Applications Of Genetically Engineered MicroRNA-34a Prodrug In Osteosarcoma

Part Ⅰ:Genetically engineered miR-34a prodrug suppresses orthotopic osteosarcoma tumor growth via the induction of apoptosis and cell cycle arrestBackground:Osteosarcoma (OS) is the most common primary malignant bone tumor in children and young adults, and microRNA-34a (miR-34a) represents a new agent to treat cancer diseases. Recently we have developed a novel approach to bioengineering miR-34a prodrug. This study is to define the effectiveness and safety profiles of biological miR-34a prodrug in an orthotopic OS xenograft tumor mouse model.Methods:MTT assay was used to examine antiproliferative activity against OS cells, and Matrigel invasion assay was employed to determine invasion capacity. Impact of miR-34a on apoptosis and cell cycle were assessed by cytometric studies, and protein levels of several miR-34a target genes were evaluated by Western blots. Orthotopic 143B xenograft mouse model was established to define the efficacy of miR-34a prodrug formulated with in vivo-jetPEI and administered intravenously. Blood chemistry profiles were determined to assess safety profiles.Results:Biologic miR-34a prodrug significantly reduced the proliferation of human OS 143B and MG-63 cells in a dose dependent manner and to a much greater degree than the controls, which was attributable to the induction of apoptosis and G2 cell cycle arrest. Inhibition of cell growth and invasion were associated with reduction of protein levels of miR-34a target genes including SIRT1, BCL2, c-MET, and CDK.6. Furthermore, therapeutic doses of miR-34a prodrug significantly repressed the growth of orthotopic 143B xenograft tumors and were well tolerated in the animals.Conclusions:Bioengineered miR-34a prodrug is effective to control OS tumor growth which involves the induction of apoptosis and cell cycle arrest.Part Ⅱ:Combination therapy with bioengineered miR-34a prodrug and doxorubicin synergistically suppresses osteosarcoma growthOsteosarcoma (OS) is the most common form of primary malignant tumor in a bone and prevalent among children and young adults. Recently we have established a novel approach to bioengineering large quantity of microRNA-34a (miR-34a) prodrug for miRNA replacement therapy. This study is to evaluate combination treatment with miR-34a prodrug and doxorubicin chemotherapy, which may synergistically suppress human OS cell growth via RNA interference and DNA intercalation. Synergistic effects were indeed obvious between miR-34a prodrug and doxorubicin for the suppression of OS cell proliferation, as defined by Chou-Talalay method. The strongest antiproliferative synergism was achieved when both agents were administered simultaneously to the cells at early stage, which was associated with much greater degrees of late apoptosis, necrosis, and G2 cell cycle arrest. Alteration of OS cellular processes and invasion capacity was also linked to the reduction of protein levels of miR-34a targeted (proto-)oncogenes including SIRT1, c-MET, and CDK6. Moreover, orthotopic OS xenograft tumor growth was repressed to a significantly greater degree by miR-34a prodrug and doxorubicin co-administered intravenously to mouse models. In addition, multiple doses of miR-34a prodrug and doxorubicin had no or minimal effects on mouse blood chemistry profiles. The results demonstrate that combination of doxorubicin chemotherapy and miR-34a replacement therapy produces synergistic antiproliferative effects and is more efficacious than monotherapy for the suppression of OS xenograft tumor growth. These findings support the development of mechanism-based combination therapy to combat OS and bioengineered miR-34a prodrug represents a new natural miRNA agent.