College of Life Science, Jilin University, Changchun, Jilin, China.
PLoS One. 2012;7(1):e29551. doi: 10.1371/journal.pone.0029551. Epub 2012 Jan 5.
microRNAs (miRNAs) are small and non-coding RNAs which play critical roles in physiological and pathological processes. A number of methods have been established to detect and quantify miRNA expression. However, method for high-throughput miRNA function detection is still lacking.
We describe an adeno-associated virus (AAV) vector-based microRNA (miRNA) sensor (Asensor) array for high-throughput functional miRNA profiling. Each Asensor contains a Gaussia luciferase (Gluc) and a firefly luciferase (Fluc) expression cassette to sense functional miRNA and to serve as an internal control respectively. Using this array, we acquired functional profiles of 115 miRNAs for 12 cell lines and found "functional miRNA signatures" for several specific cell lines. The activities of specific miRNAs including the let-7 family, miR-17-92 cluster, miR-221, and miR-222 in HEK 293 cells were compared with their expression levels determined by quantitative reverse transcriptase polymerase chain reaction (QRT-PCR). We also demonstrate two other practical applications of the array, including a comparison of the miRNA activity between HEK293 and HEK293T cells and the ability to monitor miRNA activity changes in K562 cells treated with 12-O-tetradecanoylphorbol-13-acetate (TPA).
CONCLUSIONS/SIGNIFICANCE: Our approach has potential applications in the identification of cell types, the characterization of biological and pathological processes, and the evaluation of responses to interventions.
microRNAs（miRNAs）是在生理和病理过程中发挥关键作用的小非编码 RNA。已经建立了许多方法来检测和定量 miRNA 的表达。然而，高通量 miRNA 功能检测的方法仍然缺乏。
我们描述了一种基于腺相关病毒（AAV）载体的 microRNA（miRNA）传感器（Asensor）阵列，用于高通量功能 miRNA 分析。每个 Asensor 包含一个海肾荧光素酶（Gluc）和一个萤火虫荧光素酶（Fluc）表达盒，分别用于感应功能性 miRNA 和作为内部对照。使用该阵列，我们获得了 12 种细胞系中 115 种 miRNA 的功能谱，并发现了几种特定细胞系的“功能性 miRNA 特征”。特定 miRNAs 的活性，包括 let-7 家族、miR-17-92 簇、miR-221 和 miR-222 在 HEK 293 细胞中的活性，与通过定量逆转录聚合酶链反应（QRT-PCR）确定的表达水平进行了比较。我们还展示了该阵列的另外两个实际应用，包括 HEK293 和 HEK293T 细胞之间 miRNA 活性的比较，以及监测 12-O-十四烷酰佛波醇-13-乙酸酯（TPA）处理的 K562 细胞中 miRNA 活性变化的能力。
结论/意义：我们的方法在鉴定细胞类型、描述生物学和病理过程以及评估对干预的反应方面具有潜在的应用价值。
