CAJ | 학술논문

目的探讨重症肌无力伴胸腺增生(MGH)患者胸腺内B细胞趋化因子13(CXCL13)表达的微小RNA(miRNAs)调控机制.方法收集26例胸腺标本,分别来源于自2012年3月至2013年8月在广西医科大学第一附属医院心胸外科住院确诊为重症肌无力并行胸腺完整切除术的13例患者(MGH组),以及同期因先天性心脏病行开胸治疗并在手术过程中切除胸腺的13例患者(对照组).应用miRNAs基因芯片筛选MGH组与对照组之间表达有差异的miRNA;应用生物信息学预测方法并结合miRNAs芯片检测结果寻找靶向B细胞趋化因子13 (CXCL13)的miRNA;对CXCL13 mRNA和预测的miRNAs在MGH组与对照组胸腺内的表达进行实时荧光定量PCR(qRT-PCR)分析,然后进行双荧光报告分析微小RNA-548k(miR-548k)模拟物组与空白对照组的CXCL13-3,非翻译区(UTR)双荧光载体荧光素酶活性.结果 (1)miRNAs芯片筛选结果显示,在MGH患者胸腺内呈显著差异表达的miRNAs有33个,其中miR-548k是下调最显著的一个(1.98倍).(2)生物信息学预测显示,miR-548k与CXCL 13的3'UTR有明显的相互作用.(3)qRT-PCR定量分析发现,MGH组胸腺内miR-548k的表达量较对照组明显下调(0.55±0.20 vs 1.33±0.36),差异有统计学意义(P＜0.05);MGH组胸腺内CXCL 13的表达量较对照组明显上调(4.93±1.95 vs 1.04±0.20),差异有统计学意义(P＜0.05);MGH组胸腺内miR-548k与CXCL13表达呈明显负相关(r=-0.93,P=0.003).(4)双荧光报告分析显示,miR-548k模拟物组的CXCL13-3'UTR双荧光载体荧光素酶活性较空白对照组下降约61.5％(0.385±0.016 vs 1.000±0.050),差异有统计学意义(P＜0.05).结论 MGH患者胸腺内miR-548k的低表达很可能通过转录后基因沉默机制使其靶基因CXCL13过表达,从而参与重症肌无力的发生发展.
목적 탐토중증기무력반흉선증생(MGH)환자흉선내B세포추화인자13(CXCL13)표체적미소RNA(miRNAs)조공궤제.방법 수집26례흉선표본,분별래원우자2012년3월지2013년8월재엄서의과대학제일부속의원심흉외과주원학진위중증기무력병행흉선완정절제술적13례환자(MGH조),이급동기인선천성심장병행개흉치료병재수술과정중절제흉선적13례환자(대조조).응용miRNAs기인심편사선MGH조여대조조지간표체유차이적miRNA;응용생물신식학예측방법병결합miRNAs심편검측결과심조파향B세포추화인자13 (CXCL13)적miRNA;대CXCL13 mRNA화예측적miRNAs재MGH조여대조조흉선내적표체진행실시형광정량PCR(qRT-PCR)분석,연후진행쌍형광보고분석미소RNA-548k(miR-548k)모의물조여공백대조조적CXCL13-3,비번역구(UTR)쌍형광재체형광소매활성.결과 (1)miRNAs심편사선결과현시,재MGH환자흉선내정현저차이표체적miRNAs유33개,기중miR-548k시하조최현저적일개(1.98배).(2)생물신식학예측현시,miR-548k여CXCL 13적3'UTR유명현적상호작용.(3)qRT-PCR정량분석발현,MGH조흉선내miR-548k적표체량교대조조명현하조(0.55±0.20 vs 1.33±0.36),차이유통계학의의(P＜0.05);MGH조흉선내CXCL 13적표체량교대조조명현상조(4.93±1.95 vs 1.04±0.20),차이유통계학의의(P＜0.05);MGH조흉선내miR-548k여CXCL13표체정명현부상관(r=-0.93,P=0.003).(4)쌍형광보고분석현시,miR-548k모의물조적CXCL13-3'UTR쌍형광재체형광소매활성교공백대조조하강약61.5％(0.385±0.016 vs 1.000±0.050),차이유통계학의의(P＜0.05).결론 MGH환자흉선내miR-548k적저표체흔가능통과전록후기인침묵궤제사기파기인CXCL13과표체,종이삼여중증기무력적발생발전.
Objective To explore the microRNAs regulation of CXC chemokine ligand 13 (CXCL13) in patients with myasthenia gravis combined with thymic hyperplasia (MGH).Methods Thirteen MGH tissues and 13 normal thymus tissues,collected in our hospital from March 2012 to August 2013,were used in our study.Total RNAs from these tissues were extracted by trizol and hybridized with the microarray.The miRNAs targeting CXCL13 gene-3'untranslated region were predicted by using bioinformatics.Real-time fluorogenic quantitative PCR (QRT-PCR) was employed to detect the expressions ofCXCL13 mRNAs and microRNAs in thymus tissues.Luciferase assay was used to analyze the miRNAs modulated CXCL13 expression.Results The miRNA microarray chip analysis identified 33 miRNAs differentially expressed in MGH tissues as compared with those in the control group,miR-548k was one of most obvious down-regulated miRNAs (1.98 fold).Bioinformatical analysis indicated that miR-548k can target CXCL13 3' UTR.QRT-PCR showed that the expression of CXCL13 mRNA was up-regulated and miR-548k was down-regulated in thymus hyperplasia tissues of MGH group as compared with those in the control group(4.93±l.95 vs.1.04±0.20; 0.55±0.20 vs.1.33±0.36,P＜0.05); and they showed a negative correlation (r=-0.93,P=0).003).As compared with that in the control group (1.000±0.050),the luciferase activity of pmiR-RB-REPORTTM-CXCL13-3'UTR treated with miR-548k mimics (0.385±0.016) decreased 61.5％,with significant difference (P＜0.05).Conclusion MiR-548k inhibits CXCL13 expression by post-transcriptional gene silencing to promote MG development and progression.