Journal of Molecular Medicine

, Volume 96, Issue 1, pp 85–96 | Cite as

Plasma circular RNA profiling of patients with gastric cancer and their droplet digital RT-PCR detection

  • Tianwen Li
  • Yongfu Shao
  • Liyun Fu
  • Yi Xie
  • Linwen Zhu
  • Weiliang Sun
  • Rui Yu
  • Bingxiu Xiao
  • Junming GuoEmail author
Original Article


To observe the diagnostic values of circular RNAs (circRNAs), their expression profiles between gastric cancer patients’ plasma and healthy controls were first assessed by circRNA microarray. Then, circRNA levels were measured by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and RT-droplet digital PCR (RT-ddPCR), respectively. A total of 343 differentially expressed circRNAs were found. The top 10 elevated circRNAs in patients were hsa_circ_0088300, hsa_circ_0075825, hsa_circ_0019172, hsa_circ_0000220, hsa_circ_0035277, hsa_circ_0000301, hsa_circ_0000189, hsa_circ_0090080, hsa_circ_0001888, and hsa_circ_0001874. The top 10 reduced circRNAs were hsa_circ_0004771, hsa_circ_0001190, hsa_circ_0061276, hsa_circ_0092337, hsa_circ_0058495, hsa_circ_0061274, hsa_circ_0075829, hsa_circ_0080845, hsa_circ_0001006, and hsa_circ_0003764. In cancer and dysplasia tissues, hsa_circ_0001017 and hsa_circ_0061276 were downregulated. Their levels were significantly associated with distal metastasis. The area under receiver operating characteristic curve in combinative use was 0.966 with 95.5% sensitivity and 95.7% specificity. Patients with low plasma hsa_circ_0001017 or hsa_circ_0061276 had a much shorter overall survival than those with high levels. Patients whose plasma hsa_circ_0001017 or hsa_circ_0061276 levels recovered to normal after operation had a longer disease-free survival. Finally, the in vitro model indicated gastric cancer cells secreting circRNAs into plasma. In conclusion, RT-ddPCR is a potent non-invasive and absolute quantification method for simultaneous detection of multiple circRNAs. Hsa_circ_0001017 and hsa_circ_0061276 are new potential biomarkers for gastric cancer.

Key message

  • A total of 343 circRNAs are differentially expressed between gastric cancer patients’ plasma and healthy controls.

  • Hsa_circ_0001017 and hsa_circ_0061276 are downregulated in gastric cancer tissues.

  • The RT-ddPCR is a potent method for simultaneous detection of multiple circRNAs in plasma.

  • Hsa_circ_0001017 and hsa_circ_0061276 are potential biomarkers for gastric cancer.


Biomarker  Gastric cancer  Non-invasive testing Digital polymerase chain reaction 



This study was supported by grants from the Applied Research Project on Nonprofit Technology of Zhejiang Province (no. 2016C33177), the Scientific Innovation Team Project of Ningbo (no. 2017C110019), National Natural Science Foundation of China (no. 81772279), and the K.C. Wong Magna Fund in Ningbo University.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.

Supplementary material

109_2017_1600_MOESM1_ESM.pdf (292 kb)
ESM 1 (PDF 291 kb)


