荧光定量PCR检测血清微小RNA-21方法的建立及对乳腺癌诊断的初步应用

Establishment of real-time PCR for detecting serum microRNA-21 and its preliminary application in breast cancer

目的 建立一种SYBR Green Ⅰ荧光定量PCR检测血清miR-21方法,并初步探讨其对乳腺癌诊断的应用价值.方法 用Trizol试剂提取血清总RNA.用茎环引物将miR-16（作为miR-21内参基因）与miR-21分别逆转录成相应cDNA.再用SYBR Green Ⅰ荧光定量RT-PCR对cDNA进行扩增、检测.然后,通过信噪比（signal to noise ratio,SNR）分析试验的准确性；通过熔解曲线评价试验的特异性；通过标准曲线的R2评估试验的精确性；通过批内和批间差异计算试验的稳定性.另外,用自建方法检测33例乳腺癌患者、18例乳腺良性疾病和49名健康人群血清miR-21和miR-16水平,并根据乳腺癌组与健康对照组中miR-21相对表达量确定临界值,以评价其对乳腺癌诊断的敏感度、特异度.结果 通过PCR退火与延伸在温度和时间上的优化,本试验所建立方法SNR≥99.36％；熔解曲线为单峰；标准曲线R3=0.994 8；批内CV＜ 1.5％,批间CV＜ 4％.以miR-16为内参,用自建SYBR Green Ⅰ荧光定量PCR检测乳腺癌组、良性疾病组与健康对照组血清miR-21的相对表达量分别为20.83±18.18、20.86±10.11和9.33±4.44,经Kruskal Wallis检验,3组间表达量差异有统计学意义（x2=16.92,P＜0.001）,且健康对照组与乳腺癌组、健康对照组与良性疾病组间的差异均有统计学意义（Z值分别为-2.58、-4.42,P均≤0.01）,而乳腺癌组与良性疾病组血清miR-21表达量差异无统计学意义（Z=-0.51,P=0.608）.以miR-21相对表达量18.32为临界值,其对乳腺癌诊断的敏感度为51.5％（17/33）,特异度为93.9％（46/49）.结论 建立了一种较敏感、特异、稳定的SYBRGreen Ⅰ荧光定量PCR检测血清miR-21方法,该方法对乳腺癌的诊断可能有一定价值.

Objective To establish a SYBR green Ⅰ real-time PCR method for detecting serum miR-21 and preliminarily explore its value in diagnosing breast cancer.Methods Total RNA was extracted from serum by Trizol reagent.Then miR-16 （ internal reference gene for miR-21 ） and miR-21 were reverse transcribed into corresponding cDNA by stem-loop RT primers.Their cDNA were amplified and detected by using SYBR green Ⅰ real-time PCR.The accuracy of assay was analyzed by signal to noise ratio （SNR） ; the specificity of assay was evaluated by melting curve; the precision of assay was assessed by R2 of standard curve and the stability of assay was calculated by intra-assay and inter-assay variation.Furthermore,the level of miR-21 and miR-16 were detected by this method among the serum samples of 33 breast cancer patients,18 benign breast disease patients and 49 healthy individuals.And the sensitivity and specificity in breast cancer diagnosis were evaluated according to cut-off value which was defined by relative expressions of miR-21 between breast cancer patients and healthy population.Results Through optimization of temperature and time in the annealing and extension stage during PCR,SNR was ≥99.36% ; peak of melting curve was single; R2 of standard curve was 0.994 8 and Coefficient of Variance （CV） of intra-assay 〈 1.5%,CV of inter-assay 〈4%.They indicated that this method was accurate,specific,precise and stable.When miR-16 was taken as internal reference,the relative expressions of serum miR-21 detected by SYBR green Ⅰ realtime PCR among the serum samples of breast cancer patients,benign breast disease patients and healthy population were 20.83 ± 18.18,20.86 ± 10.11 and 9.33 ±4.44,which had statistical significance among of them （x2 =16.92,P 〈 0.001 ）.There was statistical significance in healthy people vs breast cancer patients （ Z =- 2.58,P ≤ 0.01 ） and healthy people vs benign breast disease patients （ Z =- 4.42,P ≤0.01 ）,but was not between breast cancer patients and benign breast cancer patients （Z =-0.51,P =0.608）.When the value of 18.32 for the relative expressions of miR-21 was defined as cut-off value,the sensitivity and specificity of this method to diagnose breast cancer were 51.5% （17/33）and 93.9% （46/49）,respectively.Conclusions A sensitive,specific and stable SYBR green Ⅰ real-time PCR for detecting serum miR-21 has been established.This method may have some diagnostic value for breast cancer.