Department of Orthopedic Trauma and Microsurgery, Zhongnan Hospital of Wuhan University, Wuhan, Hubei 430071, China.
Department of Biochemistry & Molecular Medicine, UC Davis School of Medicine, Sacramento, CA 95817, USA.
Theranostics. 2021 Mar 4;11(10):4858-4871. doi: 10.7150/thno.56596. eCollection 2021.
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 . Recombinant RNAs produced and folded in living cells shall better recapitulate biologic RNAs. Herein, we developed a novel platform for 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. 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. This novel ncRNA bioengineering technology and resulting recombinant ncRNAs are unique additions to conventional technologies and tools for basic research and drug development.
非编码 RNA（ncRNA），如 microRNA（miR 或 miRNAs），通过转录后基因调控在细胞过程的控制中发挥重要作用。然而，ncRNA 研究仅限于利用合成的 RNA 制剂。在活细胞中产生和折叠的重组 RNA 将更好地重现生物 RNA。在此，我们开发了一种用于发酵生产携带有效载荷 miRNA 或 siRNA 的人源化重组 ncRNA 分子（即 hBERA）的新型平台。通过阴离子交换 FPLC 方法纯化靶向 hBERA。在人癌细胞中研究 hBERA/miRNAs 的功能，并在原位骨肉瘤异种移植自发肺转移小鼠模型中测定抗肿瘤活性。鉴定适当的人 tRNA 与最佳 hsa-pre-miR-34a 结合作为新的完全人源化 ncRNA 载体，以容纳弹头 miRNA 或 siRNA。鉴定了一组 30 个靶向 hBERA，它们均异质过表达（每个占总细菌 RNA 的>40%），从而实现了大规模生产（从 1L 细菌培养物中获得 8-31mg 的单个 hBERA）。模型 hBERA/miR-34a-5p 和 miR-124-3p 被选择性地加工成弹头 miRNA 在人癌细胞中调节靶基因表达，增强细胞凋亡并抑制侵袭性。此外，生物工程 miR-34a-5p 和 miR-124-3p 制剂均可显著减少原位骨肉瘤异种移植肿瘤生长和自发肺转移。这种新型 ncRNA 生物工程技术和由此产生的重组 ncRNA 是对常规技术和工具的独特补充，可用于基础研究和药物开发。
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