Skip to main content

Advertisement

SpringerLink
Log in

Regulating A549 cells growth by ASO inhibiting miRNA expression

  • Published:
Molecular and Cellular Biochemistry Aims and scope Submit manuscript

Abstract

MicroRNAs (miRNAs) have a profound impact on cell processes, including proliferation, apoptosis, and stress responses. We aimed to explore the role of antisense oligonucleotide (ASO) to induce proliferation or apoptosis of A549 cancer cells by inhibiting the expression of miRNAs. After A549/HBE/293T cells were treated with ASO, cells proliferation/apoptosis, and their relevant oncogenes/tumor suppressor genes were detected by light and electron microscopy, real-time PCR, enzyme-linked immunosorbent assay, etc. The results showed that ASO could inhibit the expression of miRNAs effectively. miR-16, miR-17, miR-34a–c, and miR-125 served as tumor suppressor miRNAs, while miR-20, miR-106, and miR-150 acted as oncogenic miRNAs. Our results also indicated that miR-16/34a–c, miR-17-5p, miR-125, miR-106, and miR-150 were the upstream factors, which could regulate the expression of BCL-2, E2F1, E2F3, RB1, and P53, respectively. After A549 cells treated with ASO for 24 h and different concentrations of anti-cancer drug (cisplatin or demethylcantharidin) were added into culture medium, the results indicated the percentage of alive cells in group treated with both ASO-106 (or ASO-150) and anti-cancer drug was lower than that in group treated with ASO, or anti-cancer drug, or both ASO-16 (or ASO-34a) and anti-cancer drug. In conclusion, ASO (specific to oncogenic miRNAs) could induce A549 cells apoptosis by inhibiting oncogenic miRNAs, and could increase chemotherapy sensitivity of A549 cells to anti-cancer drug, which holds great promise to lung cancer therapy.

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
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

Abbreviations

ASO:

Antisense oligonucleotide

miRNAs:

MicroRNAs

nt:

Nucleotide

RISC:

RNA-induced silencing complex

anti-miRNA:

Anti-microRNA

siRNA:

Small interfering RNA

DMC:

Demethylcantharidin

A549 Cells:

Human lung adenocarcinoma epithelial cell line

HBE Cells:

Normal human bronchial epithelium cells

293T Cells:

Human embryonic kidney cells

MTT:

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

DMSO:

Dimethyl sulfoxide

ELISA:

Enzyme-linked immunosorbent assay

PI:

Propidium iodide

References

  1. Kusenda B, Mraz M, Mayer J, Pospisilova S (2006) MicroRNA biogenesis, functionality and cancer relevance. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub 150:205–215

    CAS  PubMed  Google Scholar 

  2. Place RF, Li LC, Pookot D, Noonan EJ, Dahiya R (2008) MicroRNA-373 induces expression of genes with complementary promoter sequences. Proc Natl Acad Sci USA 105:1608–1613

    Article  CAS  PubMed  Google Scholar 

  3. Vasudevan S, Tong Y, Steitz JA (2007) Switching from repression to activation: microRNAs can up-regulate translation. Science 318:1931–1934

    Article  CAS  PubMed  Google Scholar 

  4. Schickel R, Boyerinas B, Park SM, Peter ME (2008) MicroRNAs: key players in the immune system, differentiation, tumorigenesis and cell death. Oncogene 27:5959–5974

    Article  CAS  PubMed  Google Scholar 

  5. Lau NC, Lim LP, Weinstein EG, Bartel DP (2001) An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans. Science 294:858–862

    Article  CAS  PubMed  Google Scholar 

  6. Bartel DP (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116:281–297

    Article  CAS  PubMed  Google Scholar 

  7. Zhu S, Wu H, Wu F, Nie D, Sheng S, Mo YY (2008) MicroRNA-21 targets tumor suppressor genes in invasion and metastasis. Cell Res 18:350–359

    Article  CAS  PubMed  Google Scholar 

  8. Ma L, Teruya-Feldstein J, Weinberg RA (2007) Tumour invasion and metastasis initiated by microRNA-10b in breast cancer. Nature 449:682–688

