实时荧光RT-PCR定量检测前列腺癌组织中AMACR基因表达

Quantitative detection of AMACR mRNA in prostate cancer tissues by real-time fluorescent quantitative reverse transcription polymerase chain reaction (FQ-RT-PCR) and the clinical significance

目的建立实时荧光定量逆转录聚合酶链反应(FQ-RT-PCR)检测AMACR mRNA的方法学并检测其在前列腺癌(PCa)组织中的表达。方法在AMACR基因的2、3外显子之间设计一对引物及MGB探针,将PCR扩增产物与pMD18-T载体连接,构建重组质粒作为定量检测的标准品,建立实时荧光定量RT-PCR方法,然后对32例PCa、60例良性前列腺增生(BPH)及34例其它肿瘤组织进行AMACR mRNA的定量检测。结果重组质粒经PCR扩增及序列测定,表明克隆成功,该方法灵敏度高,线性范围为5～5×108copies/reaction。AMACR mRNA在PCa组织中表达量显著高于BPH组织(P<0.01)及其他类型肿瘤组(P<0.01)。ROC曲线分析结果显示,曲线下面积(AUC-ROC)为0.890,AMACR mRNA诊断前列腺癌的敏感度和特异度分别为81.3%和86.7%。PCa组织中AMACR mRNA表达量及检测阳性率与不同临床分期和病理分级之间的差异尚不具有统计学意义(P值均>0.05)。结论实时荧光定量RT-PCR检测AMACR mRNA的方法具有敏感、特异、准确、重复性好等特点。AMACR mRNA在前列腺组织中的表达对PCa的诊断具有一定的临床应用价值。

Objectives To establish the real-time fluorescent quantitative reverse transcription polymerase chain reaction（FQ-RT- PCR） for the expression of AMACR mRNA in the prostate tissues. Methods A Taqman-MGB probe and a pair of primers were designed between exon 2 and 3. The PCR products were inserted into the pMD18-T vector and transfected into DH5 α as the standard substance for the standard curve. AMACR mRNAin the tissues of 32 prostate cancer （Pca）, 60 benign prostate hypertrophy （BPH） and 34other tumor tissues was detected by real-time FQ-RT-PCR. Results FQ-RT-PCR for AMACR mRNA was successfully established. The sensitivity was 5 copies/reaction, the linear range was from 5～5× 10^8copies/reaction. The expression of AMACR mRNA in PCa tissues was notably higher in PCa tissues than that in BPH tissues（P〈0.0l） and in other tumor tissues （P〈0.01）. The ROC-AUC was 0.890. The sensitivity and specificity were 81.3% and 86.7% respectively. The difference of the expression of AMACR mRNA among different clinic~ stages and different extents of differentiation was not significant statistically （P〉0.05）. Conclusion Real-time FQ-RT-PCR for AMACR mRNA was sensitive, specific, rapid and efficient method. The detection of the expression of AMACR mRNA by real-time FQ- RT-PCR in prostate tissues may be of great value in the diagnosis of prostate cancer.