摘要
目的寻找在肝细胞肝癌(HCC)中差异表达的环状RNA(circRNA)并探讨其对HCC的诊断价值。方法选取行根治性切除术的HCC患者256例(HCC组),其中发现集25例、训练集126例、验证集105例;乙型肝炎相关肝硬化患者100例(肝硬化组),其中训练集50例、验证集50例;健康体检者125名(正常对照组),其中发现集25名、训练集50名、验证集50名。采用沉淀法分离血浆外泌体,采用实时定量聚合酶链反应(RT-qPCR)检测外泌体circRNA的表达。采用Logistic回归分析构建HCC诊断模型,并采用受试者工作特征(ROC)曲线分析模型的诊断效能。结果与正常肝细胞系L02比较,肝癌细胞系Hep3B有102个exo-circRNA表达升高(P<0.05)。与exoRBase数据库比对后,鉴定出4个HCC患者表达量升高的外泌体circRNA(circ-0000690、circ_0001359、circ_0000396和circ_0000722)。肝癌组circ_0000690、circ_0001359和circ_0000396表达量显著高于正常对照组(P<0.05),circ0000722表达量2个组之间差异无统计学意义(P>0.05)。无论是训练集还是验证集,肝癌组circ_0000690表达量均高于肝硬化组和正常对照组(P<0.001);正常对照组、肝硬化组及肝癌组circ_0001359表达量依次升高(P<0.05);肝癌组与肝硬化组之间circ_0000396表达量差异无统计学意义(P>0.05),但均高于正常对照组(P<0.05)。建立的联合检测模型为:Logit(P)=1.110×circ_0000690+0.822×circ_0001359+0.622×circ_0000396-4.153。ROC曲线分析结果显示,在训练集中,circ_0000690、circ_0001359、circ_0000396及联合检测模型诊断HCC的曲线下面积(AUC)分别为0.802、0.726、0.621、0.859,诊断早期HCC(巴塞罗那分期0+A期)的AUC分别为0.767、0.698、0.611、0.847,诊断甲胎蛋白(AFP)阴性(<20 ng/mL)HCC的AUC分别为0.810、0.695、0.588、0.894;在验证集中,诊断HCC的AUC分别为0.752、0.663、0.615、0.847,诊断早期HCC的AUC分别为0.763、0.673、0.591、0.845,诊断AFP阴性HCC的AUC分别为0.702、0.670、0.641、0.840。结论基于3种circ RNA(circ_0000690、circ_0001359和circ_0000396)构建的联合检测模型对HCC具有较优异的诊断效能,亦适用于早期HCC和AFP阴性HCC的鉴别诊断。
Objective To investigate plasma exosome-derived circular RNA(exo-circRNA)with diagnostic potential for hepatocellular carcinoma(HCC).Methods A total of 256 patients with HCC who received curative resection(25 cases belonged to discovery cohort,126 cases belonged to training cohort and 105 cases belonged to validation cohort),100 patients with liver cirrhosis(50 cases belonged to training cohort and 50 cases belonged to validation cohort)and 125 healthy subjects(healthy control group,25 individuals belonged to discovery cohort,50 individuals belonged to training cohort and 50 individuals belonged to validation cohort)were enrolled.The plasma exosome was isolated by precipitation,and the expression level of exo-circRNA was determined by real-time quantitative polymerase chain reaction(RT-q PCR).The new exo-circRNA model for HCC diagnosis was constructed using Logistic regression analysis,and the diagnostic performance of this exo-circRNA model was evaluated by receiver operating characteristic(ROC)curve.Results Compared with normal liver cell line L02,102 exocircRNA expression in liver cancer cell line Hep3 B were increased(P<0.05).After conducting integrative analysis with exoRBase database,4 circRNA with increased expression in HCC patients were identified(circ_0000690,circ_0001359,circ_0000396 and circ_0000722).The expressions of circ_0000690,circ_0001359 and circ_0000396 were increased in HCC group compared with those in healthy control group(P<0.05).There was no statistical significance in the expression of circ_0000722 between the 2 groups(P>0.05).The expression of circ_0000690 was higher in HCC group compared to those in liver cirrhosis group and healthy control group in both training and validation cohorts(P<0.001).The expression of circ_0001359 showed escalating pattern in healthy control group,liver cirrhosis group and HCC group(P<0.05).There was no statistical significance in the expression of circ_0000396 between HCC group and liver cirrhosis group(P>0.05),but they were still higher than those in healthy control group(P<0.05).The established diagnostic model was Logit(P)=1.110×circ_0000690+0.822×circ_0001359+0.622×circ_0000396-4.153.ROC curve analysis results showed that,in the training cohort,the areas under curves(AUC)for diagnosing HCC of circ_0000690,circ_0001359,circ_0000396 and integrated model were 0.802,0.726,0.621 and 0.859,respectively.Meanwhile,the AUC for diagnosing early HCC(defined as Barcelona stage 0+A)of circ_0000690,circ_0001359,circ_0000396 and integrated model were 0.767,0.698,0.611 and 0.847,respectively.The AUC for diagnosing alpha-fetoprotein(AFP)-negative(<20 ng/mL)HCC were 0.810,0.695,0.588 and 0.894,respectively.In the validation cohort,the AUC for diagnosing HCC of circ_0000690,circ_0001359,circ_0000396 and integrated model were 0.752,0.663,0.615 and 0.847,respectively.The AUC for diagnosing early HCC of circ_0000690,circ_0001359,circ_0000396 and integrated model were 0.763,0.673,0.591 and 0.845,respectively.Moreover,the AUC for diagnosing AFP-negative HCC of circ_0000690,circ_0001359,circ_0000396 and integrated model were 0.702,0.670,0.641 and 0.840,respectively.Conclusions The plasma exo-circRNA model established in this study has potential diagnostic value for HCC,especially AFP-negative and early HCC.
作者
孟俊
王俊青
费晓春
顾志冬
MENG Jun;WANG Junqing;FEI Xiaochun;GU Zhidong(Department of Clinical Laboratory,Ruijin Hospital,Shanghai Jiao Tong University School of Medicine,Shanghai 200025,China;Department of General Surgery,Ruijin Hospital,Shanghai Jiao Tong University School of Medicine,Shanghai 200025,China;Department of Pathology,Ruijin Hospital,Shanghai Jiao Tong University School of Medicine,Shanghai 200025,China)
出处
《检验医学》
CAS
2022年第1期1-10,共10页
Laboratory Medicine
基金
上海市科学技术委员会资助项目(18441905400)。