    Article  CAS  PubMed  Google Scholar 

  9. Ho PT, Parkinson DR (1997) Antisense oligonucleotides as therapeutics for malignant diseases. Semin Oncol 24:187–202

    CAS  PubMed  Google Scholar 

  10. Crooke ST (1998) Molecular mechanisms of antisense drugs: RNase H. Antisense Nucleic Acid Drug Dev 8:133–134

    CAS  PubMed  Google Scholar 

  11. Stahel RA, Zangemeister-Wittke U (2003) Antisense oligonucleotides for cancer therapy—an overview. Lung Cancer 41:S81–S88

    Article  PubMed  Google Scholar 

  12. Wacheck V, Zangemeister-Wittke U (2006) Antisense molecules for targeted cancer therapy. Crit Rev Oncol Hematol 59:65–73

    Article  CAS  PubMed  Google Scholar 

  13. Visone R, Croce CM (2009) MiRNAs and cancer. Am J Pathol 174:1131–1138

    Article  CAS  PubMed  Google Scholar 

  14. Ren H, Tian L, Gu Q, Zhu W (2006) Secalonic acid D; A cytotoxic constituent from marine lichen-derived fungus Gliocladium sp. T31. Arch Pharm Res 29:59–63

    Article  CAS  PubMed  Google Scholar 

  15. O’Donovan DJ, Katkin JP, Tamura T, Smith CV, Welty SE (2000) Attenuation of hyperoxia-induced growth inhibition in H441 cells by gene transfer of mitochondrially targeted glutathione reductase. Am J Respir Cell Mol Biol 22:732–738

    PubMed  Google Scholar 

  16. Trotter PJ, Orchard MA, Walker JH (1995) Ca2+ concentration during binding determines the manner in which annexin V binds to membranes. Biochem J 308:591–598

    CAS  PubMed  Google Scholar 

  17. Fan T, Li R, Todd NW, Qiu Q, Fang HB, Wang H et al (2007) Up-regulation of 14-3-3zeta in lung cancer and its implication as prognostic and therapeutic target. Cancer Res 67:7901–7906

    Article  CAS  PubMed  Google Scholar 

  18. Videhult P, Yachnin J, Jerremalm E, Lewensohn R, Ehrsson H (2002) Synthesis and cytotoxicity of the dihydrated complex of oxaliplatin. Cancer Lett 80:191–194

    Article  Google Scholar 

  19. Efferth T, Rauh R, Kahl S, Tomicic M, Böchzelt H, Tome ME et al (2005) Molecular modes of action of cantharidin in tumor cells. Biochem Pharmacol 69:811–818

    Article  CAS  PubMed  Google Scholar 

  20. To KK, Ho YP, Au-Yeung SC (2005) Synergistic interaction between platinum-based antitumor agents and demethylcantharidin. Cancer Lett 223:227–237

    Article  CAS  PubMed  Google Scholar 

  21. Cheng AM, Byrom MW, Shelton J, Ford LP (2005) Antisense inhibition of human miRNAs and indications for an involvement of miRNA in cell growth and apoptosis. Nucleic Acids Res 33:1290–1297

    Article  CAS  PubMed  Google Scholar 

  22. van den Berg A, Kroesen BJ, Kooistra K, de Jong D, Briggs J, Blokzijl T et al (2003) High expression of B-cell receptor inducible gene BIC in all subtypes of Hodgkin lymphoma. Genes Chromosomes Cancer 37:20–28

    Article  PubMed  Google Scholar 

  23. Metzler M, Wilda M, Busch K, Viehmann S, Borkhardt A (2004) High expression of precursor microRNA-155/BIC RNA in children with Burkitt lymphoma. Genes Chromosomes Cancer 39:167–169

    Article  CAS  PubMed  Google Scholar 

  24. Kluiver J, Poppema S, de Jong D, Blokzijl T, Harms G, Jacobs S, Kroesen BJ et al (2005) BIC and miR-155 are highly expressed in Hodgkin, primary mediastinal and diffuse large B cell lymphomas. J Pathol 207:243–249

