-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 pulpo-sus cell apoptosis,reducing extracellular matrix degradation,and promoting extracellular matrix synthesis.
