RNA interference targeting leptin gene effect on hepatic stellate cells

Abstract

To construct the specific siRNA expression vectors and investigate their effect on leptin and collagen I in HSC, which provide a new approach to the prevent and treat hepatic fibrosis. The five siRNAs against leptin gene were transcript synthesized intracellularly by expression templates of plasmid vector psiRNA-hH1 neo. The recombinant leptin siRNA plasmid vectors could express in eukaryocyte, and then to evaluate them by using enzyme cutting and sequencing. The recombinant plasmids were transfected into HSCs using Lipofectamine methods respectively. The cells were selected after growing in DMEM containing 300 μg/mL G418 for about 4 weeks. Gene expression of leptin and collagen I were showed by Western blot analysis and reverse transcription polymerase chain reaction (RT-PCR). Identification by enzyme cutting and sequencing showed that the leptin siRNA expression vectors were constructed successfully, and leptin siRNA could inhibit the leptin and collagen I gene expression effectively. It was concluded that RNA interference-mediated silencing of leptin gene diminished leptin and collagen I gene expression in HSCs. Furthermore, attenuated the extracellular matrix over-deposition at the same time. Leptin gene is ideal targets of gene therapy for liver fibrosis.

Keywords

siRNA / hepatic stellate cells / leptin / collagen

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