    Article  CAS  PubMed  Google Scholar 

  25. Eis PS, Tam W, Sun L, Chadburn A, Li Z, Gomez MF et al (2005) Accumulation of miR-155 and BIC RNA in human B cell lymphomas. Proc Natl Acad Sci USA 102:3627–3632

    Article  CAS  PubMed  Google Scholar 

  26. Iorio MV, Ferracin M, Liu CG, Veronese A, Spizzo R, Sabbioni S et al (2005) MicroRNA gene expression deregulation in human breast cancer. Cancer Res 65:7065–7070

    Article  CAS  PubMed  Google Scholar 

  27. Voorhoeve PM, le Sage C, Schrier M, Gillis AJ, Stoop H, Nagel R et al (2006) A genetic screen implicates miRNA-372 and miRNA-373 as oncogenes in testicular germ cell tumors. Cell 124:1169–1181

    Article  CAS  PubMed  Google Scholar 

  28. Uziel T, Karginov FV, Xie S, Parker JS, Wang YD, Gajjar A et al (2009) The miR-17∼92 cluster collaborates with the Sonic Hedgehog pathway in medulloblastoma. Proc Natl Acad Sci USA 106:2812–2817

    Article  CAS  PubMed  Google Scholar 

  29. Zhou L, Qi X, Potashkin JA, Abdul-Karim FW, Gorodeski GI (2008) MicroRNAs miR-186 and miR-150 down-regulate expression of the pro-apoptotic purinergic P2X7 receptor by activation of instability sites at the 3′-untranslated region of the gene that decrease steady-state levels of the transcript. J Biol Chem 283:28274–28286

    Article  CAS  PubMed  Google Scholar 

  30. Nakao M, Horiike S, Fukushima-Nakase Y, Nishimura M, Fujita Y, Taniwaki M et al (2004) Novel loss-of-function mutations of the haematopoiesis-related transcription factor, acute myeloid leukaemia 1/runt-related transcription factor 1, detected in acute myeloblastic leukaemia and myelodysplastic syndrome. Br J Haematol 125:709–719

    Article  CAS  PubMed  Google Scholar 

  31. Scherr M, Venturini L, Battmer K, Schaller-Schoenitz M, Schaefer D, Dallmann I et al (2007) Lentivirus-mediated antagomir expression for specific inhibition of miRNA function. Nucleic Acids Res 35:e149

    Article  PubMed  Google Scholar 

  32. Tan LP, Wang M, Robertus JL, Schakel RN, Gibcus JH, Diepstra A et al (2009) miRNA profiling of B-cell subsets: specific miRNA profile for germinal center B cells with variation between centroblasts and centrocytes. Lab Invest 89:708–716

    Article  CAS  PubMed  Google Scholar 

  33. Calin GA, Dumitru CD, Shimizu M, Bichi R, Zupo S, Noch E et al (2002) Frequent deletions and down-regulation of micro-RNA genes miR15 and miR16 at 13q14 in chronic lymphocytic leukemia. Proc Natl Acad Sci USA 99:15524–15529

    Article  CAS  PubMed  Google Scholar 

  34. Trimarchi JM, Lees JA (2002) Sibling rivalry in the E2F family. Nat Rev Mol Cell Biol 3:11–20

    Article  CAS  PubMed  Google Scholar 

  35. Dews M, Homayouni A, Yu D, Murphy D, Sevignani C, Wentzel E et al (2006) Augmentation of tumor angiogenesis by a Myc-activated microRNA cluster. Nat Genet 38:1060–1065

    Article  CAS  PubMed  Google Scholar 

  36. Kato M, Paranjape T, Ullrich R, Nallur S, Gillespie E, Keane K et al (2009) The mir-34 microRNA is required for the DNA damage response in vivo in C. elegans and in vitro in human breast cancer cells. Oncogene 28:2419–2424

