Skip to main content

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

Abstract

Purpose

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.

Methods

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.

Results

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).

Conclusion

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

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Abbreviations

HCC:

Hepatocellular carcinoma

AFP:

Alpha-fetoprotein

miRNA:

microRNA

HCSCs:

Hepatic cancer stem cells

HNSCs:

Hepatic normal stem cells

DEN:

Diethylnitrosamine

HAI:

Histologic activity index

ROC:

Receiver operating characteristic curve

AUC:

Area under ROC

EMT:

Epithelial-to-mesenchymal transition

References

  • Bao L, Yan Y, Xu C, Ji W, Shen S, Xu G, Zeng Y, Sun B, Qian H, Chen L, Wu M, Su C, Chen J (2013) MicroRNA-21 suppresses PTEN and hSulf-1 expression and promotes hepatocellular carcinoma progression through AKT/ERK pathways. Cancer Lett 337:226–236

    CAS  Article  PubMed  Google Scholar 

  • Clevers H (2011) The cancer stem cell: premises, promises and challenges. Nat Med 17:313–319

    CAS  Article  PubMed  Google Scholar 

  • El-Serag HB (2011) Hepatocellular carcinoma. N Engl J Med 365:1118–1127

    CAS  Article  PubMed  Google Scholar 

  • Filipe V, Hawe A, Jiskoot W (2010) Critical evaluation of Nanoparticle Tracking Analysis (NTA) by NanoSight for the measurement of nanoparticles and protein aggregates. Pharm Res 27:796–810

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  • Forner A, Llovet JM, Bruix J (2012) Hepatocellular carcinoma. Lancet 379:1245–1255

    Article  PubMed  Google Scholar 

  • Giordano S, Columbano A (2013) MicroRNAs: New tools for diagnosis, prognosis, and therapy in hepatocellular carcinoma? Hepatology 57:840–847

    CAS  Article  PubMed  Google Scholar 

  • Gramantieri L, Fornari F, Callegari E, Sabbioni S, Lanza G, Croce CM, Bolondi L, Negrini M (2008) MicroRNA involvement in hepatocellular carcinoma. J Cell Mol Med 12:2189–2204

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  • Huang YS, Dai Y, Yu XF, Bao SY, Yin YB, Tang M, Hu CX (2008) Microarray analysis of microRNA expression in hepatocellular carcinoma and non-tumorous tissues without viral hepatitis. J Gastroenterol Hepatol 23:87–94

    Article  PubMed  Google Scholar 

  • Hubscher SG (1998) Histological grading and staging in chronic hepatitis: clinical applications and problems. J Hepatol 29:1015–1022

    CAS  Article  PubMed  Google Scholar 

  • Hung CS, Liu HH, Liu JJ, Yeh CT, Chang TC, Wu CH, Ho YS, Wei PL, Chang YJ (2013) MicroRNA-200a and -200b mediated hepatocellular carcinoma cell migration through the epithelial to mesenchymal transition markers. Ann Surg Oncol 20(Suppl 3):S360–S368

    Article  PubMed  Google Scholar 

  • Kaneda K, Uenishi T, Takemura S, Yamamoto T, Sakata C, Sakae M, Urata Y, Ohata K, Tanaka S, Wakasa K, Kubo S (2013) Histologically proven hepatocellular carcinoma associated with burned-out nonalcoholic steatohepatitis. Osaka City Med J 59:99–104

    PubMed  Google Scholar 

  • Kanwar SS, Dunlay CJ, Simeone DM, Nagrath S (2014) Microfluidic device (ExoChip) for on-chip isolation, quantification and characterization of circulating exosomes. Lab Chip 14:1891–1900

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  • Kitai S, Kudo M, Minami Y, Haji S, Osaki Y, Oka H, Seki T, Kasugai H, Sasaki Y, Matsunaga T (2008) Validation of a new prognostic staging system for hepatocellular carcinoma: a comparison of the biomarker-combined Japan Integrated Staging Score, the conventional Japan Integrated Staging Score and the BALAD Score. Oncology 75(Suppl 1):83–90

    Article  PubMed  Google Scholar 

  • Ladeiro Y, Couchy G, Balabaud C, Bioulac-Sage P, Pelletier L, Rebouissou S, Zucman-Rossi J (2008) MicroRNA profiling in hepatocellular tumors is associated with clinical features and oncogene/tumor suppressor gene mutations. Hepatology 47:1955–1963

