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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。
Objective To establish a real-time fluorescence quantitative reverse transcription-polymerase chain reaction (FQ-RT-PCR) method for the detection of AMACR mRNA and its expression in prostate cancer (PCa) tissues. Methods A pair of primers and MGB probes were designed between exon 2 and exon 2 of AMACR gene. The PCR products were ligated with pMD18-T vector to construct a recombinant plasmid for quantitative detection. Real-time fluorescence quantitative RT- PCR method, and then 32 cases of PCa, 60 cases of benign prostatic hyperplasia (BPH) and 34 cases of other tumor tissue AMACR mRNA quantitative detection. Results The recombinant plasmids were successfully amplified by PCR and sequencing. The results showed that the method was highly sensitive and the linear range was 5-5 × 108 copies / reaction. The expression of AMACR mRNA in PCa tissues was significantly higher than that in BPH tissues (P <0.01) and other types of tumors (P <0.01). The ROC curve analysis showed that the area under the curve (AUC-ROC) was 0.890. The sensitivity and specificity of AMACR mRNA in diagnosing prostate cancer were 81.3% and 86.7%, respectively. The difference of AMACR mRNA expression level, positive rate of detection and clinical stage and pathological grade in PCa was not statistically significant (P> 0.05). Conclusion The real-time fluorescent quantitative RT-PCR detection of AMACR mRNA is sensitive, specific, accurate and reproducible. The expression of AMACR mRNA in prostate tissue has certain clinical value in the diagnosis of PCa.