Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

MicroRNA-regulated non-viral vectors with improved tumor specificity in an orthotopic rat model of hepatocellular carcinoma

Abstract

In hepatocellular carcinoma (HCC), tumor specificity of gene therapy is of utmost importance to preserve liver function. MicroRNAs (miRNAs) are powerful negative regulators of gene expression and many are downregulated in human HCC. We identified seven miRNAs that are also downregulated in tumors in a rat hepatoma model (P<0.05) and attempted to improve tumor specificity by constructing a panel of luciferase-expressing vectors containing binding sites for these miRNAs. Attenuation of luciferase expression by the corresponding miRNAs was confirmed across various cell lines and in mouse liver. We then tested our vectors in tumor-bearing rats and identified two miRNAs, miR-26a and miR-122, that significantly decreased expression in liver compared with the control vector (6.40 and 0.26%, respectively; P<0.05). In tumor, miR-122 had a nonsignificant trend towards decreased (50%) expression, whereas miR-26 had no significant effect on tumor expression. To our knowledge, this is the first work using differentially expressed miRNAs to de-target transgene expression in an orthotopic hepatoma model and to identify miR-26a, in addition to miR-122, for de-targeting liver. Considering the heterogeneity of miRNA expression in human HCC, this information will be important in guiding development of more personalized vectors for the treatment of this devastating disease.

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

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5

References

  1. El-Serag HB, Rudolph KL . Hepatocellular carcinoma: epidemiology and molecular carcinogenesis. Gastroenterology 2007; 132: 2557–2576.

    Article  CAS  Google Scholar 

  2. Bartel DP . MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 2004; 116: 281–297.

    Article  CAS  Google Scholar 

  3. Yekta S, Shih I-H, Bartel DP . MicroRNA-directed cleavage of HOXB8 mRNA. Science 2004; 304: 594–596.

    Article  CAS  Google Scholar 

  4. Zeng Y, Yi R, Cullen BR . MicroRNAs and small interfering RNAs can inhibit mRNA expression by similar mechanisms. Proc Natl Acad Sci USA 2003; 100: 9779–9784.

    Article  CAS  Google Scholar 

  5. John B, Enright AJ, Aravin A, Tuschl T, Sander C, Marks DS . Human microRNA targets. PLoS Biol 2004; 2: e363.

    Article  Google Scholar 

  6. Lim LP, Lau NC, Garrett-Engele P, Grimson A, Schelter JM, Castle J et al. Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs. Nature 2005; 433: 769–773.

    Article  CAS  Google Scholar 

  7. Calin GA, Sevignani C, Dumitru CD, Hyslop T, Noch E, Yendamuri S et al. Human microRNA genes are frequently located at fragile sites and genomic regions involved in cancers. Proc Natl Acad Sci USA 2004; 101: 2999–3004.

    Article  CAS  Google Scholar 

  8. Croce CM, Calin GA . miRNAs, cancer, and stem cell division. Cell 2005; 122: 6–7.

    Article  CAS  Google Scholar 

  9. Kutay H, Bai S, Datta J, Motiwala T, Pogribny I, Frankel W et al. Downregulation of miR-122 in the rodent and human hepatocellular carcinomas. J Cell Biochem 2006; 99: 671–678.

    Article  CAS  Google Scholar 

  10. Varnholt H, Drebber U, Schulze F, Wedemeyer I, Schirmacher P, Dienes H-P et al. MicroRNA gene expression profile of hepatitis C virus-associated hepatocellular carcinoma. Hepatology 2008; 47: 1223–1232.

    Article  CAS  Google Scholar 

  11. Huang Y-S, Dai Y, Yu X-F, Bao S-Y, Yin Y-B, Tang M et al. Microarray analysis of microRNA expression in hepatocellular carcinoma and non-tumorous tissues without viral hepatitis. J Gastroenterol Hepatol 2008; 23: 87–94.

