实验动物与比较医学2024，Vol.44Issue(3)：270-278,9.DOI:10.12300/j.issn.1674-5817.2024.003
微RNA-887-3p能抑制大鼠椎间盘纤维环细胞中MDM4表达和细胞增殖并促进细胞凋亡
MicroRNA-887-3p Inhibited MDM4 Expression and Proliferation but Promoted Apoptosis of Intervertebral Disc Annulus Fibrosus Cells in Rats
摘要
Abstract
Objective To investigate the effects of microRNA(miRNA,miR)-887-3p on the proliferation and apoptosis of rat intervertebral disc annulus fibrosus cells and its underlying molecular mechanism.Methods Annulus fibrosus tissues were obtained from 8-week-old SPF-grade SD male rats,centrifuged to prepare and identify annulus fibrosus cells.Rats in the experiment were randomly divided into four groups:a Normal group consisting of primary annulus fibrosus cells without any treatment;a Control group treated with 10 ng/mL interleukin-1β(IL-1β)for 24 hours to establish a degenerative cell model;an interference group(miR-887-3p inhibitor)transfected with miR-887-3p inhibitor using Lipo3000 based on the Control group;and an overexpression group(miR-887-3p mimics)transfected with miR-887-3p mimics using Lipo3000 based on the Control group.CCK-8 assay was used to assess cell viability;flow cytometry was used to measure cell apoptosis rates;real-time fluorescence quantitative PCR(qPCR)was used to detect the expression levels of miR-887-3p and murine double minute 4(MDM4)mRNA;Western blotting was used to measure the protein expression levels of MDM4,Bcl-2,and Caspase-3.Results Immunofluorescence staining of isolated and cultured cells revealed a Collagen I positive rate of over 90％in rat intervertebral disc annulus fibrosus cells,indicating a cell purity level greater than 90％.Real-time fluorescence qPCR results showed that after establishing an annulus fibrosus degenerative cell model using IL-1β,the expression level of miR-887-3p significantly increased compared to the Normal group(P＜0.001).Compared to the Control group,transfection with miR-887-3p inhibitor resulted in a significant decrease in its expression level(P＜0.001).The CCK-8 assay showed that compared to the Normal group,cell viability significantly decreased in the Control group(P＜0.001).Compared to the Control group,cell proliferation ability significantly increased after miR-887-3p inhibition,and significantly decreased after overexpression of miR-887-3p.Flow cytometry results revealed that compared to the Normal group,the apoptosis rate in the Control group significantly increased(P＜0.001).Compared to the Control group,the cell apoptosis rate significantly decreased in the miR-887-3p interference group(P＜0.001)and increased in the overexpression group(P＜0.001).Western blotting analysis showed that compared to the Normal group,Bcl-2 expression level significantly decreased(P＜0.001)and Caspase-3 expression level significantly increased(P＜0.001)in the Control group.Compared to the Control group,Bcl-2 and MDM4 expression levels significantly increased(P＜0.01),and Caspase-3 expression level significantly decreased(P＜0.01)in the miR-887-3p interference group;whereas in the overexpression group,Bcl-2 and MDM4 expression levels significantly decreased(P＜0.05),and Caspase-3 levels significantly increased(P＜0.05).Real-time fluorescence qPCR and protein immunoblotting results showed that after interfering with miR-887-3p,the expression of MDM4 protein and mRNA increased(P＜0.001);after overexpressing miR-887-3p,their expression decreased(protein,P＜0.01;mRNA,P＜0.001).Conclusion MiR-887-3p may modulate the cell proliferation and apoptosis of rat intervertebral disc annulus fibrosus cells by regulating MDM4 expression,thereby influencing the development and progression of disc degeneration.关键词
微RNA-887-3p/椎间盘纤维环细胞/退变模型/细胞增殖/细胞凋亡/大鼠Key words
miR-887-3p/Intervertebral disc annulus fibrosus cells/Degenerative model/Cell proliferation/Apoptosis/Rats分类
医药卫生引用本文复制引用
朱晓雨,袁韩涛,李四波..微RNA-887-3p能抑制大鼠椎间盘纤维环细胞中MDM4表达和细胞增殖并促进细胞凋亡[J].实验动物与比较医学,2024,44(3):270-278,9.基金项目
上海市浦东新区科技发展基金-民生科研专项"椎间盘纤维环细胞中hsa-miR-887-3p在凋亡中的作用及复合胶原蛋白海绵修复大鼠纤维环损伤的研究"(PKJ2019-Y18) （PKJ2019-Y18）
上海市浦东新区卫生健康委学科带头人培养计划-李四波(PWRd2019-10) （PWRd2019-10）
浦东新区中央财政支持中医药传承创新发展示范试点项目"石印玉全国名中医传承工作室"(YC-2023-0120)、"中医高峰学科(中西医结合骨伤科)"(YC-2023-0601) （YC-2023-0120）
财政部国家中医药管理局医疗服务与保障能力提升补助资金(重点科室部分)项目"中西医协同重点科室建设"(沪卫中管便函[2023]46号) （重点科室部分）
