Skip to main content

Advertisement

SpringerLink
Log in

Applying Subtractive Hybridization Technique to Enrich and Amplify Tumor-Specific Transcripts of Esophageal Squamous Cell Carcinoma

  • Published:
Pathology & Oncology Research

Abstract

Subtractive hybridization (SH) as an efficient and powerful approach can be applied to isolate differentially expressed transcripts as well as detect of involved mRNAs in various cellular processes, particularly diseases and malignancies. This procedure leads to the enrichment of specific low copy transcripts of tumor cells. Having developed a new approach for SH to isolate tumor specific transcripts, we facilitated discovery of uniquely expressed genes in esophageal squamous cell carcinoma (ESCC). Total RNA was extracted from the fresh tumoral and their adjacent normal tissues, and purified using the Switch Mechanism At the 5ʹ end of Reverse Transcript (SMART) method. Following cDNA synthesis of normal mRNAs using magnetic beads, it was hybridized with tumor mRNAs. To enhance efficiency of subtraction, hybridization was repeated three rounds. Finally, amplification of subtracted tumor-specific transcripts was carried out using in vitro transcription. The subtracted tumoral mRNAs was analyzed quantitatively using real-time PCR for both tumor-specific and housekeeping genes. The subtracted mRNA was confirmed as tumor-specific mRNA pool using RT-PCR and quantitative real-time PCR assessment. The elevated level of tumor-specific transcripts such as MAGE-A4 and CD44 as well as declined copy number of housekeeping genes such as GAPDH, β actin and β2-microglobulin, were confirmed in subtracted tumoral mRNA. The presence of tumor genes was confirmed after the SH procedure. The designed SH method in combination with SMART technique can isolate and amplify high quality tumor-specific transcripts even from small amount of tumor tissues. Removal of common transcripts from the extracted tumoral mRNAs using SH, leads to the enrichment of tumor-specific transcripts. The isolated transcripts are of interest because of their probable roles in ESCC progression and development. In addition, these tumor-specific mRNAs can be applied for future vaccine cancer studies.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1

Similar content being viewed by others

References

  1. Moghbeli M, Moghbeli F, Forghanifard MM, Garayali A, Abbaszadegan MR (2013) Cancer stem cell markers in esophageal cancer. Am J Cancer Sci 2(1):37–50

    Google Scholar 

  2. Taleb S, Abbaszadegan MR, Moghbeli M, Roudbari NH, Forghanifard MM (2014) HES1 as an independent prognostic marker in esophageal squamous cell carcinoma. J Gastrointest Cancer 45(4):466–471

    Article  CAS  PubMed  Google Scholar 

  3. Gholamin M, Moaven O, Memar B, Farshchian M, Naseh H, Malekzadeh R, et al. (2009) Overexpression and interactions of interleukin-10, transforming growth factor, and vascular endothelial growth factor in esophageal squamous cell carcinoma. World J Surg 33(7):1439–1445

    Article  PubMed  Google Scholar 

  4. Gholamin M, Moaven O, Farshchian M, Mahmoudi M, Sankian M, Memar B, et al. (2010) Induction of cytotoxic T lymphocytes primed with tumor RNA-loaded dendritic cells in esophageal squamous cell carcinoma: preliminary step for DC vaccine design. BMC Cancer 10(1):261

    Article  PubMed  PubMed Central  Google Scholar 

  5. Kahkhaie KR, Moaven O, Abbaszadegan MR, Montazer M, Gholamin M (2014) Specific MUC1 splice variants are correlated with tumor progression in esophageal cancer. World J Surg 38(8):2052–2057

    Article  PubMed  Google Scholar 

  6. Napier KJ, Scheerer M, Misra S (2014) Esophageal cancer: A Review of epidemiology, pathogenesis, staging workup and treatment modalities. World J Gastrointest Oncol 6(5):112

    Article  PubMed  PubMed Central  Google Scholar 

  7. Kurtz DM, Gambhir SS (2014) Tracking cellular and immune therapies in cancer. Emerging Applications of Molecular Imaging to Oncology, 124:257-296