    CAS  Article  PubMed  Google Scholar 

  • Lan SH, Wu SY, Zuchini R, Lin XZ, Su IJ, Tsai TF, Lin YJ, Wu CT, Liu HS (2014) Autophagy suppresses tumorigenesis of hepatitis B virus-associated hepatocellular carcinoma through degradation of microRNA-224. Hepatology 59:505–517

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  • Leung TW, Tang AM, Zee B, Lau WY, Lai PB, Leung KL, Lau JT, Yu SC, Johnson PJ (2002) Construction of the Chinese University Prognostic Index for hepatocellular carcinoma and comparison with the TNM staging system, the Okuda staging system, and the Cancer of the Liver Italian Program staging system: a study based on 926 patients. Cancer 94:1760–1769

    Article  PubMed  Google Scholar 

  • Li QJ, Zhou L, Yang F, Wang GX, Zheng H, Wang DS, He Y, Dou KF (2012) MicroRNA-10b promotes migration and invasion through CADM1 in human hepatocellular carcinoma cells. Tumour Biol J Int Soc Oncodevelopmental Biol Med 33:1455–1465

    CAS  Article  Google Scholar 

  • Liang HH, Wei PL, Hung CS, Wu CT, Wang W, Huang MT, Chang YJ (2013) MicroRNA-200a/b influenced the therapeutic effects of curcumin in hepatocellular carcinoma (HCC) cells. Tumour Biol J Int Soc Oncodevelopmental Biol Med 34:3209–3218

    CAS  Article  Google Scholar 

  • Liu C, Yu J, Yu S, Lavker RM, Cai L, Liu W, Yang K, He X, Chen S (2010) MicroRNA-21 acts as an oncomir through multiple targets in human hepatocellular carcinoma. J Hepatol 53:98–107

    CAS  Article  PubMed  Google Scholar 

  • Liu WH, Tao KS, You N, Liu ZC, Zhang HT, Dou KF (2011) Differences in the properties and mirna expression profiles between side populations from hepatic cancer cells and normal liver cells. PLoS One 6:e23311

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  • Liu WH, You N, Zhang N, Yan HT, Wang T, Huang Z, Liu HB, Tang LJ (2012) Interpretation of interlocking key issues of cancer stem cells in malignant solid tumors. Cell Oncol 35:397–409

    Article  Google Scholar 

  • Llovet JM, Bru C, Bruix J (1999) Prognosis of hepatocellular carcinoma: the BCLC staging classification. Semin Liver Dis 19:329–338

    CAS  Article  PubMed  Google Scholar 

  • Magistri P, Leonard SY, Tang CM, Chan JC, Lee TE, Sicklick JK (2014) The glypican 3 hepatocellular carcinoma marker regulates human hepatic stellate cells via Hedgehog signaling. J Surg Res 187:377–385

    CAS  Article  PubMed  Google Scholar 

  • Murakami Y, Yasuda T, Saigo K, Urashima T, Toyoda H, Okanoue T, Shimotohno K (2006) Comprehensive analysis of microRNA expression patterns in hepatocellular carcinoma and non-tumorous tissues. Oncogene 25:2537–2545

    CAS  Article  PubMed  Google Scholar 

  • Petrelli A, Perra A, Schernhuber K, Cargnelutti M, Salvi A, Migliore C, Ghiso E, Benetti A, Barlati S, Ledda-Columbano GM, Portolani N, De Petro G, Columbano A, Giordano S (2012) Sequential analysis of multistage hepatocarcinogenesis reveals that miR-100 and PLK1 dysregulation is an early event maintained along tumor progression. Oncogene 31:4517–4526

  • Petrelli A, Perra A, Cora D, Sulas P, Menegon S, Manca C, Migliore C, Kowalik MA, Ledda-Columbano GM, Giordano S, Columbano A (2014) MicroRNA/gene profiling unveils early molecular changes and nuclear factor erythroid related factor 2 (NRF2) activation in a rat model recapitulating human hepatocellular carcinoma (HCC). Hepatology 59:228–241

    CAS  Article  PubMed  Google Scholar 

  • Png KJ, Halberg N, Yoshida M, Tavazoie SF (2012) A microRNA regulon that mediates endothelial recruitment and metastasis by cancer cells. Nature 481:190–194

    CAS  Article  Google Scholar 

  • Ray K (2013) Liver cancer: The promise of new approaches in the management of hepatocellular carcinoma—adding to the toolbox? Nat Rev Gastroenterol Hepatol 10:195

