Functional microRNA screening using a comprehensive lentiviral human microRNA expression library

Abstract

BackgroundMicroRNAs (miRNAs) are a class of small regulatory RNAs that target sequences in messenger RNAs (mRNAs) to inhibit their protein output. Dissecting the complexities of miRNA function continues to prove challenging as miRNAs are predicted to have thousands of targets, and mRNAs can be targeted by dozens of miRNAs.ResultsTo systematically address biological function of miRNAs, we constructed and validated a lentiviral miRNA expression library containing 660 currently annotated and 422 candidate human miRNA precursors. The miRNAs are expressed from their native genomic backbone, ensuring physiological processing. The arrayed layout of the library renders it ideal for high-throughput screens, but also allows pooled screening and hit picking. We demonstrate its functionality in both short- and long-term assays, and are able to corroborate previously described results of well-studied miRNAs.ConclusionsWith the miRNA expression library we provide a versatile tool for the systematic elucidation of miRNA function.

An Image-Based miRNA Screen Identifies miRNA-135s As Regulators of CNS Axon Growth and Regeneration by Targeting Krüppel-like Factor 4

“…In contrast, decreasing the availability of miR-135s further inhibits RGC axon regeneration. The developmental and regeneration-promoting effects of miR-135s both rely in part on silencing of KLF4, a well known intrinsic inhibitor of axon regeneration (Moore et al, 2009;Qin et al, 2013). Together, these data identify novel neuronal functions for miR-135s and highlight the potential of applying miRNAs to injured CNS neurons as an approach for facilitating CNS axon regeneration.…”

“…However, for many of these targets (e.g., PTK2, TAF4), knock-down had been reported to reduce neurite growth or neuron migration (data not shown). KLF4 was particularly interesting because knock-down of KLF4 in neurons, similar to overexpression of miR-135s, enhances axon growth, leading process length, and neuronal migration (Moore et al, 2009;Qin and Zhang, 2012). Furthermore, recent work in vascular smooth muscle and hepatocellular carcinoma cells links miRNA-135a to KLF4 (Lin et al, 2016;Yao et al, 2016).…”

A simple High-Throughput Technology Enables Gain-of-Function Screening of Human microRNAs

“…For example, the barcode of a miR whose overexpression inhibits cell growth would decrease over time; Izumiya et al identified candidate pancreatic cancer suppressive miRs on this basis (22). Conversely, by determining barcodes enriched in migrated cells vs. the starting cell population, candidate miRs that promoted invasiveness of cancer cell lines were identified (23,26). Although these studies successfully employed pooled viral miR libraries in functional screens, their viral miR barcode quantification methods required custom-made microarrays (22,24), bead-based detection systems (20,21) or high-throughput sequencing (23,26).…”

“…Conversely, by determining barcodes enriched in migrated cells vs. the starting cell population, candidate miRs that promoted invasiveness of cancer cell lines were identified (23,26). Although these studies successfully employed pooled viral miR libraries in functional screens, their viral miR barcode quantification methods required custom-made microarrays (22,24), bead-based detection systems (20,21) or high-throughput sequencing (23,26). Those approaches require access to costly high-throughput infrastructure and sophisticated informatics analyses, thus restricting the accessibility of these assays.…”

An In Vivo Functional Screen Uncovers miR-150-Mediated Regulation of Hematopoietic Injury Response

“…Indeed, in vitro screens based on miRNA expression libraries have proven highly useful (Voorhoeve et al, 2006; Izumiya et al, 2010; Huang et al, 2008; Poell et al, 2011). For instance, miR-372/373 were found to be potent oncogenes that have a stronger effect than p53 knockdown in relieving oncogene-induced senescence (Voorhoeve et al, 2006).…”