Abstract:

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-qPCR）. 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 exo-circRNA expression in liver cancer cell line Hep3B 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.

Key words: Circular RNA, circ_0000690, circ_0001359, circ_0000396, circ_0000722, Exosome, Alpha-fetoprotein, Hepatocellular carcinoma