    Article  PubMed  Google Scholar 

  • Rekker K, Saare M, Roost AM, Kubo AL, Zarovni N, Chiesi A, Salumets A, Peters M (2014) Comparison of serum exosome isolation methods for microRNA profiling. Clin Biochem 47:135–138

    CAS  Article  PubMed  Google Scholar 

  • Salnikov AV, Kusumawidjaja G, Rausch V, Bruns H, Gross W, Khamidjanov A, Ryschich E, Gebhard MM, Moldenhauer G, Buchler MW, Schemmer P, Herr I (2009) Cancer stem cell marker expression in hepatocellular carcinoma and liver metastases is not sufficient as single prognostic parameter. Cancer Lett 275:185–193

    CAS  Article  PubMed  Google Scholar 

  • Szabo G, Bala S (2013) MicroRNAs in liver disease. Nat Rev Gastroenterol Hepatol 10:542–552

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  • Tomimaru Y, Eguchi H, Nagano H, Wada H, Kobayashi S, Marubashi S, Tanemura M, Tomokuni A, Takemasa I, Umeshita K, Kanto T, Doki Y, Mori M (2012) Circulating microRNA-21 as a novel biomarker for hepatocellular carcinoma. J Hepatol 56:167–175

    CAS  Article  PubMed  Google Scholar 

  • Wang T, You N, Tao K, Wang X, Zhao G, Xia N, Li N, Tang L, Liu W, Dou K (2012) Notch is the key factor in the process of fetal liver stem/progenitor cells differentiation into hepatocytes. Dev Growth Differ 54:605–617

    CAS  Article  PubMed  Google Scholar 

  • Wang T, Chen T, Liang HY, Yan HT, Lin N, Liu LY, Luo H, Huang Z, Li NL, Liu WH, Tang LJ (2014) Notch inhibition promotes fetal liver stem/progenitor cells differentiation into hepatocytes via the inhibition of HNF-1beta. Cell Tissue Res 357:173–184

    CAS  Article  PubMed  Google Scholar 

  • Wei L, Lian B, Zhang Y, Li W, Gu J, He X, Xie L (2014) Application of microRNA and mRNA expression profiling on prognostic biomarker discovery for hepatocellular carcinoma. BMC Genom 15(Suppl 1):S13

    Article  Google Scholar 

  • Wicha MS, Liu S, Dontu G (2006) Cancer stem cells: an old idea—a paradigm shift. Cancer Res 66:1883–1890; discussion 1895–1886

  • Witwer KW (2015) Circulating MicroRNA biomarker studies: pitfalls and potential solutions. Clin Chem 61:56–63

    CAS  Article  PubMed  Google Scholar 

  • Xu T, Zhu Y, Wei QK, Yuan Y, Zhou F, Ge YY, Yang JR, Su H, Zhuang SM (2008) A functional polymorphism in the miR-146a gene is associated with the risk for hepatocellular carcinoma. Carcinogenesis 29:2126–2131

    CAS  Article  PubMed  Google Scholar 

  • Xu G, Zhang Y, Wei J, Jia W, Ge Z, Zhang Z, Liu X (2013) MicroRNA-21 promotes hepatocellular carcinoma HepG2 cell proliferation through repression of mitogen-activated protein kinase-kinase 3. BMC Cancer 13:469

    PubMed Central  Article  PubMed  Google Scholar 

  • Yeh TS, Wang F, Chen TC, Yeh CN, Yu MC, Jan YY, Chen MF (2014) Expression profile of microRNA-200 family in hepatocellular carcinoma with bile duct tumor thrombus. Ann Surg 259:346–354

    Article  PubMed  Google Scholar 

  • Yuan JH, Yang F, Chen BF, Lu Z, Huo XS, Zhou WP, Wang F, Sun SH (2011) The histone deacetylase 4/SP1/microrna-200a regulatory network contributes to aberrant histone acetylation in hepatocellular carcinoma. Hepatology 54:2025–2035

    CAS  Article  PubMed  Google Scholar 

Download references

Acknowledgments

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

Conflict of interest

The authors indicate no potential conflicts of interest.

Author information

Authors and Affiliations

Authors

Corresponding authors

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

Additional information

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

Electronic supplementary material

Below is the link to the electronic supplementary material.

432_2015_1943_MOESM1_ESM.tif

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 ×

432_2015_1943_MOESM2_ESM.tif

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

432_2015_1943_MOESM3_ESM.tif

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

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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). https://doi.org/10.1007/s00432-015-1943-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00432-015-1943-0

Keywords

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