  8. Forghanifard MM, Gholamin M, Farshchian M, Moaven O, Memar B, Forghani MN, et al. (2011) Cancer-testis gene expression profiling in esophageal squamous cell carcinoma: identification of specific tumor marker and potential targets for immunotherapy. Cancer Biol Ther 12(3):191–197

    Article  CAS  PubMed  Google Scholar 

  9. Bringmann A, Held SAE, Heine A, Brossart P (2010) RNA vaccines in cancer treatment. BioMed Res Int 2010

  10. Moingeon P (2001) Cancer vaccines. Vaccine 19(11):1305–1326

    Article  CAS  PubMed  Google Scholar 

  11. Mitchell DA, Nair SK (2000) RNA-transfected dendritic cells in cancer immunotherapy. J Clin Investig 106(9):1065

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Forghanifard MM, Amani J, Gheybi E, Abbaszadegan MR (2012) In silico analysis of chimeric polytope of cancer/testis antigens for dendritic cell-based immune-gene therapy applications. Gene Ther Mol Biol 14:87–96

    Google Scholar 

  13. Silva JM, Videira M, Gaspar R, Préat V, Florindo HF (2013) Immune system targeting by biodegradable nanoparticles for cancer vaccines. J Control Release 168(2):179–199

    Article  CAS  PubMed  Google Scholar 

  14. Matera L (2010) The choice of the antigen in the dendritic cell-based vaccine therapy for prostate cancer. Cancer Treat Rev 36(2):131–141

    Article  CAS  PubMed  Google Scholar 

  15. Cronwright G, Le Blanc K, Gotherstrom C, Darcy P, Ehnman M, Brodin B (2005) Cancer/testis antigen expression in human mesenchymal stem cells: down-regulation of SSX impairs cell migration and matrix metalloproteinase 2 expression. Cancer Res 65(6):2207–2215

    Article  CAS  PubMed  Google Scholar 

  16. Gholamin M, Moaven O, Farshchian M, Rajabi-Mashhadi MT, Mahmoudi M, Sankian M, et al. (2010) Highly efficient transfection of dendritic cells derived from esophageal squamous cell carcinoma patient: optimization with green fluorescent protein and validation with tumor RNA as a tool for immuno-genetherapy. Iran J Biotechnol 8(2):121–126

    CAS  Google Scholar 

  17. Garg NK, Dwivedi P, Prabha P, Tyagi RK (2013) RNA pulsed dendritic cells: an approach for cancer immunotherapy. Vaccine 31(8):1141–1156

    Article  CAS  PubMed  Google Scholar 

  18. Sagerstrm CG, Sun BI, Sive HL (1997) Subtractive cloning: past, present, and future. Annu Rev Biochem 66(1):751–783

    Article  Google Scholar 

  19. Wu N, Matand K, Williams S (2009) A novel mRNA level subtraction method for quick identification of target-orientated uniquely expressed genes between peanut immature pod and leaf. Biological Procedures Online 12(1):44–55

    Article  PubMed  PubMed Central  Google Scholar 

  20. Cho T-J, Park S-S (1998) A simulation of subtractive hybridization. Nucleic Acids Res 26(6):1440–1448

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Lin T-Y, Ying, S-Y (2003) Subtractive hybridization for the identification of differentially expressed genes using uraci-DNA glycosylase and mung-bean nuclease. Generation of cDNA Libraries. Springer, p 239-251

  22. Wang Z, Jones, MGK (2003) cDNA generation on paramagnetic beads. Generation of cDNA Libraries. Springer, p 25-31

  23. Barooei R, Mahmoudian RA, Abbaszadegan MR, Mansouri A, Gholamin M (2015) Evaluation of thymic stromal lymphopoietin (TSLP) and its correlation with lymphatic metastasis in human gastric cancer. Med Oncol 32(8):1–8

    Article  CAS  Google Scholar 

  24. Moghbeli M, Forghanifard MM, Aarabi A, Mansourian A, Abbaszadegan MR (2014) Clinicopathological sex-related relevance of Musashi1 mRNA expression in esophageal squamous cell carcinoma patients. Pathol Oncol Res 20(2):427–433