    Article  Google Scholar 

  12. Gramantieri L, Ferracin M, Fornari F, Veronese A, Sabbioni S, Liu C-G et al. Cyclin G1 is a target of miR-122a, a microRNA frequently down-regulated in human hepatocellular carcinoma. Cancer Res 2007; 67: 6092–6099.

    Article  CAS  Google Scholar 

  13. Engels BM, Hutvagner G . Principles and effects of microRNA-mediated post-transcriptional gene regulation. Oncogene 2006; 25: 6163–6169.

    Article  CAS  Google Scholar 

  14. Jiang J, Gusev Y, Aderca I, Mettler TA, Nagorney DM, Brackett DJ et al. Association of MicroRNA expression in hepatocellular carcinomas with hepatitis infection, cirrhosis, and patient survival. Clin Cancer Res 2008; 14: 419–427.

    Article  CAS  Google Scholar 

  15. Li W, Xie L, He X, Li J, Tu K, Wei L et al. Diagnostic and prognostic implications of microRNAs in human hepatocellular carcinoma. Int J Cancer 2008; 123: 1616–1622.

    Article  CAS  Google Scholar 

  16. Kota J, Chivukula RR, O'Donnell KA, Wentzel EA, Montgomery CL, Hwang H-W et al. Therapeutic microRNA delivery suppresses tumorigenesis in a murine liver cancer model. Cell 2009; 137: 1005–1017.

    Article  CAS  Google Scholar 

  17. Ji J, Wang XW . New kids on the block: diagnostic and prognostic microRNAs in hepatocellular carcinoma. Cancer Biol Ther 2009; 8: 1686–1693.

    Article  Google Scholar 

  18. Ding B, Li T, Zhang J, Zhao L, Zhai G . Advances in liver-directed gene therapy for hepatocellular carcinoma by non-viral delivery systems. Curr Gene Ther 2012; 12: 92–102.

    Article  CAS  Google Scholar 

  19. Brown BD, Cantore A, Annoni A, Sergi LS, Lombardo A, Valle della P et al. A microRNA-regulated lentiviral vector mediates stable correction of hemophilia B mice. Blood 2007; 110: 4144–4152.

    Article  CAS  Google Scholar 

  20. Brown BD, Venneri MA, Zingale A, Sergi Sergi L, Naldini L . Endogenous microRNA regulation suppresses transgene expression in hematopoietic lineages and enables stable gene transfer. Nat Med 2006; 12: 585–591.

    Article  CAS  Google Scholar 

  21. Brown BD, Gentner B, Cantore A, Colleoni S, Amendola M, Zingale A et al. Endogenous microRNA can be broadly exploited to regulate transgene expression according to tissue, lineage and differentiation state. Nat Biotechnol 2007; 25: 1457–1467.

    Article  CAS  Google Scholar 

  22. Lu J, Getz G, Miska EA, Alvarez-Saavedra E, Lamb J, Peck D et al. MicroRNA expression profiles classify human cancers. Nature 2005; 435: 834–838.

    Article  CAS  Google Scholar 

  23. Ylösmäki E, Hakkarainen T, Hemminki A, Visakorpi T, Andino R, Saksela K . Generation of a conditionally replicating adenovirus based on targeted destruction of E1A mRNA by a cell type-specific microRNA. J Virol 2008; 82: 11009–11015.

    Article  Google Scholar 

  24. Kelly EJ, Hadac EM, Greiner S, Russell SJ . Engineering microRNA responsiveness to decrease virus pathogenicity. Nat Med 2008; 14: 1278–1283.

    Article  CAS  Google Scholar 

  25. Cawood R, Chen HH, Carroll F, Bazan-Peregrino M, van Rooijen N, Seymour LW . Use of tissue-specific microRNA to control pathology of wild-type adenovirus without attenuation of its ability to kill cancer cells. PLoS Pathog 2009; 5: e1000440.

    Article  Google Scholar 

  26. Sakurai F, Katayama K, Mizuguchi H . MicroRNA-regulated transgene expression systems for gene therapy and virotherapy. Front Biosci 2011; 17: 2389–2401.

