脊柱结核患者椎间盘病灶组织中环状RNA表达的初步筛选和功能分析

Preliminary screening and functional analysis of circular RNA expression in intervertebral disc lesions of patients with spinal tuberculosis

目的利用基因芯片技术筛选脊柱结核患者椎间盘病灶组织中差异表达的环状RNA(circRNA)并分析其在脊柱结核发生发展中可能的分子作用机制。方法选取2019年1月至2020年12月就诊于宁夏医科大学总医院脊柱骨科手术治疗的脊柱结核患者3例作为实验组,腰椎间盘退变手术治疗的患者3例作为对照组,收集术中切除的间盘组织,利用Arraystar Human circRNA Array V2芯片检测并采用独立样本t检验进行数据分析筛选出两组差异表达的circRNA;用生物信息学软件预测差异circRNA可能靶向作用的微小RNA(miRNA);对差异表达的circRNA分子的亲本基因进行基因本体(GO)生物学过程富集分析和京都基因与基因组百科全书(KEGG)通路富集分析。结果脊柱结核患者的间盘病灶组织中差异表达的circRNA(差异倍数>2,P<0.05)共有1 396条,其中有757种表达上调,639种表达下调;hsacirc0031968、hsacirc0001806、hsacirc0077607、hsacirc0071312、hsacirc0062683表达显著上调;hsacirc0004183、hsacirc0000367、hsacirc0000691、hsacirc0006220、hsacirc0043278表达显著下调;对所有差异表达的circRNA的亲本基因进行GO分析发现其主要参与细胞蛋白分解代谢、三磷酸鸟苷(GTP)酶结合、组蛋白修饰、转录因子结合、蛋白激酶激活等过程;经KEGG通路分析发现其主要富集在内质网中的蛋白质加工、细胞凋亡、蛋白水解、环鸟苷酸-蛋白激酶G、腺苷酸活化蛋白激酶、EB病毒感染等信号通路。结论脊柱结核患者椎间盘病灶组织中差异表达的circRNA可能与脊柱结核的发生和发展密切相关。

Objective Using gene chip technology to screen the differentially expressed circular RNA(circRNA)in intervertebral disc lesions of patients with spinal tuberculosis and analyze its possible molecular mechanism in the occurrence and development of spinal tuberculosis.Methods Three patients diagnosed as spinal tuberculosis and hospitalized for surgical treatment in the Department of Spinal Orthopedics,General Hospital of Ningxia Medical University from January 2019 to December 2020 were selected as the experimental group,and three patients with lumbar intervertebral disc degeneration matched with age and lesion segment during the same period were selected as the control group.Collect the intervertebral disc tissues resected during the operation,use the Arraystar Human circRNA Array V2 chip and independent sample t test to detect the circRNA in the experimental group and the control group,and screen out the differentially expressed circRNA in patients with spinal tuberculosis and lumbar degeneration;Bioinformatics software was used to predict microRNAs(miRNAs)that may be targeted by differential circRNAs.Perform gene ontology(GO)biological process enrichment analysis and kyoto encyclopedia of genes and genomes(KEGG)pathway enrichment analysis on the parental genes of differentially expressed circRNA molecules.Results There were 1396 differentially expressed circRNAs(fold change>2,P<0.05)in the intervertebral disc lesions of patients with spinal tuberculosis,of which 757 were up-regulated and 639 were down-regulated.hsa_circ_0031968,hsa_circ_0001806,hsa_circ_0077607,hsa_circ_0071312,hsa_circ_0062683 were significantly up-regulated expression.hsa_circ_0004183,hsa_circ_0000367,hsa_circ_0000691,hsa_circ_0006220,hsa_circ_0043278 were significantly down-regulated expression.GO analysis of the parental genes corresponding to each differentially expressed circRNA revealed that it was mainly involved in cellular protein catabolism,GTPase binding,histone modification,transcription factor binding,protein kinase activation and other processes.KEGG Pathway analysis showed that protein processing,apoptosis,proteolysis,Cyclic guanosine mono phosphate(cGMP)-Protein kinase G(PKG),AMP-activated protein kinase(AMPK),Epstein-Barr(EB)virus infection and other signal pathways were mainly concentrated in endoplasmic reticulum.Conclusion The differentially expressed circRNA in the intervertebral disc lesions of patients with spinal tuberculosis may be closely related to the occurrence and development of spinal tuberculosis,and it is expected to become a potential clinical diagnosis or treatment target for spinal tuberculosis in the future.