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

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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.

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Abbreviations

miRNA:

microRNA

HSC:

Hepatic stellate cell

3′UTR:

3′untranslated region

SD:

Standard deviation

CCK-8:

Cell counting kit-8

CD1:

Cyclin D1

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Acknowledgements

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

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Authors and Affiliations

  1. Digestive Disease Laboratory and Department of Gastroenterology, Xinhua Hospital, School of Medicine, Shanghai Jiaotong University, No. 1665 Kongjiang Road, Shanghai, 200092, China

    Can-Jie Guo, Qin Pan, Guang-Yu Chen & Ding-Guo Li

  2. Central South University, Changsha, China

    Bo Jiang

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  1. Can-Jie Guo
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  2. Qin Pan
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  3. Bo Jiang
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  4. Guang-Yu Chen
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  5. Ding-Guo Li
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Correspondence to Qin Pan or Ding-Guo Li.

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Guo, CJ., Pan, Q., Jiang, B. et al. Effects of upregulated expression of microRNA-16 on biological properties of culture-activated hepatic stellate cells. Apoptosis 14, 1331–1340 (2009). https://doi.org/10.1007/s10495-009-0401-3

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