Development of RNA aptamers as molecular probes for HER2+ breast cancer study using cell-SELEX

Document Type : Original Article


1 Biotechnology Research Center, Nanotechnology Research Center, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran

2 Targeted Drug Delivery Research Center, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran

3 Biotechnology Research Center, Pharmacy School, Mashhad University of Medical Sciences, Mashhad, Iran

4 Nanotechnology Research Center, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran

5 Pharmaceutical Research Center, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran


Objective(s): Development of molecules that specifically recognize cancer cells is one of the major areas in cancer research. Human epidermal growth factor receptor 2 (HER2) is specifically expressed on the surface of breast cancer cells. HER2 is associated with an aggressive phenotype and poor prognosis. In this study we aimed to isolate RNA aptamers that specifically bind to HER2 overexpressing TUBO cell line.
Materials and Methods: Panel of aptamers was selected using cell-based systematic evolution of ligands by exponential enrichment (cell-SELEX).
Results: Binding studies showed that selected aptamers can identify TUBO cell line with high affinity and selectivity. Our preliminary investigation of the target of aptamers suggested that aptamers bind with HER2 proteins on the surface of TUBO cells.
Conclusion: We believe the selected aptamers could be useful ligands for targeted breast cancer therapy. 


1. Incorvati JA, Shah S, Mu Y, Lu J. Targeted therapy for HER2 positive breast cancer. J Hematol Oncol 2013, 6:1-9.
2. Li Q, Wu M, Wang H, Xu G, Zhu T, Zhang Y, et al. Ezrin silencing by small hairpin RNA reverses metastatic behaviors of human breast cancer cells. Cancer Lett2008; 261:55-63.
3. Yarden Y. Biology of HER2 and its importance in breast cancer. Oncology2001; 61:1-13.
4. Pal S, Pegram M. HER2 targeted therapy in breast cancer...beyond Herceptin. Rev Endocr Metab Disord2007; 8:269-277.
5. Levy-Nissenbaum E, Radovic-Moreno AF, Wang AZ, Langer R, Farokhzad OC. Nanotechnology and aptamers: applications in drug delivery. Trends Biotechnol 2008; 26:442-449.
6. Proske D, Blank M, Buhmann R, Resch A. Aptamers--basic research, drug development, and clinical applications. Appl Microbiol Biotechnol 2005; 69:367-374.
7. Famulok M, Hartig JS, Mayer G. Functional aptamers and aptazymes in biotechnology, diagnostics, and therapy. Chem Rev2007; 107:3715-3743.
8. Cox JC, Rudolph P, Ellington AD. Automated RNA selection. Biotechnol Prog1998; 14:845-850.
9. Farokhzad OC, Karp JM, Langer R. Nanoparticle-aptamer bioconjugates for cancer targeting. Expert Opin Drug Deliv2006; 3:311-324.
10. Farokhzad OC, Cheng J, Teply BA, Sherifi I, Jon S, Kantoff PW, et al. Targeted nanoparticle-aptamer bioconjugates for cancer chemotherapy in vivo. Proc Natl Acad Sci USA2006; 103:6315-6320.
11. Dua P, Kim S, Lee D-k. Nucleic acid aptamers targeting cell-surface proteins. Methods2011; 54:215-225.
12. Guo KT, Ziemer G, Paul A, Wendel HP. CELL-SELEX: Novel perspectives of aptamer-based therapeutics. Int J Mol Sci 2008; 9:668-678.
13. Stoltenburg R, Reinemann C, Strehlitz B. SELEX-A (r)evolutionary method to generate high-affinity nucleic acid ligands. Biomol Eng2007; 24:381-403.
14.  Kim Y, Liu C, Tan W. Aptamers generated by Cell SELEX for biomarker discovery. Biomark Med 2009; 3:193-202.
15. Yang Y, Yang D, Schluesener HJ, Zhang Z. Advances in SELEX and application of aptamers in the central nervous system. Biomol Eng2007; 24:583-592.
16. Penichet ML, Challita PM, Shin SU, Sampogna SL, Rosenblatt JD, Morrison SL. In vivo properties of three human HER2/neu-expressing murine cell lines in immunocompetent mice. Lab Anim Sci 1999; 49:179-188.
17. Jalali SA, Sankian M, Tavakkol-Afshari J, Jaafari MR. Induction of tumor-specific immunity by multi-epitope rat HER2/neu-derived peptides encapsulated in LPD Nanoparticles. Nanomedicine 2012; 8:692-701.
18. Hall B, Micheletti JM, Satya P, Ogle K, Pollard J, Ellington AD. Design, Synthesis, and Amplification of DNA Pools for In Vitro Selection. In Curr Protoc Mol Biol. Volume 9: John Wiley & Sons, Inc.; 2009: 9.2.1-9.2.28.
19. Mayer G, Piasecki S, Hall B, Ellington A. Nucleic Acid Pool Preparation and Characterization. In: Nucleic Acid and Peptide Aptamers. Humana Press; 2009: p. 3-18: Methods in Molecular Biology.
20. Ellington A. PJ. Purification of Oligonucleotides Using Denaturing Polyacrylamide Gel Electrophoresis. In Curr: 42 Vol: John Wiley & Sons, Inc. Protoc Mol Biolp.2.12.11-
