食管鳞状细胞癌患者血清全基因组microRNA测序和分析

Sequencing and identification of microRNAs from a genome-wide expression profile in serum of esophageal squamous cell carcinoma

目的通过测序、分析食管鳞状细胞癌(ESCC)患者血清全基因组微小核糖核酸(miRNA)表达谱,筛选出表达上调的miRNA,探讨其作为ESCC分子标志物的可能性。方法收集未经治疗的199例ESCC患者血清,同时以107例年龄、性别匹配的健康人血清作对照。用Solexa测序技术对血清miRNA进行测序和表达量测定,用实时荧光定量PCR技术对表达上调的miR-223验证,并与血清CEA含量比较。结果 Solexa测序结果显示,ESCC患者和健康人混合血清小分子RNA中miRNA含量所占比例分别是75.46%和81.44%,22种miRNA在患者血清中表达量是健康人对照的2倍以上;PCR分析证实,miR-223在患者血清中的浓度[M(P25,P75)]显著高于健康人对照[893.8(684.8,1 200.7)fmol/L vs 445.6(347.7,664.0)fmol/L,t=10.03,P<0.01];用于ESCC诊断的miR-223 ROC曲线下面积(AUCROC)为0.84(95%CI,0.79～0.89),明显大于CEA的0.55(95%CI,0.48～0.62),P<0.01;当cut off值为710.2 fmol/L时,miR-223诊断ESCC的敏感性和特异性分别为70%、80%;ESCC患者年龄、性别、病理类型、TNM分期、吸烟史、饮酒史与血清miR-223水平无明显相关性(P>0.05)。结论 ESCC患者血清miRNA表达谱与健康人存在差异,miR-223在患者血清中浓度显著升高,是ESCC潜在的分子标志物。

Objective To sequence and investigate microRNAs from a genome-wide serum microRNA（miRNA） expression profile to identify differentially expressed miRNAs in esophageal squamous cell carcinoma（ESCC） and explore the potential as a novel molecular biomarkers for ESCC.Methods Serum samples were taken from 199 ESCC patients and 107 age-and gender-matched healthy controls.The serum miRNAs were screened and sequenced by Solexa sequencing technology,and the expression levels of miRNAs were quantified.The upregulated miR-223 was confirmed by real-time quantitative PCR（RT-qPCR） assay.Results The Solexa data showed that the proportions of miRNA in the small RNAs of ESCC patients and healthy controls were 75.46% and 81.44% respectively,and 22 miRNAs were upregulated in ESCC more than double the controls.RT-qPCR confirmed that the concentration of miR-223 was significantly elevated in the serum from ESCC patients as compared with the controls[893.8（684.8,1 200.7） fmol/L vs 445.6（347.7,664.0） fmol/L,P0.01].The area under ROC curve（AUC） of miR-223 for diagnosis of ESCC was 0.84（95% CI 0.79 to 0.89）,which was significantly greater than that of carcinoembryonic antigen（0.55 for AUC,95%CI 0.48 to 0.62,P0.01）.When the cut-off value was defined as 710.2 fmol/L,sensitivity and specificity of miR-223 were high up to 70% and 80% for ESCC prediction.Conclusion The expression profile of serum miRNAs of ESCC patients was distinctive from healthy individuals and miR-223 concentration was significantly elevated in ESCC serum,which hold potential as novel noninvasive biomarker for diagnosis of ESCC.