Apoptosis

, 14:1331 | Cite as

Effects of upregulated expression of microRNA-16 on biological properties of culture-activated hepatic stellate cells

Original Paper

Abstract

In our previous studies, we identified miR-16 as being downregulated during activation of hepatic stellate cells (HSCs) by microarray hybridization. However, the roles and related mechanisms of miR-16 in HSCs are not understood. In this study, The miRNA RNAi technique was used to analyze the effects of miR-16 on biological properties of HSCs in vitro. The lentiviral vector encoding miR-16 was constructed and transfected. Furthermore, the expression level of miR-16 was measured by real-time PCR. Cellular growth and proliferation capacity were assayed using the cell counting kit-8 (CCK-8). The apoptosis rate and cell-cycle distribution were measured by flow cytometry. Cell morphological characteristics were identified by phase-contrast microscopy, fluorescence microscopy and electron microscopy. The underlying mechanisms related to the changes in biological properties were assessed. The identity of the recombinant plasmid was confirmed by restriction endonuclease analysis and DNA sequencing. Virus titer was 108 > ifu/m. Restoring the intracellular miRNAs by miR-16 administration greatly reduced the expression levels of cyclin D1 (CD1). Cell-cycle arrest and typical features of apoptosis were detected in activated HSCs treated with pLV-miR-16. Our results indicate that transduction of miR-16 offers a feasible approach to significantly inhibit HSC proliferation and increase the apoptosis index. Thus, targeted transfer of miR-16 into HSC may be useful for the treatment of hepatic fibrosis.

Keywords

Liver fibrosis Hepatic stellate cells rno-miR-16 Lentivirus 

Abbreviations

miRNA

microRNA

HSC

Hepatic stellate cell

3′UTR

3′untranslated region

SD

Standard deviation

CCK-8

Cell counting kit-8

CD1

Cyclin D1

Notes

Acknowledgements

This work is supported by the Foundation of Shanghai Commission of Science Technology of Research Program (O7JC14044).

