Skip to main content

Advertisement

SpringerLink
Log in

Hepatic Stellate Cell-Specific Gene Silencing Induced by an Artificial MicroRNA for Antifibrosis In Vitro

  • Original Article
  • Published:
Digestive Diseases and Sciences Aims and scope Submit manuscript

Abstract

Background

We previously reported that the anti-transforming growth factor-beta1 (TGF-β1) ribozymes directed by T7 and CMV promoters could reverse the character of activated hepatic stellate cells (HSCs) in vitro and improve fibrotic pathology in vivo. However, nonspecific elimination of the effects of TGF-β1 without selectivity might have unfavorable consequences, such as overwhelming inflammation, tissue necrosis, etc.

Aims

To establish an activated-HSC-specific gene silencing method and validate its feasibility for antifibrosis in vitro.

Methods

An artificial intronic microRNA (miRNA) expression system was established, containing three parts: (1) a 1,074-bp SM-α actin promoter SMP8, which is a kind of RNA polymerase II promoter and has no activity in normal liver-derived cells but is switched on during the activation of HSCs, (2) intron1 modified by inserting an artificial pre-miRNA sequence against TGF-β1, and (3) report gene enhanced green fluorescent proteins (EGFP). The feasibility of this system for artificial microRNA expression was validated through microRNA detection by real-time polymerase chain reaction (PCR). Alteration of biological characteristics of HSCs with the anti-TGF-β1 miRNAs was preliminarily evaluated by measuring the expression levels of TGF-β1 and its downstream molecules, including collagen I, matrix metalloproteinase 2 (MMP2), tissue inhibitor of metalloproteinase 1 (TIMP-1), etc.

Results

The microRNA expression system could successfully produce mature anti-TGF-β1 miRNAs in an activated-HSC-specific manner. The microRNA-induced inhibition rate of TGF-β1 reached 70% and above. Accompanied by TGF-β1 suppression, its downstream targets such as collagen I, MMP2, TIMP-1, etc. were also significantly downregulated in vitro.

Conclusions

Activated-HSC-cell-specific gene silencing could be induced well by the artificial intronic microRNA expression system to realize antifibrosis in vitro.

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
Fig. 6

Similar content being viewed by others

References

  1. Gressner AM, Weiskirchen R. Modern pathogenetic concepts of liver fibrosis suggest stellate cells and TGF-beta as major players and therapeutic targets. J Cell Mol Med. 2006;10:76–99.

    Article  CAS  PubMed  Google Scholar 

  2. Bataller R, Brenner DA. Liver fibrosis. J Clin Invest. 2005;115:209–218.

    CAS  PubMed  Google Scholar 

  3. Bataller R, Paik YH, Lindquist JN, Lemasters JJ, Brenner DA. Hepatitis C virus core and nonstructural proteins induce fibrogenic effects in hepatic stellate cells. Gastroenterology. 2004;126:529–540.

    Article  CAS  PubMed  Google Scholar 

  4. Bridle KR, Crawford DH, Ramm GA. Identification and characterization of the hepatic stellate cell transferrin receptor. Am J Pathol. 2003;162:1661–1667.

    CAS  PubMed  Google Scholar 

  5. Iredale JP. Models of liver fibrosis: Exploring the dynamic nature of inflammation and repair in a solid organ. J Clin Invest. 2007;117:539–548.

    Article  CAS  PubMed  Google Scholar 

  6. Qi Z, Atsuchi N, Ooshima A, Takeshita A, Ueno H. Blockade of type beta transforming growth factor signaling prevents liver fibrosis and dysfunction in the rat. Proc Natl Acad Sci USA. 1999;96:2345–2349.

    Article  CAS  PubMed  Google Scholar 

  7. Zardi EM, Dobrina A, Ambrosino G, Margiotta D, Polistina F, Afeltra A. New therapeutic approaches to liver fibrosis: A practicable route? Curr Med Chem. 2008;15:1628–1644.

    Article  CAS  PubMed  Google Scholar 

  8. Song YH, Chen XL, Kong XJ, et al. Ribozymes against TGFbeta1 reverse character of activated hepatic stellate cells in vitro and inhibit liver fibrosis in rats. J Gene Med. 2005;7:965–976.

    Article  CAS  PubMed  Google Scholar 

  9. Xu Y, Pasche B. TGF-beta signaling alterations and susceptibility to colorectal cancer. Hum Mol Genet. 2007;16(1):R14–20.

    Article  CAS  PubMed  Google Scholar 

  10. Schaffert D, Wagner E. Gene therapy progress and prospects: Synthetic polymer-based systems. Gene Ther. 2008;15:1131–1138.

    Article  CAS  PubMed  Google Scholar 

  11. Zhang M, Smith EP, Kuroda H, Banach W, Chernausek SD, Fagin JA. Targeted expression of a protease-resistant IGFBP-4 mutant in smooth muscle of transgenic mice results in IGFBP-4 stabilization and smooth muscle hypotrophy. J Biol Chem. 2002;277:21285–21290.

