Jiang Bei, Bai Chong, Pan Jie, Shen Bin, Li Lijun
Department of Spine Surgery, Zhejiang Rongjun Hospital, School of Medicine, Jiaxing University, Jiaxing, China.
Department of Spine Surgery, Shanghai East Hospital, School of Medicine, Tongji University, Shanghai, China.
Front Med (Lausanne). 2024 Oct 8;11:1446294. doi: 10.3389/fmed.2024.1446294. eCollection 2024.
This study aimed to discover micro-ribonucleic acids (microRNAs) involved in the degeneration of cartilage endplates through next-generation sequencing and lay the foundation for further research.
The cartilage endplate was obtained from patients who underwent interbody fusion surgery at the Department of Spine Surgery, Shanghai East Hospital Affiliated to Tongji University, from 1 January 2020 to 1 January 2023. Total RNA was extracted from the cartilage endplate tissue. Discover differential genes through NGS. To annotate gene functions, all target genes were aligned against the Kyoto Encyclopedia of Genes (KEGG) and Gene Ontology (GO) databases. The GO enrichment and KEGG enrichment analyses of target genes were performed using phyper, a function of R. The -value was corrected using the Bonferroni method, and a corrected -value of ≤0.05 was taken as the threshold. GO terms or KEGG terms fulfilling this condition were defined as significantly enriched terms. The screened miRNAs and their target protein were verified using quantitative polymerase chain reaction (qPCR) and Western blotting (WB).
RNA was extracted from normal and degenerated cartilage endplate tissues for NGS. Eight downregulated differentially expressed genes (DEGs) and 22 upregulated DEGs were found. The KEGG pathway analysis of these target genes revealed that differential microRNAs and target genes were enriched in different signaling pathways, and the regulated signaling pathways were mainly mitochondrial autophagy and autophagy. The qPCR results demonstrated a significant upregulation of miR-25-3p and miR-345-5p in degenerative cartilage endplate tissues ( ≤ 0.001). Western blot analysis revealed that BRD4 exhibited a marked increase in protein expression levels in degenerative cartilage endplate tissues ( ≤ 0.0001), while BECN1 showed a significant decrease in protein expression levels within these samples ( ≤ 0.0001).
We found that DEG hsa-miR-25-3p and hsa-miR-345-5p can be used as diagnostic and therapeutic targets for IDD. The significant target proteins of miR-25-3p and miR-345-5p were BRD4 and BECN1, respectively.
本研究旨在通过下一代测序发现参与软骨终板退变的微小核糖核酸（微小RNA），为进一步研究奠定基础。
收集2020年1月1日至2023年1月1日在同济大学附属东方医院脊柱外科接受椎间融合手术患者的软骨终板。从软骨终板组织中提取总RNA。通过NGS发现差异基因。为注释基因功能，将所有靶基因与京都基因与基因组百科全书（KEGG）和基因本体论（GO）数据库进行比对。使用R语言的phyper函数对靶基因进行GO富集和KEGG富集分析。采用Bonferroni方法校正P值，以校正后的P值≤0.05为阈值。满足此条件的GO术语或KEGG术语定义为显著富集术语。使用定量聚合酶链反应（qPCR）和蛋白质印迹法（WB）验证筛选出的微小RNA及其靶蛋白。
从正常和退变的软骨终板组织中提取RNA进行NGS。发现8个下调的差异表达基因（DEG）和22个上调的DEG。对这些靶基因的KEGG通路分析表明，差异微小RNA和靶基因富集于不同的信号通路，调控的信号通路主要是线粒体自噬和自噬。qPCR结果显示，退变软骨终板组织中miR-25-3p和miR-345-5p显著上调（P≤0.001）。蛋白质印迹分析显示，退变软骨终板组织中BRD4蛋白表达水平显著升高（P≤0.0001），而BECN1蛋白表达水平显著降低（P≤0.0001）。
我们发现DEG hsa-miR-25-3p和hsa-miR-345-5p可作为椎间盘退变的诊断和治疗靶点。miR-25-3p和miR-345-5p的显著靶蛋白分别为BRD4和BECN1。