    Article  CAS  PubMed  Google Scholar 

  37. Zenz T, Mohr J, Eldering E, Kater AP, Bühler A, Kienle D et al (2009) miR-34a as part of the resistance network in chronic lymphocytic leukemia. Blood 113:3801–3808

    Article  CAS  PubMed  Google Scholar 

  38. Yamakuchi M, Ferlito M, Lowenstein CJ (2008) miR-34a repression of SIRT1 regulates apoptosis. Proc Natl Acad Sci USA 105:13421–13426

    Article  CAS  PubMed  Google Scholar 

  39. Tong AW, Nemunaitis J (2008) Modulation of miRNA activity in human cancer: a new paradigm for cancer gene therapy? Cancer Gene Ther 15:341–355

    Article  CAS  PubMed  Google Scholar 

  40. Dykxhoorn DM, Chowdhury D, Lieberman J (2008) RNA interference and cancer: endogenous pathways and therapeutic approaches. Adv Exp Med Biol 615:299–329

    Article  PubMed  Google Scholar 

  41. Stenvang J, Kauppinen S (2008) MicroRNAs as targets for antisense-based therapeutics. Expert Opin Biol Ther 8:59–81

    Article  CAS  PubMed  Google Scholar 

  42. Blenkiron C, Miska EA (2007) miRNAs in cancer: approaches, aetiology, diagnostics and therapy. Hum Mol Genet 16:R106–R113

    Article  CAS  PubMed  Google Scholar 

  43. Meister G, Landthaler M, Dorsett Y, Tuschl T (2004) Sequence-specific inhibition of microRNA- and siRNA-induced RNA silencing. RNA 10:544–550

    Article  CAS  PubMed  Google Scholar 

  44. Davis S, Lollo B, Freier S, Esau C (2006) Improved targeting of miRNA with antisense oligonucleotides. Nucleic Acids Res 34:2294–2304

    Article  CAS  PubMed  Google Scholar 

  45. Takamizawa J, Konishi H, Yanagisawa K, Tomida S, Osada H, Endoh H et al (2004) Reduced expression of the let-7 microRNAs in human lung cancers in association with shortened postoperative survival. Cancer Res 64:3753–3756

    Article  CAS  PubMed  Google Scholar 

  46. Kovalchuk O, Filkowski J, Meservy J, Ilnytskyy Y, Tryndyak VP, Chekhun VF et al (2008) Involvement of microRNA-451 in resistance of the MCF-7 breast cancer cells to chemotherapeutic drug doxorubicin. Mol Cancer Ther 7:2152–2159

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This study was supported by National Natural Science Foundation of China (No. 30801324), Foundation of Shan Dong Science and Technology Committee (No. 2007BS03048, 2009ZRB01212), and Yantai Science and Technology Committee (No. 2007153), China. We also thank Wenbo Liu, Xiuwen Wang, Cheng Yang, Xiaodong Song, Jinjin Zhang, and Lixia Zhang (Experimental Central Lab of Binzhou Medical University) for their help of electron microscopy and flow cytometry analysis.

Author information

Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Institute of Medical Molecular Genetics, Binzhou Medical University, No. 346 Guan Hai Road, Lai Shan District, 264003, Yan Tai City, Shan Dong Province, People’s Republic of China

    Ping-Yu Wang, You-Jie Li, Shuai Zhang, Zun-Ling Li, Zhen Yue & Shu-Yang Xie

  2. Medical School of Shandong University, 250012, Jinan, People’s Republic of China

    Ning Xie

Authors
  1. Ping-Yu Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. You-Jie Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Shuai Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Zun-Ling Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Zhen Yue
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Ning Xie
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Shu-Yang Xie
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Shu-Yang Xie.

Additional information

Ping-Yu Wang and You-Jie Li contributed equally to this work.

Electronic supplementary material

Below is the link to the electronic supplementary material.

(DOC 1608 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Wang, PY., Li, YJ., Zhang, S. et al. Regulating A549 cells growth by ASO inhibiting miRNA expression. Mol Cell Biochem 339, 163–171 (2010). https://doi.org/10.1007/s11010-009-0380-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11010-009-0380-2

Keywords

Search

Navigation