Artificial MicroRNA-Mediated Tgfbr2 and Pdgfrb Co-Silencing Ameliorates Carbon Tetrachloride–Induced Hepatic Fibrosis in Mice

Abstract

Hepatic stellate cells (HSCs) are the primary cell type responsible for liver fibrogenesis. Transforming growth factor beta 1 (TGF-β1) and platelet-derived growth factor (PDGF) are key profibrotic cytokines that regulate HSC activation and proliferation with functional convergence. Dual RNA interference against their receptors may achieve therapeutic effects. A novel RNAi strategy based on HSC-specific GFAP promoter-driven and lentiviral-expressed artificial microRNAs (amiRNAs) was devised that consists of an microRNA-30a backbone and effective shRNAs against mouse Pdgfrβ and Tgfbr2. Then, its antifibrotic efficacy was tested in primary and cultured HSCs and in mice affected with carbon tetrachloride–induced hepatic fibrosis. The study shows that amiRNA-mediated Pdgfrβ and Tgfbr2 co-silencing inhibits HSC activation and proliferation. After recombinant lentiviral particles were delivered into the liver via tail-vein injection, therapeutic amiRNAs were preferentially expressed in HSCs and efficiently co-knocked down in situ Tgfbr2 and Pdgfrβ expression, which correlates with downregulated expression of target or effector genes of their signaling, which include Pai-1, P70S6K, and D-cyclins. amiRNA-based HSC-specific co-silencing of Tgfbr2 and Pdgfrβ significantly suppressed hepatic expression of fibrotic markers α-Sma and Col1a1, extracellular matrix regulators Mmps and Timp1, and phenotypically ameliorated liver fibrosis, as indicated by reductions in serum alanine aminotransferase activity, collagen deposition, and α-Sma-positive staining. The findings provide proof of concept for the use of amiRNA-mediated co-silencing of two profibrogenic pathways in liver fibrosis treatment and highlight the therapeutic potential of concatenated amiRNAs for gene therapy.