    Article  CAS  PubMed  Google Scholar 

  25. Alsadat Mahmoudian R, Abbaszadegan MR, Mansouri A, Gholamin M (2015) Amplification of Tumor Transcripts from Limited Quantity of Esophageal Squamous Cell Carcinoma Tissue Samples. Am J Cancer Sci 4(1):54–62

    Google Scholar 

  26. Mansouri A, Foroughmand AM, Abbaszadegan MR, Memar B, Mahmoudian RA, Gholamin M (2015) Expression analysis of CD44 isoforms S and V3, in patients with esophageal squamous cell carcinoma. Iran J Basic Med Sci 18(4):380

    PubMed  PubMed Central  Google Scholar 

  27. Forghanifard MM, Gholamin M, Moaven O, Farshchian M, Ghahraman M, Aledavood A, et al. (2014) Neoantigen in esophageal squamous cell carcinoma for dendritic cell-based cancer vaccine development. Med Oncol 31(10):1–7

    Article  Google Scholar 

  28. Nair SK, Morse M, Boczkowski D, Cumming RI, Vasovic L, Gilboa E, et al. (2002) Induction of tumor-specific cytotoxic T lymphocytes in cancer patients by autologous tumor RNA-transfected dendritic cells. Ann Surg 235(4):540

    Article  PubMed  PubMed Central  Google Scholar 

  29. Hossein Naseh M, Bahram Memar M, Bagheri R, MontazerMD M, AbbaszadeganPhD MR (2013) A Cancer-array approach elucidates the immune escape mechanism and defects in the DNA repair system in esophageal squamous cell carcinoma. Arch Iran Med 16(8):463

    PubMed  Google Scholar 

  30. Amatschek S, Koenig U, Auer H, Steinlein P, Pacher M, Gruenfelder A, et al. (2004) Tissue-wide expression profiling using cDNA subtraction and microarrays to identify tumor-specific genes. Cancer Res 64(3):844–856

    Article  CAS  PubMed  Google Scholar 

  31. Even-Desrumeaux K, Baty D, Chames P (2011) State of the art in tumor antigen and biomarker discovery. Cancer 3(2):2554–2596

    Article  Google Scholar 

  32. Srinivasan R, Wolchok JD (2004) Tumor antigens for cancer immunotherapy: therapeutic potential of xenogeneic DNA vaccines. J Transl Med 2(1):12

    Article  PubMed  PubMed Central  Google Scholar 

  33. Heiser A, Dahm P, Yancey DR, Maurice MA, Boczkowski D, Nair SK, et al. (2000) Human dendritic cells transfected with RNA encoding prostate-specific antigen stimulate prostate-specific CTL responses in vitro. J Immunol 164(10):5508–5514

    Article  CAS  PubMed  Google Scholar 

  34. Müller MR, Grünebach F, Nencioni A, Brossart P (2003) Transfection of dendritic cells with RNA induces CD4-and CD8-mediated T cell immunity against breast carcinomas and reveals the immunodominance of presented T cell epitopes. J Immunol 170(12):5892–5896

    Article  PubMed  Google Scholar 

  35. Gilboa E, Vieweg J (2004) Cancer immunotherapy with mRNA transfected dendritic cells. Immunol Rev 199(1):251–263

    Article  CAS  PubMed  Google Scholar 

  36. Caspi RR (2008) Immunotherapy of autoimmunity and cancer: the penalty for success. Nat Rev Immunol 8(12):970–976

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Yang F, Yang X-F (2005) New concepts in tumor antigens: their significance in future immunotherapies for tumors. Cell Mol Immunol 2(5):331–341

    CAS  PubMed  Google Scholar 

  38. Ghafouri-Fard S, Modarressi M-H (2009) Cancer-testis antigens: potential targets for cancer immunotherapy. Arch Iran Med 12(4):395–404

    PubMed  Google Scholar 

  39. Amos SM, Duong CPM, Westwood JA, Ritchie DS, Junghans RP, Darcy PK, et al. (2011) Autoimmunity associated with immunotherapy of cancer. Blood 118(3):499–509

    Article  CAS  PubMed  Google Scholar 

  40. Kaufman HL, Wolchok JD (2006) Is tumor immunity the same thing as autoimmunity? Implications for cancer immunotherapy. J Clin Oncol 24(15):2230–2232

    Article  CAS  PubMed  Google Scholar 

  41. Blumberg B, Belmonte, JCI. (1999) Subtractive hybridization and construction of cDNA libraries. Molecular Embryology. Springer, p 555–574.

