膀胱癌患者血清microRNA检测中内参基因的筛选及验证  

Identification and validation of suitable reference genes for the investigation of serum microRNA in bladder cancer patients

作　　者：刘益民[1] 杜鲁涛[1] 王丽丽[1] 蒋秀梅[1] 李娟[1] 曲爱林 王海燕[1] 郑桂喜[1] 张欣[1] 杨咏梅[1] 王传新[1] 

机构地区：[1]山东大学齐鲁医院检验科,山东济南250012

出　　处：《山东大学学报（医学版）》2014年第5期86-91,共6页Journal of Shandong University:Health Sciences

基　　金：国家自然科学基金(81271916)

摘　　要：目的筛选并验证适用于膀胱癌患者血清microRNA(miRNA)定量检测的内参基因。方法选取100例肌层浸润性膀胱癌患者、105例非肌层浸润性膀胱癌患者及139例健康对照者血清,通过Miseq测序筛选出表达较为稳定的miRNA,采用实时荧光定量PCR(qRT-PCR)法检测上述筛选出的miRNA及U6表达水平,利用geNorm及NormFinder软件对其稳定性进行分析,并在新的实验组中对软件所推荐内参基因及组合进行验证,选取miR-148b-3p作为靶分子,以进一步分析所选择内参基因的可靠性。结果采用qRT-PCR法对M iseq测序结果进行验证,筛选出hsa-miR-193a-5p、hsa-miR-16-5p、U6、hsa-miR-191-5p和hsa-let-7d-3p作为候选内参基因,经分析和验证后证实,hsa-miR-193a-5p作为内参基因最稳定,hsa-miR-193a-5p和hsa-miR-16-5p的组合为最佳内参组合。同时,miR-148b-3p在膀胱癌患者血清中明显上调并具有一定的诊断价值。结论在对膀胱癌血清miRNA检测时,hsa-miR-193a-5p为最稳定内参基因,hsa-miR-193a-5p和hsa-miR-16-5p为最佳组合,联合应用可使结果更为准确可靠。Objective To identify the most stable reference genes for the quantification of serum miRNA in patients with bladder cancer. Methods A total of 100 muscle-invasive bladder cancer（M IBC） patients,105 non-muscle-invasive bladder cancer（NM IBC） patients,and 139 healthy controls were included in our study. MiSeq sequencing was performed,then the expressions of selected miRNAs by sequencing and U6 were examined by real time quantitative polymerase chain reaction（qRT-PCR）,and the stability was analyzed with geNorm and NormFinder. The availability of the most stable reference genes was validated again in a newcohort. Furthermore,the miR-148b-3p was selected as a target gene to determine the effect of candidate normalizers on miRNA quantification. Results After confirmation by qRT-PCR,5 genes（hsa-miR-193a-5p,hsa-miR-16-5p,U6,hsa-miR-191-5p and hsa-let-7d-3p） were selected as candidate reference genes. The analysis and validation results showed that hsa-miR-193a-5p was the most stably expressed,and the combination of reference genes hsa-miR-193a-5p and hsa-miR-16-5p was suggested. Furthermore,the expression of miR-148b-3p in bladder cancer serum was significantly up-regulated and could be valuable in the diagnosis of bladdercancer. Conclusion Hsa-miR-193a-5p is the most stable and normalization of miRNA data using a combination of hsamiR-193a-5p and hsa-miR-16-5p as reference genes may produce reliable and accurate results for serum miRNAs study in bladder cancer.

关 键 词：膀胱癌 MICRORNA QRT-PCR 内参基因 

分 类 号：R446.119[医药卫生—诊断学] R737.14[医药卫生—临床医学]