    Article  CAS  PubMed  Google Scholar 

  12. George J, Roulot D, Koteliansky VE, Bissell DM. In vivo inhibition of rat stellate cell activation by soluble transforming growth factor beta type II receptor: A potential new therapy for hepatic fibrosis. Proc Natl Acad Sci USA. 1999;96:12719–12724.

    Article  CAS  PubMed  Google Scholar 

  13. Shi R, Chiang VL. Facile means for quantifying microRNA expression by real-time PCR. Biotechniques. 2005;39:519–525.

    Article  CAS  PubMed  Google Scholar 

  14. Zeng Y, Wagner EJ, Cullen BR. Both natural and designed micro RNAs can inhibit the expression of cognate mRNAs when expressed in human cells. Mol Cell. 2002;9:1327–1333.

    Article  CAS  PubMed  Google Scholar 

  15. Zeng Y, Cullen BR. Sequence requirements for micro RNA processing and function in human cells. RNA. 2003;9:112–123.

    Article  CAS  PubMed  Google Scholar 

  16. Zhou H, Xia XG, Xu Z. An RNA polymerase II construct synthesizes short-hairpin RNA with a quantitative indicator and mediates highly efficient RNAi. Nucleic Acids Res. 2005;33:e62.

    Article  PubMed  Google Scholar 

  17. Buck AH, Santoyo-Lopez J, Robertson KA, Kumar DS, Reczko M, Ghazal P. Discrete clusters of virus-encoded micrornas are associated with complementary strands of the genome and the 7.2-kilobase stable intron in murine cytomegalovirus. J Virol. 2007;81:13761–13770.

    Article  CAS  PubMed  Google Scholar 

  18. Castoldi M, Schmidt S, Benes V, et al. A sensitive array for microRNA expression profiling (miChip) based on locked nucleic acids (LNA). RNA. 2006;12:913–920.

    Article  CAS  PubMed  Google Scholar 

  19. Rojkind M, Giambrone MA, Biempica L. Collagen types in normal and cirrhotic liver. Gastroenterology. 1979;76:710–719.

    CAS  PubMed  Google Scholar 

  20. Rayburn ER, Zhang R. Antisense, RNAi, and gene silencing strategies for therapy: Mission possible or impossible? Drug Discov Today. 2008;13:513–521.

    Article  CAS  PubMed  Google Scholar 

  21. Kim D, Rossi J. RNAi mechanisms and applications. Biotechniques. 2008;44:613–616.

    Article  CAS  PubMed  Google Scholar 

  22. Huang C, Li M, Chen C, Yao Q. Small interfering RNA therapy in cancer: mechanism, potential targets, and clinical applications. Expert Opin Ther Targets. 2008;12:637–645.

    Article  CAS  PubMed  Google Scholar 

  23. Rockey DC. Gene therapy for hepatic fibrosis-bringing treatment into the new millennium. Hepatology. 1999;30:816–818.

    Article  CAS  PubMed  Google Scholar 

  24. Spankuch B, Strebhardt K. RNA interference-based gene silencing in mice: The development of a novel therapeutical strategy. Curr Pharm Des. 2005;11:3405–3419.

    Article  PubMed  Google Scholar 

  25. Xia XG, Zhou H, Xu Z. Transgenic RNAi: Accelerating and expanding reverse genetics in mammals. Transgenic Res. 2006;15:271–275.

    Article  CAS  PubMed  Google Scholar 

  26. Cai X, Zhou J, Chang Y, Sun X, Li P, Lin J. Targeting gene therapy for hepatocarcinoma cells with the E. coli purine nucleoside phosphorylase suicide gene system directed by a chimeric alpha-fetoprotein promoter. Cancer Lett. 2008;264:71–82.

    Article  CAS  PubMed  Google Scholar 

  27. Wang J, Niu W, Nikiforov Y, et al. Targeted overexpression of IGF-I evokes distinct patterns of organ remodeling in smooth muscle cell tissue beds of transgenic mice. J Clin Invest. 1997;100:1425–1439.

    Article  CAS  PubMed  Google Scholar 

  28. Moreira RK. Hepatic stellate cells and liver fibrosis. Arch Pathol Lab Med. 2007;131:1728–1734.

    CAS  PubMed  Google Scholar 

  29. Vogel S, Piantedosi R, Frank J, et al. An immortalized rat liver stellate cell line (HSC-T6): A new cell model for the study of retinoid metabolism in vitro. J Lipid Res. 2000;41:882–893.

    CAS  PubMed  Google Scholar 

  30. Kim Y, Ratziu V, Choi SG, et al. Transcriptional activation of transforming growth factor beta1 and its receptors by the Kruppel-like factor Zf9/core promoter-binding protein and Sp1. Potential mechanisms for autocrine fibrogenesis in response to injury. J Biol Chem. 1998;273:33750–33758.

