RNA interference (RNAi) denotes sequence-specific mRNA degradation induced by short interfering double-stranded RNA (siRNA) and has become a revolutionary tool for functional annotation of mammalian genes, as well as for development of novel therapeutics. The practical applications of RNAi are usually achieved by expressing short hairpin RNAs (shRNAs) or siRNAs in cells. However, a major technical challenge is to simultaneously express multiple siRNAs to silence one or more genes. We previously developed pSOS system, in which siRNA duplexes are made from oligo templates driven by opposing U6 and H1 promoters. While effective, it is not equipped to express multiple siRNAs in a single vector. Gibson DNA Assembly (GDA) is an in vitro recombination system that has the capacity to assemble multiple overlapping DNA molecules in a single isothermal step. Here, we developed a GDA-based pSOK assembly system for constructing single vectors that express multiple siRNA sites. The assembly fragments were generated by PCR amplifications from the U6-H1 template vector pB2B. GDA assembly specificity was conferred by the overlapping unique siRNA sequences of insert fragments. To prove the technical feasibility, we constructed pSOK vectors that contain four siRNA sites and three siRNA sites targeting human and mouse β-catenin, respectively. The assembly reactions were efficient, and candidate clones were readily identified by PCR screening. Multiple β-catenin siRNAs effectively silenced endogenous β-catenin expression, inhibited Wnt3A-induced β-catenin/Tcf4 reporter activity and expression of Wnt/β-catenin downstream genes. Silencing β-catenin in mesenchymal stem cells inhibited Wnt3A-induced early osteogenic differentiation and significantly diminished synergistic osteogenic activity between BMP9 and Wnt3A in vitro and in vivo. These findings demonstrate that the GDA-based pSOK system has been proven simplistic, effective and versatile for simultaneous expression of multiple siRNAs. Thus, the reported pSOK system should be a valuable tool for gene function studies and development of novel therapeutics.

Osteosarcoma (OS) is the most common primary malignant tumor of bone with a high propensity for lung metastasis. Despite significant advances in surgical techniques and chemotherapeutic regimens over the past few decades, there has been minimal improvement in OS patient survival. There is an urgent need to identify novel antitumor agents to treat human OS. Repurposing the clinically-used drugs represents a rapid and effective approach to the development of new anticancer agents. The anthelmintic drug niclosamide has recently been identified as a potential anticancer agent in human cancers. Here, we investigate if niclosamide can be developed as an anti-OS drug. We find that niclosamide can effectively inhibit OS cell proliferation and survival at low micromolar concentrations. Cell migration and wounding closure are significantly inhibited by niclosamide. Niclosamide induces cell apoptosis and inhibits cell cycle progression in OS cells. Analysis of niclosamide's effect on 11 cancer-related signal pathway reporters reveals that three of them, the E2F1, AP1, and c-Myc-responsive reporters, are significantly inhibited. To a lesser extent, the HIF1α, TCF/LEF, CREB, NFκB, Smad/TGFβ, and Rbpj/Notch pathway reporters are also inhibited, while the NFAT and Wnt/β-catenin reporters are not significantly affected by niclosamide treatment. We demonstrate that the expression of c-Fos, c-Jun. E2F1, and c-Myc in OS cells is effectively inhibited by niclosamide. Furthermore, niclosamide is shown to effectively inhibit tumor growth in a mouse xenograft tumor model of human osteosarcoma cells. Taken together, these results strongly suggest that niclosamide may exert its anticancer activity in OS cells by targeting multiple signaling pathways. Future investigations should be directed to exploring the antitumor activity in clinically relevant OS models and ultimately in clinical trials.

Sustained, high level transgene expression in mammalian cells, especially stem cells, may be desired in many cases for studying gene functions. Traditionally, stable transgene expression has been accomplished by using retroviral or lentiviral vectors. However, such viral vector-mediated transgene expression is often at low levels and can be reduced over time due to low copy numbers and/or chromatin remodeling repression. The piggyBac transposon has emerged as a promising non-viral vector system for efficient gene transfer into mammalian cells. Despite its inherent advantages over lentiviral and retroviral systems, piggyBac system has not been widely used, at least in part due to the limited availability of piggyBac vectors with manipulation flexibilities. Here, we seek to optimize piggyBac-mediated transgene expression and generate a more efficient, user-friendly piggyBac system. By engineering a panel of versatile piggyBac vectors and constructing recombinant adenoviruses expressing piggyBac transposase (PBase), we demonstrate that adenovirus-mediated PBase expression significantly enhances the integration efficiency and expression level of transgenes in mesenchymal stem cells and osteosarcoma cells, compared to that obtained from co-transfection of the CMV-PBase plasmid. We further determine the drug selection timeline to achieve optimal stable transgene expression. Moreover, we demonstrate that the transgene copy number of piggyBac-mediated integration is approximately 10 times higher than that mediated by retroviral vectors. Using the engineered tandem expression vector, we show that three transgenes can be simultaneously expressed in a single vector with high efficiency. Thus, these results strongly suggest that the optimized piggyBac system is a valuable tool for making stable cell lines with sustained, high transgene expression.