References

  1. 1.
    Sprenger H, Kaufmann A, Garn H, Lahme B, Gemsa D, Gressner AM (1997) Induction of neutrophil-attracting chemokines in transforming rat hepatic stellate cells. Gastroenterology 113:277–285PubMedCrossRefGoogle Scholar
  2. 2.
    Kruglov EA, Correa PR, Arora G, Yu J, Nathanson MH, Dranoff JA (2007) Molecular basis for calcium signaling in hepatic stellate cells. Am J Physiol Gastrointest Liver Physiol 292:G975–G982PubMedCrossRefGoogle Scholar
  3. 3.
    Novo E, Marra F, Zamara E, Valfrè di Bonzo L, Monitillo L, Cannito S et al (2006) Overexpression of Bcl-2 by activatedhumanhepatic stellate cells: resistance to apoptosis as a mechanism of progressive hepatic fibrogenesis in humans. Gut 55:1174–1182PubMedCrossRefGoogle Scholar
  4. 4.
    Kawada N (2006) Human hepatic stellate cells are resistant to apoptosis: implications for human fibrogenic liver disease. Gut 55:1073–1074PubMedCrossRefGoogle Scholar
  5. 5.
    Lin YL, Lee TF, Huang YJ, Huang YT (2006) Antiproliferative effect of salvianolic acid A on rat hepatic stellate cells. J Pharm Pharmacol 58:933–939PubMedCrossRefGoogle Scholar
  6. 6.
    Chitwood DH, Timmermans MC (2007) Target mimics modulate miRNAs. Nat Genet 39:935–936PubMedCrossRefGoogle Scholar
  7. 7.
    Ambros V (2004) The functions of animal miRNA. Nature 431:350–355PubMedCrossRefGoogle Scholar
  8. 8.
    Guo CJ, Pan Q, Li DG, Sun H, Liu BW (2009) miR-15b and miR-16 are implicated in activation of the rat hepatic stellate cell: an essential role for apoptosis. J Hepatol 50:766–778PubMedCrossRefGoogle Scholar
  9. 9.
    Guo CJ, Pan Q, Cheng T, Jiang B, Chen GY, Li DG (2009) Changes in microRNAs associated with hepatic stellate cell activation status identify signaling pathways. FEBS J 276:5163–5176PubMedCrossRefGoogle Scholar
  10. 10.
    Friedman SL, Roll FJ (1987) Isolation and culture of hepatic lipocytes, Kupffer cells, and sinusoidal endothelial cells by density gradient centrifugation with Stractan. Anal Biochem 161:207–218PubMedCrossRefGoogle Scholar
  11. 11.
    Ying-Bin Hu, Ding-Guo Li, Han-Ming Lu (2007) Modified synthetic siRNA targeting tissue inhibitor of metalloproteinase-2 inhibits hepatic fibrogenesis in rats. J Gene Med 9:217–229CrossRefGoogle Scholar
  12. 12.
    Chen C, Ridzon DA, Broomer AJ, Zhou Z, Lee DH, Nguyen JT et al (2005) Real-time quantification of microRNAs by stem-loop RTPCR. Nucleic Acids Res 33:e179PubMedCrossRefGoogle Scholar
  13. 13.
    Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25:402–408PubMedCrossRefGoogle Scholar
  14. 14.
    Mraz M, Pospisilova S, Malinova K, Slapak I, Mayer J (2009) MicroRNAs in chronic lymphocytic leukemia pathogenesis and disease subtypes. Leuk Lymphoma 50:506–509PubMedCrossRefGoogle Scholar
  15. 15.
    Raveche ES, Salerno E, Scaglione BJ et al (2007) Abnormal microRNA-16 locus with synteny to human 13q14 linked to CLL in NZB mice. Blood 109:5079–5086PubMedCrossRefGoogle Scholar
  16. 16.
    Bottoni A, Piccin D, Tagliati F, Luchin A, Zatelli MC, degli Uberti EC (2005) miR-15a and miR-16-1 down-regulation in pituitary adenomas. J Cell Physiol 204:280–285PubMedCrossRefGoogle Scholar
  17. 17.
    Bandi N, Zbinden S, Gugger M et al (2009) miR-15a and miR-16 are implicated in cell cycle regulation in a Rb-dependent manner and are frequently deleted or down-regulated in non-small cell lung cancer. Cancer Res 69:5553–5559PubMedCrossRefGoogle Scholar
  18. 18.
    Bonci D, Coppola V, Musumeci M et al (2008) The miR-15a-miR-16-1 cluster controls prostate cancer by targeting multiple oncogenic activities. Nat Med 14:1271–1277PubMedCrossRefGoogle Scholar
  19. 19.
    Linsley PS, Schelter J, Burchard J et al (2007) Transcripts targeted by the microRNA-16 family cooperatively regulate cell cycle progression. Mol Cell Biol 27:2240–2252PubMedCrossRefGoogle Scholar
  20. 20.
    Kim MR, Kim HS, Lee MS, Lee MJ, Jang JJ (2005) Cell cycle protein profile of the hepatic stellate cells (HSCs) in dimethylnitrosamine-induced rat hepatic fibrosis. Exp Mol Med 37:335–342PubMedGoogle Scholar
  21. 21.
    Dudas J, Saile B, El-Armouche H, Aprigliano I, Ramadori G (2003) Endoreplication and polyploidy in primary culture of rat hepatic stellate cells. Cell Tissue Res 313:301–311PubMedCrossRefGoogle Scholar
  22. 22.
    Chen MH, Chen SH, Wang QF et al (2008) The molecular mechanism of gypenosides-induced G1 growth arrest of rat hepatic stellate cells. J Ethnopharmacol 117:309–317PubMedCrossRefGoogle Scholar
  23. 23.
    Chen RW, Bemis LT, Amato CM et al (2008) Truncation in CCND1 mRNA alters miR-16-1 regulation in mantle cell lymphoma. Blood 112:822–829PubMedCrossRefGoogle Scholar
  24. 24.
    Liu Q, Fu H, Sun F et al (2008) miR-16 family induces cell cycle arrest by regulating multiple cell cycle genes. Nucleic Acids Res 36:5391–5404PubMedCrossRefGoogle Scholar
  25. 25.
    Stenvang J, Kauppinen S (2008) MicroRNAs as targets for antisense-based therapeutics. Expert Opin Biol Ther 8:59–81PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2009

Authors and Affiliations

  1. 1.Digestive Disease Laboratory and Department of GastroenterologyXinhua Hospital, School of Medicine, Shanghai Jiaotong UniversityShanghaiChina
  2. 2.Central South UniversityChangshaChina

Personalised recommendations