胆道闭锁循环miRNA的高通量测序分析

High-throughput sequencing of circulating miRNAs in patients with biliary atresia

目的用高通量二代测序方法（next generation sequencing, NGS）分析胆道闭锁（biliary atresia，BA）患儿外周血中循环miRNA的表达特征，为寻找新的疾病相关无创诊断标志物提供依据。方法分别收集2013年1月至2014年5月到本中心就诊的29例BA（疾病组），10例婴儿肝炎综合征、5例Citrin基因缺陷导致的新生儿肝内胆汁淤积症（NICCD）、5例胆总管囊肿（疾病对照组）和29例健康对照（正常对照组）血浆样本。选取前期收集的疾病组和正常对照组样本各9例用于高通量测序，将两组儿童分别按性别和年龄匹配分为3组，将血浆样本按上述分组进行组内等比例混合，提取血浆混合池中的总RNA，以Illumina HiSeq 2000测序仪分析混合样品中的全部miRNA序列。组间差异比较采用t检验，靶基因预测软件TargetScan, miRanda和PITA对差异miRNA的功能进行预测；并以实时荧光定量聚合酶链反应（reverse transcription quantitative real-time polymerase chain reaction，RT-qPCR）对差异表达显著的miRNA进行验证。结果本研究成功构建BA患儿循环miRNA表达谱；BA患儿组与正常对照组中有188个循环miRNA表达存在差异（P〈0.05），包括87个上调、101个下调，56个为新发现的miRNA，其余皆为数据库中的已知miRNA。表达差异大于2倍的miRNA有146个，77个上调、69个下调。其中，表达量高且为各样本组恒定表达的miRNA有8个，均为已知miRNA，包括上调的has-miR-122-5p、-100-5p、-432-5p，下调的has-miR-140-3p、-10b-5p、-26a-5p、-126-3p、-744-5p；功能预测显示，这8个差异miRNA与肝胆发育、肝纤维化、细胞凋亡等相关；RT-qPCR验证结果表明，BA组血浆hsa-miR-122-5p、-100-5p表达量显著高于正常对照，而hsa-miR-140-3p、-10b-5p、-126-3p显著低于正常对照组（均P〈0.05），与测序结果一致。结论本研究结果全面展示了BA患儿外周血循环中miRNA的表达特征，其中一些差异性的miRNA有可能作为BA无创性诊断的候选标志物。

Objective To employ next generation high-throughput sequencing （NGS） for analyzing distinct expression profiles of circulating miRNAs in infants with biliary atresia （BA） and exploring potential non-invasive biomarkers for its diagnosis. Methods Between January 2013 and May 2014, plasma samples were collected from 29 BA infants, 10 cases of infant hepatitis syndrome, 5 with neonatal intrahepatic cholestasis disease （NICCD）, 5 choledochal cysts and 29 healthy controls. Samples collected previously from 9 BA infants and 9 age/gender-matched healthy controls were used for high-throughput sequencing and the other samples for verification. Illumina deep sequencing was employed and online software analysis used for predicting biological functions of differential miRNAs with TargetScan, miRanda and PITA. The results of sequencing were validated by reverse transcription quantitative real-time polymerase chain reaction. Results The characteristic expression profiles of circulating miRNAs were detected for BA infants. A total of 188 miRNAs in BA were differentially expressed including 87 up-regulated miRNAs and 101 down-regulated miRNAs. Among them, 56 miRNAs were never previously identified. The amount of 146 miRNAs were 2 folds or more than the controls, including 77 up-regulated and 69 down-regulated ones. Compared with healthy controls, the circulating levels of hsa-miR-122-5p, -100-5p and -432-5p became significantly elevated while hsa-miR-140-3p, -10b-5p, -26a-5p, -126-3p and -744-5p significantly decreased markedly in BA. Online software indicated that these 8 miRNAs were involved in the processes of hepatobiliary development, liver fibrogenesis and cell apoptosis. As revealed by RT-qPCR, the expressions of hsa- miR-122-5p and-100-5p were obviously up-regulated while those of hsa-miR-140-3p, -10b-5p and - 126-3p decreased（all P〈0. 05）. Conclusions Circulating miRNAs from BA infants have a unique expression profile. And differential circulating miRNAs may become potential biomarkers for non- invasive diagnosis of BA.