An Image-Based Biosensor Assay Strategy to Screen for Modulators of the microRNA 21 Biogenesis Pathway

Abstract

microRNAs (miRNAs) are evolutionary conserved, small endogenous non-coding, RNA molecules. Although their mode of action has been extensively studied, little is known about their biogenesis. As their altered expression has been implicated in many diseases, small molecules that would modulate their expression are sought after. They are generated through the concerted action of several complexes which promote their transcription, maturation, export, trafficking, and loading of mature miRNA into silencing complexes. An increasing number of studies have suggested that each of these steps serves as a regulatory junction in the process, and therefore provides an intervention point. For this purpose, we have developed a simple image-based assay strategy to screen for such modulators. Here, we describe its successful implementation which combines the use of a microRNA 21 (miR-21) synthetic mimic together with an EGFP based reporter cell line, where its expression is under the control of miR-21, to monitor EGFP expression in a format suitable for HTS. The strategy was further validated using a small panel of known gene modulators of the miRNA pathway. A screen was performed in duplicate against a library of 6,912 compounds and identified 48 initial positives exhibiting enhanced EGFP fluorescence intensity. 42 compounds were found to be inherently fluorescent in the green channel leaving the remaining 6 as potential inhibitors and with a positive rate of 0.09%. Taken together, this validated strategy offers the opportunity to discover novel and specific inhibitors of the pathway through the screening of diverse chemical libraries.

An Arrayed RNA Interference Genome-Wide Screen Identifies Candidate Genes Involved in the MicroRNA 21 Biogenesis Pathway

“…19 In brief, HeLaS3 cells were seeded into a 24-well plate at 200,000 cells per well in 500 mL of growth media containing Dulbecco's modified Eagle's medium (DMEM), high glucose with L-glutamine, D-glucose, and sodium pyruvate supplemented with 10% heat-inactivated fetal bovine serum (FBS). After 24 h, pcDNA/TO/EGFPmiR21 (Addgene, Cambridge, MA) was diluted with Opti-MEM media to 0.2 mg/well; in which Lipofectamine 2000 transfection solution was added at a final concentration of 0.4 mL/ well, and incubated at room temperature for 15 min to promote transfection reagent complex formation.…”

“…Zeocin-resistant cells were harvested and cell stocks were stored at -170°C. 19 Cells were grown at 37°C and 5% CO 2 in complete growth media containing DMEM, high glucose with L-glutamine, D-glucose, and sodium pyruvate supplemented with 10% heat-inactivated FBS, and 200 mg/mL of Zeocin. All cell culture supplies were from Life Technologies (Carlsbad, CA) and Sigma-Aldrich (St Louis, MO).…”

“…19 To validate the reporter assay for RNAi screening, our optimized conditions were tested with a focused panel targeting 17 genes: (1) known and active components of the miRNA pathway: DGCR8, DICER1, DROSHA, EIF2C1, EIF2C2, EIF2C3, EIF2C4, DHX9, MOV10, TARBP2, TNRC6A, TNRC6B, TNRC6C, XPO5, (2) cell cycle regulators as functional controls for transfection efficiency: KIF11, PLK1, and (3) an unrelated gene as a negative functional control: SCPEP1. Three siRNA duplexes per gene were obtained from the Ambion Silencer Select v4.0 library, including the Silencer Select Negative Control siRNA #1 (4390843) as the assay negative control.…”

“…19 The reporter cell line expresses a plasmid encoding EGFP fused to a sequence with perfect complementarity to miR-21 in its 3 0 UTR region, whereby miR-21 endogenously generated by the HeLa S3 cells specifically destabilizes the EGFP mRNA resulting in low-level protein expression of EGFP; hence, low fluorescence output. By knocking down genes potentially involved in the miR-21 biogenesis, the miR-21 levels will be reduced; thus, allowing for the expression and stabilization of the EGFP mRNA resulting in an increase production of the EGFP protein and its fluorescence output (Fig.…”

“…18 As such, we made use of a recently described reporter cell line of miRNA activity for chemical screening; adapting the multiplexing potential of the assay to search for modulator genes using siRNA technology to interrogate and dissect the miRNA biogenesis pathway by loss-of-function analysis. 19 Our strategy makes use of a multiplexed-approach to score for genes that modulate miR-21 biogenesis by monitoring enhanced green fluorescent protein (EGFP) expression, while simultaneously assessing cell death through imaged Hoechst-stained nuclei (NUCL), and the cellular metabolic activity through the use of the Alamar Blue (AB).…”