肛瘘患者特征性微小RNA表达的检测及分析  

Detection and analysis of the characteristic expression of microRNAs of anal fistula patients

作　　者：裘建明[1] 余吉平[1] 杨关根[1] 徐侃[1] 陶勇[1] 林阿丽[1] 王东[1] 

机构地区：[1]杭州市第三人民医院肛肠外科,310009

出　　处：《中华胃肠外科杂志》2016年第7期789-792,共4页Chinese Journal of Gastrointestinal Surgery

基　　金：浙江省自然科学基金（LY15H030001,LY15H160001）;杭州市科技局重点专科专病项目（20150733Q28）

摘　　要：目的检测并分析肛瘘患者的特征性微小RNA（miRNA）表达谱，探讨其可能的靶基因和临床意义。方法收集2014年3-12月间杭州市第三人民医院肛肠外科收治的3例肛瘘联合混合痔切除手术患者的痔核近端肛管黏膜标本（肛瘘组），另选取年龄、性别和体质量与之相匹配的行单纯混合痔手术的3例患者的肛管黏膜标本作为对照（对照组）。利用miRNA微阵列芯片检测比较两组肛管黏膜的1285个miRNAs的表达差异，并通过聚类分析差异miRNAs的聚集性（差异基因的筛选标准是：P≤0.05且变化倍数≥2或≤0.5）；采用DIANAmT、miRanda、miRDB、miRwalk等10种预测软件对差异miRNAs的靶基因进行预测，并进行综合打分，确定出预测分值最高的基因；采用基因本体（GO）富集法分析与靶基因相关的生物学过程；利用免疫组织化学方法检测两组肛管黏膜中分值最高基因的蛋白表达。结果肛瘘组患者的1285个miRNA中，发现13个miRNA与对照组存在显著差异，其中在肛瘘组上调2个，下调11个。经配对t检验显示-肛瘘组中miR-3609上调是对照组的5-98倍（P=0.0231），miR-181a-2-3p下调0.13倍（P=0.0067），是差异表达最大的miRNA。进一步聚类分析显示，上调的hsa-miR-3609和hsa-miR-6086相对表达量在两组间的变化趋势一致；下调的11个miRNAs中，miR-125b-1-3p和miR-548q表达类似，其余9个表达相似（包括：hsa-miR-1185-1-3p、hsa-miR-532-3p、hsa-miR-1233-5p、hsa-miR-769-5p、hsa-miR-149—5p、hsa-miR-99b-3p、hsa-miR。138-5p、hsa-miR-141-3p及hsa-miR-181a-2-3p）。对13个差异miRNAs进行靶基因预测分析显示，其中7个（53-8％）miRNAs能预测到靶基因，共计预测到靶基因104个；其余6个（46-2％）miRNAs未能预测到靶基因。预测分值最高的为几丁质酶基因1（CHff1），其对应的差异miRNA为hsa-miR-769-5p（r=-0.94286，P=0.0167）。基因本体（GO）富集分析结果显示，与这1Objective To detect and analyze the characteristic miRNAs profile of anal fistula and explore their possible target genes and potential clinical significance. Methods The anal mucosa close to the hemorrhoids were collected from three patients undergoing fistulectomy and hemorrhoidectomy （fistula group） as well as three patients receiving only hemorroidectomy （hemorrhoids group）, matching with fistula group in age, gender and body weight, miRNA microarray was used to compare the expression of 1 285 human miRNAs of the anal mucosa between two groups. Cluster analysis was adopted to analyze the accumulation of the differentially expressed miRNAS （P 〈 0.05, fold ≥2.0 or ≤0.5） and their target genes were predicted with 10 softwares such as DIANAmT, miRanda, miRDB, miRWalk etc. Comprehensive scoring was performed to identify genes with highest predictive score. Gene ontology （GO） concentration technique was used to analyze the target gene-associated biological process. Immunohistochemistry was used to examine protein expression of genes with the highest score. Results Among 1285 miRNAs in fistula group, 13 miRNAs were differentially expressed with those in hemorrhoid group, including 2 of up-regulation and 11 of down-regulation. Paired t test showed that in fistula group, miRNA-3609 up-regulation was 5.98 folds （P = 0.0231） and miR-181a-2-3p down- regulation was 0.13 folds （P= 0.0067） compared to those in hemorrhoid group, which had the greatest differential expression. Cluster analysis suggested that up-regulated miR-3609 and miR-6086 had similar change trend in both groups. Among 11 down-regulated miRNAs, miR-125bp-l-3p and miR-548q had similar expression and other 9 miRNAs had similar expression as well, including miR-1155-1-3p, miR- 532-3p, miR-1233-5p, miR-769-5p,, miR-149-5p, miR-99b-3p, miR-141-3p, miR-138-5p, and miR- 181a-2-3p. Target gene prediction analysis of above 13 genes showed that 7 miRNAs （53.8%） were eligible to predict their potential target genes, yielding totally

关 键 词：肛瘘 微小RNA 靶基因 

分 类 号：R657.16[医药卫生—外科学]