脊髓损伤后肌肉萎缩lncRNA⁃miRNA⁃mRNA调控网络的筛选及验证

Screening and verification of the lncRNA⁃miRNA⁃mRNA regulatory network in muscle atrophy after spinal cord injury

目的:基于生物信息学分析挖掘脊髓损伤(spinal cord injury,SCI)后肌肉萎缩的关键靶点,构建lncRNA‑miRNA‑mRNA调控网络,通过动物实验验证关键调控网络在SCI后肌肉萎缩中的表达变化,为SCI后肌肉萎缩发病机制及治疗寻求新的研究方向。方法:从GEO数据库下载GSE21497数据集,进行差异表达基因筛选、WGCNA处理,再结合在线预测数据库筛选出关键mRNA,利用DAVID数据库对关键mRNA进行GO、KEGG富集分析,构建lncRNA‑miRNA‑mRNA调控网络,挑选出关键调控基因,然后采用RT‑qPCR进行验证。结果:(1)共筛选出差异表达基因1405个,结合WGCNA和在线数据库预测,共得到30个关键mRNA;(2)GO、KEGG富集分析发现其主要在神经元的再生、保护、信号传递等功能及HIF信号通路、PD‑L1表达和PD‑1检查点通路显著富集;(3)筛选出4个(LINC00410/miR‑17‑5p/KCNK10、LINC00410/miR‑17‑5p/PCDHA3、LINC00410/miR‑20b‑5p/KCNK10、LINC00410/miR‑20b‑5p/PCDHA3)关键调控网络;(4)RT‑qPCR实验结果显示,与对照组相比,观察组模鼠miR‑17‑5p、miR‑20b‑5p表达上升,KCNK10、PCDHA3表达下降。结论:miR‑17‑5p、miR‑20b‑5p、KCNK10、PCDHA3在神经元的再生、保护、信号传递可能具有重要的调控作用,有望成为SCI后肌肉萎缩诊疗及康复的新靶点。

Objective:To find the key targets of muscle atrophy after spinal cord injury(SCI)were excavated,to construct the lncRNA‑miRNA‑mRNA regulatory network based on bioinformatics analysis,and to verify the expression changes of key regulatory networks in muscle atrophy after SCI by animal experiments,so as to seek new research directions for the pathogenesis and treatment of muscle atrophy after SCI.Methods:The GSE21497 data set was downloaded from the GEO database for differential expression gene screening and WGCNA treatment.Combined with the online prediction database,key mRNAs were screened out.GO and KEGG enrichment analyses of key mRNAs were performed using the DAVID database to construct the lncRNA‑miRNA‑mRNA regulatory network.The key regulatory genes were selected and then verified by RT‑qPCR.Results:A total of 1405 differentially expressed genes were screened,and 30 key mRNAs were predicted by the WGCNA and online database.GO and KEGG enrichment analyses showed that it was mainly enriched in the functions of neuron regeneration,protection,signal transmission,the HIF signaling pathway,PD‑L1 expression and the PD‑1 checkpoint pathway.Four key regulatory networks were identified(LINC00410/miR‑17‑5p/KCNK10,LINC00410/miR‑17‑5p/PCDHA3,LINC00410/miR‑20b‑5p/KCNK10,LINC00410/miR‑20b‑5p/PCDHA3).The results of RT‑qPCR showed that compared with the control group,the expression of miR‑17‑5p and miR‑20b‑5p in the observation group was increased,and the expression of KCNK10 and PCDHA3 was decreased.Conclusion:MiR‑17‑5p,miR‑20b‑5p,KCNK10,and PCDHA3 may play an important regulatory role in the regeneration,protection,and signal transmission of neurons,which is expected to become a new target for the diagnosis and treatment of muscle atrophy after SCI.