CAJ | 학술논문

目的筛选人退变腰椎间盘特异性表达microRNA的靶基因,并探讨JNK信号转导通路在椎间盘退变(intervertebral disc degeneration,IVDD)分子生物学机制中发挥的作用.方法对获取的8份腰椎退行性疾病患者(男6例,女2例；44～68岁)手术髓核组织和3份腰椎骨折患者(男3例,17、18、22岁)正常髓核组织依次进行髓核细胞分离、培养、染色鉴定、提取标本总RNA;采用microRNA微阵列基因表达实验和分析技术筛选差异表达的microRNAs并采用实时qPCR技术对其中高表达者进行验证；综合MicroCosm v5、TargetScan 5.1和microRNA.org等三个数据库的靶基因信息,取交集分析预测靶基因,并分析与差异基因或靶基因功能显著相关的生物信号通路；定量PCR方法验证筛选结果.结果退变组中microRNA-513a-5p和microRNA-494呈显著高表达,比值分别为2.222 2和2.948 5,并与验证结果相吻合；预测靶基因分别为MKK4和JunD,此两种靶基因分别位于JNK信号通路的上、下游,均参与JNK信号传导.结论 microRNA-513a-5p和microRNA-494在退变椎间盘中具有高表达,其对应的靶基因为MKK4和JunD.JNK信号传导通路在IVDD的发病机制中可能发挥重要正负反馈调节作用.
목적 사선인퇴변요추간반특이성표체microRNA적파기인,병탐토JNK신호전도통로재추간반퇴변(intervertebral disc degeneration,IVDD)분자생물학궤제중발휘적작용.방법 대획취적8빈요추퇴행성질병환자(남6례,녀2례；44～68세)수술수핵조직화3빈요추골절환자(남3례,17、18、22세)정상수핵조직의차진행수핵세포분리、배양、염색감정、제취표본총RNA;채용microRNA미진렬기인표체실험화분석기술사선차이표체적microRNAs병채용실시qPCR기술대기중고표체자진행험증；종합MicroCosm v5、TargetScan 5.1화microRNA.org등삼개수거고적파기인신식,취교집분석예측파기인,병분석여차이기인혹파기인공능현저상관적생물신호통로；정량PCR방법험증사선결과.결과 퇴변조중microRNA-513a-5p화microRNA-494정현저고표체,비치분별위2.222 2화2.948 5,병여험증결과상문합；예측파기인분별위MKK4화JunD,차량충파기인분별위우JNK신호통로적상、하유,균삼여JNK신호전도.결론 microRNA-513a-5p화microRNA-494재퇴변추간반중구유고표체,기대응적파기인위MKK4화JunD.JNK신호전도통로재IVDD적발병궤제중가능발휘중요정부반궤조절작용.
Objective To screen and validate differentially expressed microRNAs in human degenerative nucleus pulposus cells (NPCs) and to predict their target genes,and to investigate the role of JNK pathway in degenerative intervertebral disc disease.Methods Eight degenerative nucleus pulposus tissues were harvested from 8 patients (6 males and 2 females) with lumbar degenerative disease,and three normal nucleus pulposus tissues were harvested from 3 patients (3 males) with lumbar fracture,intraoperatively.Differentially expressed microRNAs were screened by microRNA microarray analysis and validated by real-time qPCR.Target genes of highly expressed microRNAs were predicted by analyzing information from data bases:MicroCosm v5,TargetScan 5.1 and microRNA.org.Signal pathways associated with the target genes were analyzed,and qPCR was used to validate the screening results.Results Twenty differentially expressed microRNAs were identified.The microRNA-513a-5p and microRNA-494 were highly expressed in degenerative nucleus pulposus tissue,which was corresponding to the verification results,and the ratio was 2.222 2 and 2.948 5,respectively.Target genes of microRNA-513a-5p and microRNA-494 were MKK4 and JunD,respectively.MKK4 and JunD were both involved in JNK signaling pathway and located in upstream and downstream of this pathway,respectively.Conclusion In human degenerative nucleus pulposus cells,microRNA-513a-5p and microRNA-494 are highly expressed microRNAs,whose target genes are MKK4 and JunD,respectively.Both MKK4 and JunD are involved in JNK signaling pathway.These results indicate that JNK pathway may play important role in the pathogenesis of degenerative intervertebral disc disease.