Effects of lentiviral vector-mediated shRNA silencing of TGF beta 1 on the expression of Col1a1 in rat hepatic stellate cells

摘要

The present study aimed to construct a lentiviral RNA interference (RNAi) vector targeting the transforming growth factor beta 1 (TGF beta 1) gene of rats, in order to examine its effect on silencing of the TGF beta 1 gene and on the expression of collagen type 1 alpha 1 (Col1a1) in HSC-T6 rat hepatic stellate cells. Three RNAi sites of the TGF beta 1 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 pGreen-Puro plasmids to establish the pGreenPuro/TGF beta 1 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 TGF beta 1 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/TGF beta 1 shRNA lentiviral vector. This lentiviral vector effectively silenced the TGF beta 1 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 TGF beta 1 gene of rats was successfully constructed, which effectively silenced the TGF beta 1 gene of the HSC-T6 cells and inhibited the expression of Col1a1.