  1. 1.
    Guo J, Miao Y, Xiao B, Huan R, Jiang Z, Meng D, Wang Y (2009) Differential expression of microRNA species in human gastric cancer versus non-tumorous tissues. J Gastroenterol Hepatol 24:652–657CrossRefPubMedGoogle Scholar
  2. 2.
    Li T, Mo X, Fu L, Xiao B, Guo J (2016) Molecular mechanisms of long noncoding RNAs on gastric cancer. Oncotarget 7:8601–8612PubMedPubMedCentralGoogle Scholar
  3. 3.
    Allemani C, Weir HK, Carreira H, Harewood R, Spika D, Wang XS, Bannon F, Ahn JV, Johnson CJ, Bonaventure A et al (2015) Global surveillance of cancer survival 1995-2009: analysis of individual data for 25, 676, 887 patients from 279 population-based registries in 67 countries (CONCORD-2). Lancet 385:977–1010CrossRefPubMedGoogle Scholar
  4. 4.
    Hashad D, Elbanna A, Ibrahim A, Khedr G (2016) Evaluation of the role of circulating long non-coding RNA H19 as a promising novel biomarker in plasma of patients with gastric cancer. J Clin Lab Anal 30:1100–1105CrossRefPubMedGoogle Scholar
  5. 5.
    Li Q, Shao Y, Zhang X, Zheng T, Miao M, Wang B, Ye G, Xiao B, Guo J (2015) Plasma long noncoding RNA protected by exosomes as a potential stable biomarker for gastric cancer. Tumour Biol 36:2007–2012CrossRefPubMedGoogle Scholar
  6. 6.
    Cui L, Lou Y, Zhang X, Zhou H, Deng H, Song H, Yu X, Xiao B, Wang W, Guo J (2011) Detection of circulating tumor cells in peripheral blood from patients with gastric cancer using piRNAs as markers. Clin Biochem 44:1050–1057CrossRefPubMedGoogle Scholar
  7. 7.
    Zhou H, Xiao B, Zhou F, Deng H, Zhang X, Lou Y, Gong Z, Du C, Guo J (2012) MiR-421 is a functional marker of circulating tumor cells in gastric cancer patients. Biomarkers 17:104–110CrossRefPubMedGoogle Scholar
  8. 8.
    Schneider J, Schulze G (2003) Comparison of tumor M2-pyruvate kinase (tumor M2-PK), carcinoembryonic antigen (CEA), carbohydrate antigens CA 19-9 and CA 72-4 in the diagnosis of gastrointestinal cancer. Anticancer Res 23:5089–5093PubMedGoogle Scholar
  9. 9.
    Carpelan-Holmstrom M, Louhimo J, Stenman UH, Alfthan H, Haglund C (2002) CEA, CA 19-9 and CA 72-4 improve the diagnostic accuracy in gastrointestinal cancers. Anticancer Res 22:2311–2316PubMedGoogle Scholar
  10. 10.
    Sykes PJ, Neoh SH, Brisco MJ, Hughes E, Condon J, Morley AA (1992) Quantitation of targets for PCR by use of limiting dilution. BioTechniques 13:444–449PubMedGoogle Scholar
  11. 11.
    Riva F, Bidard FC, Houy A, Saliou A, Madic J, Rampanou A, Hego C, Milder M, Cottu P, Sablin MP et al (2017) Patient-specific circulating tumor DNA detection during neoadjuvant chemotherapy in triple-negative breast cancer. Clin Chem 6:691–699CrossRefGoogle Scholar
  12. 12.
    Guttery DS, Page K, Hills A, Woodley L, Marchese SD, Rghebi B, Hastings RK, Luo J, Pringle JH, Stebbing J et al (2015) Noninvasive detection of activating estrogen receptor 1 (ESR1) mutations in estrogen receptor-positive metastatic breast cancer. Clin Chem 61:974–982CrossRefPubMedGoogle Scholar
  13. 13.
    Shen F, Du W, Kreutz JE, Fok A, Ismagilov RF (2010) Digital PCR on a SlipChip. Lab Chip 10:2666–2672CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Shen F, Sun B, Kreutz JE, Davydova EK, Du W, Reddy PL, Joseph LJ, Ismagilov RF (2011) Multiplexed quantification of nucleic acids with large dynamic range using multivolume digital RT-PCR on a rotational SlipChip tested with HIV and hepatitis C viral load. J Am Chem Soc 133:17705–17712CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Yao T, Chen Q, Fu L, Guo J (2017) Circular RNAs: biogenesis, properties, roles, and their relationships with liver diseases. Hepatol Res 47:497–504CrossRefPubMedGoogle Scholar
  16. 16.
    Li Y, Zheng Q, Bao C, Li S, Guo W, Zhao J, Chen D, Gu J, He X, Huang S (2015) Circular RNA is enriched and stable in exosomes: a promising biomarker for cancer diagnosis. Cell Res 25:981–984CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Li P, Chen S, Chen H, Mo X, Li T, Shao Y, Xiao B, Guo J (2015) Using circular RNA as a novel type of biomarker in the screening of gastric cancer. Clin Chim Acta 444:132–136CrossRefPubMedGoogle Scholar
  18. 18.
    Li F, Zhang L, Li W, Deng J, Zheng J, An M, Lu J, Zhou Y (2015) Circular RNA ITCH has inhibitory effect on ESCC by suppressing the Wnt/beta-catenin pathway. Oncotarget 6:6001–6013PubMedPubMedCentralGoogle Scholar
  19. 19.
