School of Basic Medical Sciences, Xi'an Jiaotong University, Xi'an 710061, China.
Department of Chemistry, Korea University, Seoul 02841, Korea.
Theranostics. 2023 Apr 23;13(8):2552-2561. doi: 10.7150/thno.84111. eCollection 2023.
MicroRNAs (miRNAs) play key roles in multiple biological processes, many of which exhibit distinct cell type-specific expression patterns. A miRNA-inducible expression system can be adapted as a signal-on reporter for detecting miRNA activity or as a cell type-specific gene activation tool. However, due to the inhibitory properties of miRNAs on gene expression, few miRNA-inducible expression systems are available, and the available systems are only transcriptional or post-transcriptional regulatory system with obvious leaky expression. To address this limitation, a miRNA-inducible expression system that can tightly control target gene expression is desirable. Here, by taking advantage of an enhanced LacI repression system and the translational repressor L7Ae, a miRNA-inducible dual transcriptional-translational switch system was designed called the miR-ON-D system. Luciferase activity assay, western blotting, CCK-8 assay and flow cytometry analysis were performed to characterize and validate this system. The results demonstrated that leakage expression was strongly suppressed in the miR-ON-D system. It was also validated that the miR-ON-D system could be used to detect exogenous and endogenous miRNAs in mammalian cells. Moreover, it was shown that the miR-ON-D system could be triggered by cell type-specific miRNAs to regulate the expression of biologically relevant proteins (e.g., p21 and Bax) to achieve cell type-specific reprogramming. This study established a tight miRNA-inducible expression switch system for miRNA detection and cell type-specific gene activation.
微小 RNA（miRNA）在多种生物过程中发挥着关键作用，其中许多过程表现出独特的细胞类型特异性表达模式。miRNA 诱导表达系统可作为检测 miRNA 活性的信号开启报告子，或作为细胞类型特异性基因激活工具。然而，由于 miRNA 对基因表达的抑制特性，可用的 miRNA 诱导表达系统很少，并且现有的系统仅为转录或转录后调控系统，存在明显的漏表达。为了解决这一限制，需要设计一种能够紧密控制靶基因表达的 miRNA 诱导表达系统。在这里，我们利用增强型 LacI 抑制系统和翻译抑制剂 L7Ae，设计了一种称为 miR-ON-D 的 miRNA 诱导的双转录-翻译开关系统。通过荧光素酶活性测定、Western blot、CCK-8 测定和流式细胞术分析对该系统进行了表征和验证。结果表明，miR-ON-D 系统强烈抑制了漏表达。还验证了该系统可用于检测哺乳动物细胞中外源和内源性 miRNA。此外，还表明 miR-ON-D 系统可以被细胞类型特异性 miRNA 触发，以调节生物相关蛋白（如 p21 和 Bax）的表达，从而实现细胞类型特异性重编程。本研究建立了一种用于 miRNA 检测和细胞类型特异性基因激活的紧密 miRNA 诱导表达开关系统。
