1. Department of Orthopedic Trauma and Microsurgery, Zhongnan Hospital of Wuhan University, Wuhan, Hubei 430071, China.
2. Department of Biochemistry & Molecular Medicine, UC Davis School of Medicine, Sacramento, CA 95817, USA.
3. Department of Pathology, Roswell Park Cancer Institute, Buffalo, NY 14263, USA.
4. Division of Hematology Oncology, UC Davis School of Medicine, Sacramento, CA 95817, USA.
5. Department of Internal Medicine, UC Davis School of Medicine, Sacramento, CA 95817, USA.
#These authors contributed equally to this work.
Rationale: Noncoding RNAs (ncRNAs) such as microRNAs (miRs or miRNAs) play important roles in the control of cellular processes through posttranscriptional gene regulation. However, ncRNA research is limited to utilizing RNA agents synthesized in vitro. Recombinant RNAs produced and folded in living cells shall better recapitulate biologic RNAs.
Methods: Herein, we developed a novel platform for in vivo fermentation production of humanized recombinant ncRNA molecules, namely hBERAs, carrying payload miRNAs or siRNAs. Target hBERAs were purified by anion exchange FPLC method. Functions of hBERA/miRNAs were investigated in human carcinoma cells and antitumor activities were determined in orthotopic osteosarcoma xenograft spontaneous lung metastasis mouse models.
Results: Proper human tRNAs were identified to couple with optimal hsa-pre-miR-34a as new fully-humanized ncRNA carriers to accommodate warhead miRNAs or siRNAs. A group of 30 target hBERAs were all heterogeneously overexpressed (each accounting for >40% of total bacterial RNA), which facilitated large-scale production (8-31 mg of individual hBERAs from 1L bacterial culture). Model hBERA/miR-34a-5p and miR-124-3p were selectively processed to warhead miRNAs in human carcinoma cells to modulate target gene expression, enhance apoptosis and inhibit invasiveness. In addition, bioengineered miR-34a-5p and miR-124-3p agents both reduced orthotopic osteosarcoma xenograft tumor growth and spontaneous pulmonary metastases significantly.
Conclusion: This novel ncRNA bioengineering technology and resulting recombinant ncRNAs are unique additions to conventional technologies and tools for basic research and drug development.
Keywords: ncRNA, miRNA, bioengineering, therapy, cancer