    Article  Google Scholar 

  27. Jin H, Lv S, Yang J, Wang X, Hu H, Su C et al. Use of microRNA Let-7 to control the replication specificity of oncolytic adenovirus in hepatocellular carcinoma cells. PLoS One 2011; 6: e21307.

    Article  CAS  Google Scholar 

  28. Fu X, Rivera A, Tao L, de Geest B, Zhang X . Construction of an oncolytic herpes simplex virus that precisely targets hepatocellular carcinoma cells. Mol Ther 2012; 20: 339–346.

    Article  CAS  Google Scholar 

  29. Card PB, Hogg RT, del Alcazar CRG, Gerard RD . MicroRNA silencing improves the tumor specificity of adenoviral transgene expression. Cancer Gene Ther 2012; 19: 451–459.

    Article  CAS  Google Scholar 

  30. Su H, Yang J-R, Xu T, Huang J, Xu L, Yuan Y et al. MicroRNA-101, down-regulated in hepatocellular carcinoma, promotes apoptosis and suppresses tumorigenicity. Cancer Res 2009; 69: 1135–1142.

    Article  CAS  Google Scholar 

  31. Gramantieri L, Fornari F, Callegari E, Sabbioni S, Lanza G, Croce CM et al. MicroRNA involvement in hepatocellular carcinoma. J Cell Mol Med 2008; 12: 2189–2204.

    Article  CAS  Google Scholar 

  32. Braconi C, Patel T . MicroRNA expression profiling: a molecular tool for defining the phenotype of hepatocellular tumors. Hepatology 2008; 47: 1807–1809.

    Article  CAS  Google Scholar 

  33. Wang Y, Lee ATC, Ma JZI, Wang J, Ren J, Yang Y et al. Profiling microRNA expression in hepatocellular carcinoma reveals microRNA-224 up-regulation and apoptosis inhibitor-5 as a microRNA-224-specific target. J Biol Chem 2008; 283: 13205–13215.

    Article  CAS  Google Scholar 

  34. Lagos-Quintana M, Rauhut R, Yalcin A, Meyer J, Lendeckel W, Tuschl T . Identification of tissue-specific microRNAs from mouse. Curr Biol 2002; 12: 735–739.

    Article  CAS  Google Scholar 

  35. Tada M, Hatano E, Taura K, Nitta T, Koizumi N, Ikai I et al. High volume hydrodynamic injection of plasmid DNA via the hepatic artery results in a high level of gene expression in rat hepatocellular carcinoma induced by diethylnitrosamine. J Gene Med 2006; 8: 1018–1026.

    Article  CAS  Google Scholar 

  36. Suzuki T, Sakurai F, Nakamura S-I, Kouyama E, Kawabata K, Kondoh M et al. miR-122a-regulated expression of a suicide gene prevents hepatotoxicity without altering antitumor effects in suicide gene therapy. Mol Ther 2008; 16: 1719–1726.

    Article  CAS  Google Scholar 

  37. Edge RE, Falls TJ, Brown CW, Lichty BD, Atkins H, Bell JC . A let-7 microRNA-sensitive vesicular stomatitis virus demonstrates tumor-specific replication. Mol Ther 2008; 16: 1437–1443.

    Article  CAS  Google Scholar 

  38. Wu C, Lin J, Hong M, Choudhury Y, Balani P, Leung D et al. Combinatorial control of suicide gene expression by tissue-specific promoter and microRNA regulation for cancer therapy. Mol Ther 2010; 17: 2058–2066.

    Article  Google Scholar 

  39. Sugio K, Sakurai F, Katayama K, Tashiro K, Matsui H, Kawabata K et al. Enhanced safety profiles of the telomerase-specific replication-competent adenovirus by incorporation of normal cell-specific microRNA-targeted sequences. Clin Cancer Res 2011; 17: 2807–2818.

    Article  CAS  Google Scholar 

  40. Mizuguchi Y, Mishima T, Yokomuro S, Arima Y, Kawahigashi Y, Shigehara K et al. Sequencing and bioinformatics-based analyses of the microRNA transcriptome in hepatitis B-related hepatocellular carcinoma. PLoS One 2011; 6: e15304.

