创伤性脊髓损伤后环状非编码RNA-微小RNA潜在调控网络信息挖掘

Identification of potential traumatic spinal cord injury related circular RNA-microRNA networks by sequence analysis

目的通过高通量测序获取创伤性脊髓损伤(traumatic spinal cord injury,TSCI)后环状非编码RNA(circular RNA,circRNA)与微小RNA(microRNA,miRNA)表达谱,预测潜在circRNA-miRNA调控网络。方法取48只雄性C57BL/6小鼠(体质量18~22 g)随机均分为两组(n=24),TSCI组采用Allen’s打击器械制备TSCI模型,假手术组(Sham组)仅切开椎板不损伤脊髓。术后3 d,两组取材行HE染色,观察脊髓组织结构;提取组织总RNA建库,高通量测序鉴定circRNA和miRNA差异表达谱,基因本体分析(gene ontology,GO)注释差异表达的circRNA宿主基因功能,筛选显著差异表达的miRNA,通过TargetScan和miRanda预测circRNAmiRNA靶向结合,筛选关键circRNA,构建潜在调控网络。结果HE染色示Sham组小鼠脊髓结构完整无破裂,TSCI组脊髓结构有明显损伤破裂。测序共鉴定出17440个circRNA、1228个miRNA。差异表达的circRNA宿主基因主要富集在细胞质,生物过程中差异基因主要富集在转录的正调控和蛋白磷酸化过程。差异表达最显著的miRNA为mmu-miR-21-5p,筛选出可与其靶向结合的circRNA6730,以circRNA6730为核心构建潜在circRNAmiRNA调控网络。结论通过表达谱分析和功能注释分析,显著差异表达的circRNA和miRNA有潜在的临床标志物价值,以circRNA6730为核心包含mmu-miR-21-5p的靶向互作网络可能在TSCI的发生发展过程中起重要调控作用,有助于阐明TSCI的病理生理进程机制,为临床诊疗提供新思路。

Objective To systematically profile and characterize the circular RNA(circRNA)and microRNA(miRNA)expression pattern in the lesion epicenter of spinal tissues after traumatic spinal cord injury(TSCI)and predict the structure and potential functions of the regulatory network.Methods Forty-eight adult male C57 BL/6 mice(weighing,18-22 g)were randomly divided into the TSCI(n=24)and sham(n=24)groups.Mice in the TSCI group underwent T8-10 vertebral laminectomy and Allen’s weight-drop spinal cord injury.Mice in the sham group underwent the same laminectomy without TSCI.The spinal tissues were harvested after 3 days.Some tissues were stained with HE staining to observe the structure.The others were used for sequencing.The RNA-Seq,gene ontology(GO)analysis,and circRNA-miRNA network analyses(TargetScan and miRanda)were used to profile the expression and regulation patterns of network of mice models after TSCI.Results HE staining showed the severe damage to the spinal cord in TSCI group compared with sham group.A total of 17440 circRNAs and 1228 miRNAs were identified.The host gene of significant differentially expressed circRNA enriched in the cytoplasm,associated with positive regulation of transcription and protein phosphorylation.mmu-miR-21-5 p was the most significant differentially expressed miRNA after TSCI,and circRNA6730 was predicted to be its targeted circRNA.Then a potential regulatory circRNA-miRNA network was constructed.Conclusion The significant differentially expressed circRNAs and miRNAs may play important roles after TSCI.A targeted interaction network with mmu-miR-21-5 p at the core of circRNA6730 could provide basis of pathophysiological mechanism,as well as help guide therapeutic strategies for TSCI.