  42. Mishra RN, Ramesha A, Kaul T, Nair S, Sopory SK, Reddy MK (2005) A modified cDNA subtraction to identify differentially expressed genes from plants with universal application to other eukaryotes. Anal Biochem 345(1):149–157

    Article  CAS  PubMed  Google Scholar 

  43. D’Urso OF, D’Urso PI, Storelli C, Marsigliante S (2009) Solid phase subtractive cloning in differentially expressed genes identification. Mol Biol Rep 37(3):1435–1438

    Article  PubMed  Google Scholar 

  44. Byers RJ, Hoyland JA, Dixon J, Freemont AJ (2000) Subtractive hybridization genetic takeaways and the search for meaning. Int J Exp Pathol 81(6):391–404

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. D’Urso OF, D’Urso PI, Storelli C, Marsigliante S (2010) Solid phase subtractive cloning in differentially expressed genes identification. Mol Biol Rep 37(3):1435–1438

    Article  PubMed  Google Scholar 

  46. Smith CL, Bouchard J, Surdi G, Nguyen G, Pimpis K, Tolstoi LG (2000) Comparative genomics: differential display and subtractive hybridization. Encycl Anal Chem 6:4893–4901

    Google Scholar 

  47. Seth D, Gorrell MD, McGuinness PH, Leo MA, Lieber CS, McCaughan GW, et al. (2003) SMART amplification maintains representation of relative gene expression: quantitative validation by real time PCR and application to studies of alcoholic liver disease in primates. J Biochem Biophys Methods 55(1):53–66

    Article  CAS  PubMed  Google Scholar 

  48. Hsu F-M, Cheng JC-H, Chang Y-L, Lee J-M, Koong AC, Chuang EY (2015) Circulating mRNA Profiling in Esophageal Squamous Cell Carcinoma Identifies FAM84B As A Biomarker In Predicting Pathological Response to Neoadjuvant Chemoradiation. Sci Report 5

  49. Wang J, Zhang K-Y, Liu S-M, Sen S (2014) Tumor-associated circulating microRNAs as biomarkers of cancer. Molecules 19(2):1912–1938

    Article  PubMed  Google Scholar 

Download references

Acknowledgments

The authors gratefully acknowledge the colleagues at Division of Human Genetics and Immunology Department in Mashhad University of Medical Sciences for their technical assistance.

Author information

Authors and Affiliations

  1. Division of Human Genetics, Immunology Research Center, Avicenna Research Institute, Mashhad University of Medical Sciences, Bu-Ali Sq, Khorasan Razavi, Mashhad, 9196773117, Iran

    Reihaneh Alsadat Mahmoudian & Mehran Gholamin

  2. Medical Genetics Research Center, Medical School, Mashhad University of Medical Sciences, Mashhad, Iran

    Mohammad Reza Abbaszadegan

Authors
  1. Reihaneh Alsadat Mahmoudian
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Mohammad Reza Abbaszadegan
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Mehran Gholamin
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Mehran Gholamin.

Ethics declarations

Conflict of Interest

The authors declare that they have no conflict of interest.

Funding

This study was supported by a grant from Vice chancellor of Mashhad University of Medical Sciences (#911302).

Informed Consent

Informed consent was obtained from all individual participants included in the study.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Mahmoudian, R.A., Abbaszadegan, M.R. & Gholamin, M. Applying Subtractive Hybridization Technique to Enrich and Amplify Tumor-Specific Transcripts of Esophageal Squamous Cell Carcinoma. Pathol. Oncol. Res. 23, 271–279 (2017). https://doi.org/10.1007/s12253-016-0090-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12253-016-0090-5

Keywords

Search

Navigation