    Yang W, Du WW, Li X, Yee AJ, Yang BB (2016) Foxo3 activity promoted by non-coding effects of circular RNA and Foxo3 pseudogene in the inhibition of tumor growth and angiogenesis. Oncogene 35:3919–3931CrossRefPubMedGoogle Scholar
  20. 20.
    Shao Y, Ye M, Jiang X, Sun W, Ding X, Liu Z, Ye G, Zhang X, Xiao B, Guo J (2014) Gastric juice long noncoding RNA used as a tumor marker for screening gastric cancer. Cancer 120:3320–3328CrossRefPubMedGoogle Scholar
  21. 21.
    Li P, Chen H, Chen S, Mo X, Li T, Xiao B, Yu R, Guo J (2017) Circular RNA 0000096 affects cell growth and migration in gastric cancer. Br J Cancer 116:626–633CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Janku F, Huang HJ, Fujii T, Shelton DN, Madwani K, Fu S, Tsimberidou AM, Piha-Paul SA, Wheler JJ, Zinner RG et al (2017) Multiplex KRASG12/G13 mutation testing of unamplified cell-free DNA from the plasma of patients with advanced cancers using droplet digital polymerase chain reaction. Ann Oncol 28:642–650CrossRefPubMedGoogle Scholar
  23. 23.
    Yu D, Li HX, Liu Y, Ying ZW, Guo JJ, Cao CY, Wang J, Li YF, Yang HR (2017) The reference intervals for serum C-terminal agrin fragment in healthy individuals and as a biomarker for renal function in kidney transplant recipients. J Clin Lab Anal 31:e22059CrossRefGoogle Scholar
  24. 24.
    Klapkova E, Fortova M, Prusa R, Moravcova L, Kotaska K (2016) Determination of urine albumin by new simple high-performance liquid chromatography method. J Clin Lab Anal 30:1226–1231CrossRefPubMedGoogle Scholar
  25. 25.
    Ay E, Marakoğlu K, Kizmaz M, Ünlü A (2016) Evaluation of Netrin-1 levels and albuminuria in patients with diabetes. J Clin Lab Anal 30:972–977CrossRefPubMedGoogle Scholar
  26. 26.
    Capello M, Bantis LE, Scelo G, Zhao Y, Li P, Dhillon DS, Patel NJ, Kundnani DL, Wang H, Abbruzzese JL et al (2017) Sequential validation of blood-based protein biomarker candidates for early-stage pancreatic cancer. J Natl Cancer Inst 109.
  27. 27.
    Min QH, Chen XM, Zou YQ, Zhang J, Li J, Wang Y, Li SQ, Gao QF, Sun F, Liu J et al (2017) Differential expression of urinary exosomal microRNAs in IgA nephropathy. J Clin Lab Anal.
  28. 28.
    Raitoharju E, Seppala I, Lyytikainen LP, Viikari J, Ala-Korpela M, Soininen P, Kangas AJ, Waldenberger M, Klopp N, Illig T et al (2016) Blood hsa-miR-122-5p and hsa-miR-885-5p levels associate with fatty liver and related lipoprotein metabolism—the young Finns study. Sci Rep 6:38262CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Shang X, Li G, Liu H, Li T, Liu J, Zhao Q, Wang C (2016) Comprehensive circular RNA profiling reveals that hsa_circ_0005075, a new circular RNA biomarker, is involved in hepatocellular carcinoma development. Medicine (Baltimore) 95:e3811CrossRefGoogle Scholar
  30. 30.
    Song X, Zhang N, Han P, Moon BS, Lai RK, Wang K, Lu W (2016) Circular RNA profile in gliomas revealed by identification tool UROBORUS. Nucleic Acids Res 44:e87CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Zhao ZJ, Shen J (2017) Circular RNA participates in the carcinogenesis and the malignant behavior of cancer. RNA Biol 14:514–521CrossRefPubMedGoogle Scholar
  32. 32.
    Sillence KA, Roberts LA, Hollands HJ, Thompson HP, Kiernan M, Madgett TE, Welch CR, Avent ND (2015) Fetal sex and RHD genotyping with digital PCR demonstrates greater sensitivity than real-time PCR. Clin Chem 61:1399–1407CrossRefPubMedGoogle Scholar
  33. 33.
    Huggett JF, Cowen S, Foy CA (2015) Considerations for digital PCR as an accurate molecular diagnostic tool. Clin Chem 61:79–88CrossRefPubMedGoogle Scholar
  34. 34.
    Yang Y, Shao Y, Zhu M, Li Q, Yang F, Lu X, Xu C, Xiao B, Sun Y, Guo J (2016) Using gastric juice lncRNA-ABHD11-AS1 as a novel type of biomarker in the screening of gastric cancer. Tumour Biol 37:1183–1188CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany 2017

Authors and Affiliations

  1. 1.Department of Biochemistry and Molecular Biology, and Zhejiang Key Laboratory of Pathophysiology, Medical SchoolNingbo UniversityNingboChina
  2. 2.Department of Gastroenterology, the Affiliated Hospital of Medical School ofNingbo UniversityNingboChina
  3. 3.Department of Hepatology, Ningbo No. 2 Hospital and the Affiliated Hospital, Medical SchoolNingbo UniversityNingboChina
  4. 4.Ningbo Yinzhou People’s Hospital and the Affiliated Hospital, Medical SchoolNingbo UniversityNingboChina

Personalised recommendations