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

作　　者：朱晓雨 袁韩涛 李四波[1] ZHU Xiaoyu;YUAN Hantao;LI Sibo(Department of Spinal Surgery,Seventh People's Hospital Affiliated to Shanghai University of Traditional Chinese Medicine,Shanghai 200137,China)

机构地区：[1]上海中医药大学附属第七人民医院脊柱外科,上海200137

出　　处：《实验动物与比较医学》2024年第3期270-278,共9页Laboratory Animal and Comparative Medicine

基　　金：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);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);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);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)。

摘　　要：目的探讨微RNA(microRNA,miRNA或miR)-887-3p对大鼠椎间盘纤维环细胞增殖和凋亡的影响及其潜在的分子机制。方法取8周龄SPF级雄性SD大鼠的纤维环组织,离心制备并鉴定纤维环细胞。实验随机分为4组:正常组(Normal group)是不做任何处理的原代纤维环细胞;对照组(Control group)用10 ng/mL白细胞介素-1β(interleukin-1β,IL-1β)处理纤维环细胞24 h,形成退变细胞模型;干扰组(miR-887-3p inhibitor)是在对照组的基础上用Lipo3000转染miR-887-3p抑制子;过表达组(miR-887-3p mimics)是在对照组的基础上用Lipo3000转染miR-887-3p模拟物。采用CCK-8法检测各组细胞活力;流式细胞术检测各组细胞凋亡率;实时荧光定量PCR法检测miR-887-3p和鼠双微体4(murine double minute 4,MDM4)mRNA的表达;蛋白质印迹法检测MDM4、Bcl-2和Caspase-3的蛋白表达水平。结果免疫荧光染色法鉴定分离培养的细胞发现,大鼠椎间盘纤维环细胞中Collagen I的阳性率在90%以上,提示纤维环细胞纯度大于90%。实时荧光定量PCR结果显示,利用IL-1β构建纤维环退变细胞模型后,miR-887-3p表达水平与正常组相比显著上升(P<0.001);与对照组相比,转染miR-887-3p抑制子后,miR-887-3p表达水平显著下降(P<0.001)。CCK-8法检测结果表明,与正常组相比,对照组细胞活力显著下降(P<0.001);与对照组相比,抑制miR-887-3p的表达后,细胞增殖能力显著增强;miR-887-3p过表达后,细胞增殖能力显著下降。流式细胞仪检测结果表明,与正常组相比,对照组的细胞凋亡率显著增加(P<0.001);与对照组相比,miR-887-3p干扰组的细胞凋亡率显著降低(P<0.001),miR-887-3p过表达组的细胞凋亡率显著增加(P<0.001)。蛋白质印迹法检测结果发现,与正常组相比,对照组中Bcl-2的表达水平显著降低(P<0.001),Caspase-3的表达水平显著升高(P<0.001);与对照组相比,miR-887-3p干扰组中Bcl-2和MDM4表达水平显著升高(P<0.01),Caspase-3的表达水平显著降低(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 cy

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 

分 类 号：R681.5[医药卫生—骨科学] Q95-33[医药卫生—外科学]