Effects of lentiviral vector‑mediated shRNA silencing of TGFβ1 on the expression of Col1a1 in rat hepatic stellate cells

  • Authors:
    • Gan‑Ping Bai
    • Guo‑He Yan
    • Guo‑Jian Wang
    • Ping Wan
    • Xiao‑Fei Liu
    • Heng Wang
    • Rong‑Hua Zhang
  • View Affiliations

  • Published online on: July 6, 2017     https://doi.org/10.3892/mmr.2017.6945
  • Pages: 2785-2790
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Abstract

The present study aimed to construct a lentiviral RNA interference (RNAi) vector targeting the transforming growth factor β1 (TGFβ1) gene of rats, in order to examine its effect on silencing of the TGFβ1 gene and on the expression of collagen type 1 α1 (Col1a1) in HSC‑T6 rat hepatic stellate cells. Three RNAi sites of the TGFβ1 gene were selected according to its CDs sequence. Three pairs of small interfering RNA (siRNA) of these RNAi sites were synthesized and then transfected into HSC‑T6 cells, respectively, to confirm the optimal siRNA sequence via reverse transcription‑polymerase chain reaction analysis. Subsequently, shRNA targeting the sequence of the optimal siRNA was designed, synthesized and annealed to form a double‑stranded structure. The annealed oligonucleotide fragment was cloned into pGreenPuro plasmids to establish the pGreenPuro/TGFβ1 shRNA lentiviral vector, which was then transfected into 293T cells, following identification by restriction enzyme digestion and sequencing for the production of lentiviral particles exhibiting high reactivity. These particles were used to infect HSC‑T6 cells, following which the expression of GFP in the transfected cells was observed under an inverted microscope. The effects on TGFβ1 gene silencing and the expression levels of Colla1 were detected at the mRNA and protein levels. The results provided confirmation of the optimal siRNA sequence. Enzyme digestion and sequencing verified successful construction of the pGreenPuro/TGFβ1 shRNA lentiviral vector. This lentiviral vector effectively silenced the TGFβ1 gene in the HSC‑T6 cells, and inhibited the expression of Col1a1 at the mRNA and protein levels. Taken together, the lentiviral RNAi vector targeting the TGFβ1 gene of rats was successfully constructed, which effectively silenced the TGFβ1 gene of the HSC‑T6 cells and inhibited the expression of Col1a1.
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September-2017
Volume 16 Issue 3

Print ISSN: 1791-2997
Online ISSN:1791-3004

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Spandidos Publications style
Bai GP, Yan GH, Wang GJ, Wan P, Liu XF, Wang H and Zhang RH: Effects of lentiviral vector‑mediated shRNA silencing of TGFβ1 on the expression of Col1a1 in rat hepatic stellate cells. Mol Med Rep 16: 2785-2790, 2017
APA
Bai, G., Yan, G., Wang, G., Wan, P., Liu, X., Wang, H., & Zhang, R. (2017). Effects of lentiviral vector‑mediated shRNA silencing of TGFβ1 on the expression of Col1a1 in rat hepatic stellate cells. Molecular Medicine Reports, 16, 2785-2790. https://doi.org/10.3892/mmr.2017.6945
MLA
Bai, G., Yan, G., Wang, G., Wan, P., Liu, X., Wang, H., Zhang, R."Effects of lentiviral vector‑mediated shRNA silencing of TGFβ1 on the expression of Col1a1 in rat hepatic stellate cells". Molecular Medicine Reports 16.3 (2017): 2785-2790.
Chicago
Bai, G., Yan, G., Wang, G., Wan, P., Liu, X., Wang, H., Zhang, R."Effects of lentiviral vector‑mediated shRNA silencing of TGFβ1 on the expression of Col1a1 in rat hepatic stellate cells". Molecular Medicine Reports 16, no. 3 (2017): 2785-2790. https://doi.org/10.3892/mmr.2017.6945