    Article  CAS  PubMed  Google Scholar 

  31. Zhou XM, Lin JS, Shi Y, et al. Establishment of a screening system for selection of siRNA target sites directed against hepatitis B virus surface gene. Acta Biochim Biophys Sin (Shanghai). 2005;37:310–316.

    Article  CAS  Google Scholar 

  32. Cramer P, Armache KJ, Baumli S, et al. Structure of eukaryotic RNA polymerases. Annu Rev Biophys. 2008;37:337–352.

    Article  CAS  PubMed  Google Scholar 

  33. Lee Y, Kim M, Han J, et al. MicroRNA genes are transcribed by RNA polymerase II. EMBO J. 2004;23:4051–4060.

    Article  CAS  PubMed  Google Scholar 

  34. Lin SL, Miller JD, Ying SY. Intronic microRNA (miRNA). J Biomed Biotechnol. 2006;2006:26818.

    PubMed  Google Scholar 

  35. Rajewsky N. microRNA target predictions in animals. Nat Genet. 2006;38(Suppl):S8–13.

    Article  CAS  PubMed  Google Scholar 

  36. Krek A, Grun D, Poy MN, et al. Combinatorial microRNA target predictions. Nat Genet. 2005;37:495–500.

    Article  CAS  PubMed  Google Scholar 

  37. Lewis BP, Shih IH, Jones-Rhoades MW, Bartel DP, Burge CB. Prediction of mammalian microRNA targets. Cell. 2003;115:787–798.

    Article  CAS  PubMed  Google Scholar 

  38. Zhang B, Wang Q, Pan X. MicroRNAs and their regulatory roles in animals and plants. J Cell Physiol. 2007;210:279–289.

    Article  CAS  PubMed  Google Scholar 

  39. Jones-Rhoades MW, Bartel DP, Bartel B. MicroRNAS and their regulatory roles in plants. Annu Rev Plant Biol. 2006;57:19–53.

    Article  CAS  PubMed  Google Scholar 

  40. Rhoades MW, Reinhart BJ, Lim LP, Burge CB, Bartel B, Bartel DP. Prediction of plant microRNA targets. Cell. 2002;110:513–520.

    Article  CAS  PubMed  Google Scholar 

  41. Cuellar TL, McManus MT. MicroRNAs and endocrine biology. J Endocrinol. 2005;187:327–332.

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  43. Cheng K, Yang N, Mahato RI. TGF-beta1 gene silencing for treating liver fibrosis. Mol Pharm. 2009;6:772–779.

    Article  CAS  PubMed  Google Scholar 

  44. Kim KH, Kim HC, Hwang MY, et al. The antifibrotic effect of TGF-beta1 siRNAs in murine model of liver cirrhosis. Biochem Biophys Res Commun. 2006;343:1072–1078.

    Article  CAS  PubMed  Google Scholar 

  45. Li G, Xie Q, Shi Y, et al. Inhibition of connective tissue growth factor by siRNA prevents liver fibrosis in rats. J Gene Med. 2006;8:889–900.

    Article  CAS  PubMed  Google Scholar 

  46. Xu W, Wang LW, Shi JZ, Gong ZJ. Effects of RNA interference targeting transforming growth factor-beta 1 on immune hepatic fibrosis induced by Concanavalin A in mice. Hepatobiliary Pancreat Dis Int. 2009;8:300–308.

    CAS  PubMed  Google Scholar 

  47. Schnabl B, Kweon YO, Frederick JP, Wang XF, Rippe RA, Brenner DA. The role of Smad3 in mediating mouse hepatic stellate cell activation. Hepatology. 2001;34:89–100.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

We thank Mr. James A Fagin (University of Cincinnati Medical Center, USA) for the plasmid pSMP8. Supported by grants from the National Natural Science Foundation of China (No. 30600277) and the National Basic Research Program of China (No. 2007CB512903).

Author information

Authors and Affiliations

  1. Institute of Liver Diseases, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1095# Jie Fang Da Dao, 430022, Wuhan, China

    Ying Chang, Xue-mei Sun, Xiao-kun Cai, Xing-xing He, Pei-yuan Li, Wang-xian Tang, Yu-hu Song & Ju-sheng Lin

  2. Department of Nephrology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, 430022, Wuhan, China

    Hua-jun Jiang

Authors
  1. Ying Chang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Hua-jun Jiang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Xue-mei Sun
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Xiao-kun Cai
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Xing-xing He
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Pei-yuan Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Wang-xian Tang
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Yu-hu Song
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Ju-sheng Lin
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Yu-hu Song or Ju-sheng Lin.

Additional information

Ying Chang and Hua-jun Jiang contributed equally to this work.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Chang, Y., Jiang, Hj., Sun, Xm. et al. Hepatic Stellate Cell-Specific Gene Silencing Induced by an Artificial MicroRNA for Antifibrosis In Vitro. Dig Dis Sci 55, 642–653 (2010). https://doi.org/10.1007/s10620-009-1021-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10620-009-1021-z

Keywords

Search

Navigation