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Combination of exosomes and circulating microRNAs may serve as a promising tumor marker complementary to alpha-fetoprotein for early-stage hepatocellular carcinoma diagnosis in rats



Due to unsatisfying prognosis of AFP for hepatocellular carcinoma (HCC), we aim to evaluate the prognostic value of combination of exosomes and miRNAs in detecting HCC.


HCC was induced with diethylnitrosamine in rats and using a scoring system based on histological examination six different stages (normal liver, degeneration, fibrosis, cirrhosis, early HCC and late HCC) were identified in the development of HCC. The expression levels of AFP, exosomes and miRNAs (miRNA-10b, miRNA-21, miRNA-122 and miRNA-200a) were detected in both tissue and blood samples from those six stages. Receiver operating characteristic (ROC) curve analysis was conducted to evaluate the power of each parameter and their different combinations in diagnosing HCC or cirrhosis.


A change in the expression of both exosomes and miRNAs was observed during cirrhosis, which in contrast with AFP starts showing up until the early HCC stage. Interestingly, the expressions of exosomes and the selected four miRNAs at early HCC stage obtained more remarkably alterations than the level of AFP (P < 0.05). On correlation analysis, four selected miRNAs had a significant closer relationship with exosomes when compared with AFP. The different combinations of AFP, exosomes, serous miRNAs and exosomal miRNAs had stronger power in predicting HCC than AFP (area under the curve of ROC, 0.943 vs 0.826).


To conclude, the combination of circulating miRNAs and exosomes might serve as promising biomarkers for non-virus infected HCC screening and cirrhosis discrimination.

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Hepatocellular carcinoma






Hepatic cancer stem cells


Hepatic normal stem cells




Histologic activity index


Receiver operating characteristic curve


Area under ROC


Epithelial-to-mesenchymal transition


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National Natural Science Foundation of China (No. 81302168).

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The authors indicate no potential conflicts of interest.

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Correspondence to Wei-hui Liu or Li-jun Tang.

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Wei-hui Liu, Li-na Ren, Xing Wang, Tao Wang, Ning Zhang, Yuan Gao and Hao Luo have contributed equally to this work.

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Supplementary figure 1. The expression of miRNAs in tissue samples at different time points after DEN treatment. By in situ hybridization, it was found that the expression of two miRNAs (miRNA-10b and miRNA-21) was gradually increased from 2 weeks to 22 weeks after DEN treatment, while other two tested miRNAs (miRNA-122 and miRNA-200a) were less and less expressed as HCC progressing after chemical induction. Original magnification, 100 ×


Supplementary figure 2. The relative profiles of miRNAs in blood samples at different time points during HCC development. (A) Normalized by the internal reference miRNA-484, relative expression levels of four miRNAs in sera were consecutively either upregulated or downregulated as time went by. (B) The relative folds of miRNA expression in isolated exosomes were reflected in a turning curve chart. DT = during treatment of DEN; AT = after treatment of DEN


Supplementary figure 3. Correlation analysis of serous miRNAs with exosomal miRNAs, and serous miRNAs with miRNAs in liver tissues. (A) The expression of exosomal miRNAs was closely related to that of serous miRNAs. (B) However, the relative expression folds of miRNAs in tissues were moderately correlated with those of the corresponding serous miRNAs

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Liu, Wh., Ren, Ln., Wang, X. et al. Combination of exosomes and circulating microRNAs may serve as a promising tumor marker complementary to alpha-fetoprotein for early-stage hepatocellular carcinoma diagnosis in rats. J Cancer Res Clin Oncol 141, 1767–1778 (2015).

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  • Hepatocellular carcinoma (HCC)
  • Alpha-fetoprotein (AFP)
  • Circulating miRNAs
  • Exosomes
  • Molecular diagnosis