LDR-qPCR法检测miRNA122a的表达及其与乳腺癌雌激素调控的关系  

Ligase dependent reaction-qPCR detective method for miR-122a expression: its development and application in estrogen regulation in MCF-7 cells

作　　者：恽文[1] 季明华[2] 李文静[3] 李建[4] 钟山亮[5] 唐金海[4] 赵建华[5] 

机构地区：[1]徐州医学院外科学教研室,江苏徐州221000 [2]江苏省肿瘤医院放疗科,江苏南京210009 [3]南京医科大学,江苏南京210029 [4]江苏省肿瘤医院普外科,江苏南京210009 [5]江苏省临床检验中心,江苏南京210009

出　　处：《中国肿瘤外科杂志》2012年第4期209-213,共5页Chinese Journal of Surgical Oncology

基　　金：国家自然科学基金资助项目( No30840093);江苏省社会发展科技计划项目( NoBS2007077)

摘　　要：目的建立一种新的基于连接酶依赖性反应的miR-122a实时定量PCR检测方法 (LDR-qPCR);研究乳腺癌细胞MCF-7中miR-122a的表达以及雌激素水平对其的影响。方法以乳腺癌细胞MCF-7为样本、成熟的miR-122a为检测对象,将探针的连接反应和实时荧光定量PCR技术相结合,建立LDR-qPCR法,并开展方法学评价以及不同(有或无激素)培养环境下miR-122a的表达分析。结果 LDR-qPCR法的扩增产物特异,经测序证实为miR-122a;LDR-qPCR法重复性好(cv<10%);检测下限为5pg/μL总RNA,比茎环RT-qPCR法(50pg/μL)敏感10倍。miR-122a在激素饥饿的MCF-7细胞中表达上调,约为常规培养细胞的3.65倍;经5nmol/L雌二醇处理后又出现下调(P=0.040),但未回到常规培养细胞水平。结论 LDR-qPCR法特异、灵敏,检测范围宽;乳腺癌细胞MCF-7可表达miR-122a并受雌激素水平的调节。Objective To establish a ligase dependent reaction-qPCR method for detecting miR-122a, and to investigate the estrogen regulation of miR-122a expression in MCF-7 cells. Methods We developed an assay through the combination of enzymatic probe ligation and real-time PCR amplification for the measurement of ma- ture miR-122a in breast cancer cell line MCF-7, as well as the measurement of U6 snRNA as an endogenous control. In addition, the characters of detection performance of the method were evaluated, and the method was applied to study the relativity between miR-122a expression and estrogen levels in MCF-7 cells. Results The LDR-qPCR products were confirmed by melting curve, agarose gel electrophoresis, and gene sequencing, and the coefficient of variation is lower than 10%. The LDR-qPCR is little more sensitive than stem-loop qPCR as- say by 1 dillution, with detection limit of total RNA samples being 5 pg/pA（50 pg/pA by stem-loop qPCR）. Levels of miR-122a increased approximately 3.65 fold in estrogen starvation cells, and decreased by the addi- tion of estradiol-17β in 5nmol/l（P = 0. 040）. Conclusions The LDR-qPCR method has been developed suecessfully with satisfactory assay specificity, variability and high sensitivity. Also, analysis of MCF-7 cells re- vealed that the expression of functional miR-122 is regulated by estradiol-17β.

关 键 词：荧光定量PCR miR-122a 乳腺癌 雌激素 

分 类 号：R737.9[医药卫生—肿瘤]