    Article  CAS  Google Scholar 

  41. Wei W, Xue JL, Tian L . Vector retargeting for cancer gene therapy. Ai Zheng 2009; 28: 86–90.

    PubMed  Google Scholar 

  42. Sia KC, Huynh H, Chinnasamy N, Hui KM . Lam PYP. Suicidal gene therapy in the effective control of primary human hepatocellular carcinoma as monitored by noninvasive bioimaging. Gene Ther 2011; 19: 532–542.

    Article  Google Scholar 

  43. Kim YI, Ahn B-C, Ronald JA, Katzenberg R, Singh A, Paulmurugan R et al. Intratumoral versus intravenous gene therapy using a transcriptionally targeted viral vector in an orthotopic hepatocellular carcinoma rat model. J Vasc Interv Radiol 2012; 23: 704–711.

    Article  Google Scholar 

  44. Shiba H, Misawa T, Iida T, Okamoto T, Futagawa Y, Sakurai M et al. Adenovirus vector-mediated gene therapy using iodized oil esters for hepatocellular carcinoma in rats. Anticancer Res 2008; 28: 51–53.

    CAS  PubMed  Google Scholar 

  45. Zhang Y, Ma H, Zhang J, Liu S, Liu Y, Zheng D . AAV-mediated TRAIL gene expression driven by hTERT promoter suppressed human hepatocellular carcinoma growth in mice. Life Sci 2008; 82: 1154–1161.

    Article  CAS  Google Scholar 

  46. Ahn B-C, Ronald JA, Kim YI, Katzenberg R, Singh A, Paulmurugan R et al. Potent, tumor-specific gene expression in an orthotopic hepatoma rat model using a Survivin-targeted, amplifiable adenoviral vector. Gene Ther 2011; 18: 606–612.

    Article  CAS  Google Scholar 

  47. Doench JG, Petersen CP, Sharp PA . siRNAs can function as miRNAs. Genes Dev 2003; 17: 438–442.

    Article  CAS  Google Scholar 

  48. Mukherji S, Ebert MS, Zheng GXY, Tsang JS, Sharp PA, van Oudenaarden A . MicroRNAs can generate thresholds in target gene expression. Nat Genet 2011; 43: 854–859.

    Article  CAS  Google Scholar 

  49. Tryndyak VP, Ross SA, Beland FA, Pogribny IP . Down-regulation of the microRNAs miR-34a, miR-127, and miR-200b in rat liver during hepatocarcinogenesis induced by a methyl-deficient diet. Mol Carcinog 2009; 48: 479–487.

    Article  CAS  Google Scholar 

  50. Petrelli A, Perra A, Schernhuber K, Cargnelutti M, Salvi A, Migliore C et al. Sequential analysis of multistage hepatocarcinogenesis reveals that miR-100 and PLK1 dysregulation is an early event maintained along tumor progression. Oncogene 2012; 31: 4517–4526.

    Article  CAS  Google Scholar 

  51. Brown BD, Naldini L . Exploiting and antagonizing microRNA regulation for therapeutic and experimental applications. Nat Rev Genet 2009; 10: 578–585.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We would like to acknowledge the funding from NCI ICMIC P50CA114747 (SSG), and NCI RO1 CA082214 (SSG). JAR was supported by a postdoctoral fellowship from the Canadian Institutes of Health Research (CIHR).

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to S S Gambhir.

Ethics declarations

Competing interests

The authors declare no conflict of interest.

Additional information

Supplementary Information accompanies this paper on Gene Therapy website

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Cite this article

Ronald, J., Katzenberg, R., Nielsen, C. et al. MicroRNA-regulated non-viral vectors with improved tumor specificity in an orthotopic rat model of hepatocellular carcinoma. Gene Ther 20, 1006–1013 (2013). https://doi.org/10.1038/gt.2013.24

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/gt.2013.24

Keywords

This article is cited by

Search

Quick links