液态活检miR-509-5p/miR-124比值诊断膀胱癌的临床应用

The miR-509-5p/miR-124 ratio of liquid biopsies in diagnosis of bladder cancer

背景与目的:液态活检是一种从血液、尿液等非实体生物组织中取样分析,主要用于恶性肿瘤诊断、监测及判断预后的方法。该研究通过优化尿液mi RNA的提取方法,建立标准化的液态活检手段,筛选并验证膀胱癌患者尿液mi RNA标志物以阐明其临床应用价值。方法:收集2014年1月—2015年9月膀胱癌患者及健康对照者的尿液,应用mi RNA表达谱芯片进行筛选,在78例膀胱癌患者及23例健康对照者的晨尿中进行实时荧光定量聚合酶链反应(real-time fluorescent quantitative polymerase chain reaction,RTFQ-PCR)验证,分析液态活检的mi RNA标志物与膀胱癌临床病理的关系及其诊断价值。结果:通过对6例膀胱癌及6例健康对照者的尿液mi RNA表达谱进行分析,筛选出了10个mi RNA有差异表达,结合既往文献报道的膀胱癌尿液mi RNA标志物,我们挑选了20个mi RNA进行RTFQ-PCR验证,发现mi R-509-5p/mi R-124比值在膀胱癌患者尿液中表达水平较健康对照者高,差异有统计学意义(P<0.000 1)。尿液mi R-509-5p/mi R-124比值随着患者肿瘤分期和肿瘤分级的上升而升高(P=0.003)。当界值定在0.41时,mi R-509-5p/mi R-124比值对膀胱癌的诊断灵敏度为73.1%,特异度为82.6%,曲线下面积(area under the curve,AUC)达到0.864,比尿液脱落细胞学的诊断价值高(P=0.000 2)。结论:该研究优化了尿液mi RNA的提取方法,建立了标准化的液态活检尿液mi RNA标志物的检测手段,发现mi R-509-5p/mi R-124比值可能是膀胱癌理想的诊断标志物。

Background and purpose:Liquid biopsy is a kind of blood,urine and other non-solid biological tissue sampling analysis,mainly for malignant tumor diagnosis,monitoring and predicting its prognosis.In this research,we optimized the extraction of miRNA in urine,established a standardized means of liquid biopsy,screened and verified the miRNA markers in patients with bladder cancer.Methods:From Jan.2014to Sept.2015,we used miRNA microarray in six patients with bladder cancer and six healthy controls.Samples of78cases of bladder cancer and23healthy controls were tested by real-time fluorescent quantitative polymerase chain reaction(RTFQ-PCR)to verify the relationship between miRNA markers in liquid biopsy and clinical pathological parameters.The diagnostic value of miRNA markers was also analyzed and compared.Results:We screened10miRNAs differential expression in urine.Combined with previous literature,we selected20miRNAs to verify their expression levels in bladder cancers and healthy controls.miR-509-5p/miR-124ratio in the urine was found higher in patients with bladder cancer than in healthy controls(P<0.0001).With the rise of miR-509-5p/miR-124ratio in urine,tumor stage and grade were also increased(P=0.003).When the cutoff was set at0.41,the diagnostic sensitivity and specificity of miR-509-5p/miR-124ratio were73.1%and82.6%,respectively.The AUC of miR-509-5p/miR-124ratio to detect bladder cancer was0.864,higher than that of urinary exfoliated cells(P=0.0002).Conclusion:We optimized the extraction of miRNAs in urine,established a standardized liquid biopsy of miRNA markers.The miR-509-5p/miR-124ratio could be an ideal diagnostic marker for bladder cancer.