21. Sefah K, Shangguan D, Xiong X, O'Donoghue MB, Tan W. Development of DNA aptamers usingCell-SELEX. Nat Protoc 2010; 5:1169-1185.
22. Sioud M, Meng L, Sefah K, Colon D, Chen H, O'Donoghue M, et al. Using Live Cells to Generate Aptamers for Cancer Study. In RNA Therapeutics. Volume 629: Humana Press; 353-365: Methods in Molecular Biology.
24. Kang HS, Huh YM, Kim S, Lee Dk. Isolation of RNA aptamers targeting HER-2-overexpressing breast cancer cells using cell-SELEX. Bull Korean Chem Soc2009; 30:1827-1831.
25. Zuker M. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 2003; 31:3406-3415.
26. Sefah K, Meng L, Lopez-Colon D, Jimenez E, Liu C, Tan W. DNA Aptamers as Molecular Probes for Colorectal Cancer Study. PLoS One2010; 5:e14269.
27. Kang D, Wang J, Zhang W, Song Y, Li X, Zou Y, et al. Selection of DNA Aptamers against Glioblastoma Cells with High Affinity and Specificity. PLoS One2012; 7:e42731.
28. Sefah K, Tang ZW, Shangguan DH, Chen H, Lopez-Colon D, Li Y, et al. Molecular recognition of acute myeloid leukemia using aptamers. Leukemia2009; 23:235-244.
29. Akiyama T, Sudo C, Ogawara H, Toyoshima K, Yamamoto T. The product of the human c-erbB-2 gene: a 185-kilodalton glycoprotein with tyrosine kinase activity. Science 1986; 232:1644-1646.
30. Disis ML, Calenoff E, McLaughlin G, Murphy AE, Chen W, Groner B, et al. Existent T-cell and antibody immunity to HER-2/neu protein in patients with breast cancer. Cancer Res1994; 54:16-20.
31. Neve RM, Sutterluty H, Pullen N, Lane HA, Daly JM, Krek W, et al. Effects of oncogenic ErbB2 on G1 cell cycle regulators in breast tumour cells. Oncogene2000; 19:1647-1656.
32. Slamon DJ, Clark GM, Wong SG, Levin WJ, Ullrich A, McGuire WL. Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science1987; 235:177-182.
33. Kim MY, Jeong S. In vitro selection of RNA aptamer and specific targeting of ErbB2 in breast cancer cells. Nucleic Acid Ther 2011; 21:173-178.
34. Mahlknecht G, Maron R, Mancini M, Schechter B, Sela M, Yarden Y. Aptamer to ErbB-2/HER2 enhances degradation of the target and inhibits tumorigenic growth. Proc Natl Acad Sci USA 2013; 110:8170-8175.
35. Thiel KW, Hernandez LI, Dassie JP, Thiel WH, Liu X, Stockdale KR, et al. Delivery of chemo-sensitizing siRNAs to HER2+-breast cancer cells using RNA aptamers. Nucleic Acids Res2012; 40:6319-6337.
36. Dastjerdi K, Tabar GH, Dehghani H, Haghparast A. Generation of an enriched pool of DNA aptamers for an HER2-overexpressing cell line selected by Cell SELEX. Biotechnol Appl Biochem 2011; 58:226-230.
37. Thiel KW, Hernandez LI, Dassie JP, Thiel WH, Liu X, Stockdale KR, et al. Delivery of chemo-sensitizing siRNAs to HER2+-breast cancer cells using RNA aptamers. Nucleic Acids Res 40:6319-6337.
38. Seavey MM, Pan ZK, Maciag PC, Wallecha A, Rivera S, Paterson Y, et al. A novel human Her-2/neuchimeric molecule expressed by Listeria monocytogenes can elicit potent HLA-A2 restricted CD8-positive T cell responses and impact the growth and spread of Her-2/neu-positive breast tumors. Clin Cancer Res2009; 15:924-932.
39. Ray P, White RR. Aptamers for Targeted Drug Delivery. Pharmaceuticals2010; 3:1761-1778.
40. Germer K, Leonard M, Zhang X. RNA aptamers and their therapeutic and diagnostic applications. Int J Biochem Mol Biol2013; 4:27-40.
41. Disis ML, Grabstein KH, Sleath PR, Cheever MA. Generation of immunity to the HER-2/neu oncogenic protein in patients with breast and ovarian cancer using a peptide-based vaccine. Clin Cancer Res 1999; 5:1289-1297.
42. Svoboda P, Cara AD. Hairpin RNA: a secondary structure of primary importance. Cel Mol Life SciCMLS 2006; 63:901-908.
43. Mallikaratchy P, Tang Z, Kwame S, Meng L, Shangguan D, Tan W. Aptamer Directly Evolved from Live Cells Recognizes Membrane Bound Immunoglobin Heavy Mu Chain in Burkitt'sLymphoma Cells. Mol Cell Proteomics2007; 6:2230-2238.
44. Jimenez E, Sefah K, Lopez-Colon D, Van Simaeys D,Chen HW, Tockman MS, et al. Generation of lung adenocarcinoma DNA aptamers for cancer studies. PLoS One2012; 7:e46222.
45. Shangguan D, Li Y, Tang Z, Cao ZC, Chen HW, Mallikaratchy P, et al. Aptamers evolved from live cells as effective molecular probes for cancer study. Proc Natl Acad Sci USA2006; 103:11838-11843.
46. Shangguan D, Cao Z, Meng L, Mallikaratchy P, Sefah K, Wang H, et al. Cell-specific aptamerprobes for membrane protein elucidation in cancer cells. J Proteome Res2008; 7:2133-2139.
47. Tang Z, Parekh P, Turner P, Moyer RW, Tan W. Generating aptamers for recognition of virus-infected cells. Clin Chem2009; 55:813-822.
48. Ara MN, Hyodo M, Ohga N, Hida K, Harashima H. Development of a Novel DNA Aptamer Ligand Targeting to Primary Cultured Tumor Endothelial Cells by a Cell-Based SELEX Method. PLoS One2012; 7:e50174.