A High-Throughput Screening Identifies MicroRNA Inhibitors That Influence Neuronal Maintenance and/or Response to Oxidative Stress

Abstract

Small non-coding RNAs (sncRNAs), including microRNAs (miRNAs) are important post-transcriptional gene expression regulators relevant in physiological and pathological processes. Here, we combined a high-throughput functional screening (HTFS) platform with a library of antisense oligonucleotides (ASOs) to systematically identify sncRNAs that affect neuronal cell survival in basal conditions and in response to oxidative stress (OS), a major hallmark in neurodegenerative diseases. We considered hits commonly detected by two statistical methods in three biological replicates. Forty-seven ASOs targeting miRNAs (miRNA-ASOs) consistently decreased cell viability under basal conditions. A total of 60 miRNA-ASOs worsened cell viability impairment mediated by OS, with 36.6% commonly affecting cell viability under basal conditions. In addition, 40 miRNA-ASOs significantly protected neuronal cells from OS. In agreement with cell viability impairment, damaging miRNA-ASOs specifically induced increased free radical biogenesis. miRNAs targeted by the detrimental ASOs are enriched in the fraction of miRNAs downregulated by OS, suggesting that the miRNA expression pattern after OS contributes to neuronal damage. The present HTFS highlighted potentially druggable sncRNAs. However, future studies are needed to define the pathways by which the identified ASOs regulate cell survival and OS response and to explore the potential of translating the current findings into clinical applications.

Keywords: expression profiles; high-throughput screening; miRNAs; mitochondrial function; neurodegeneration; non-coding RNAs; oxidative stress; small RNA sequencing.

Figure 1

Scheme of the Experimental Design for the HTFS

Figure 2

Cell Viability Determinations in the HTFS under Basal Conditions and in Response to Oxidative Stress Cell viability determinations in every plate are shown in mock-transfected cells, cells transfected with a negative control sncRNA-ASO (C−), and cells transfected with the positive control sncRNA-ASO (C+), under basal conditions and following treatment with 3 mM MPP+. Each dot represents the percentage of cell viability determinations in a well, with 100% considered to be the mock-transfected cells under basal conditions. All values are represented relative to a mock-transfected well under basal conditions. Differences between conditions were determined by using linear mixed-effects models, where experimental conditions to be compared are included in the model as fixed affects, and plates are included as random effects to account for between-plate variability. A Bonferroni correction was applied to account for multiple comparisons (number of comparisons = 9). Significant differences are found between MPP⁺ treatment and basal conditions, in each experimental condition (Mock, C−, and C+; *p < 0.001), and in C+ versus mock and C− under basal conditions and after exposure to MPP⁺ (+p < 0.001).

Figure 3

Validation of HTFS Hits Based on the Evaluation of ROS Levels Plots of relative ROS levels in cells transfected with sncRNA-ASOs in basal conditions (A) and after exposure to 3 mM MPP⁺ (B). ROS levels in each biological replicate are represented by a dot and expressed relative to a control mock-transfected well. In the plots, numbers, and colors correspond to the diverse transfected sncRNA-ASOs: blue, sncRNA-ASOs that impair cell viability in basal conditions (asterisks label miRNA-ASOs that impair cell viability both under basal conditions and in response to MPP+); green, sncRNA-ASOs that worsen cell viability in response to MPP+; orange, sncRNA-ASOs that protect against MPP+; black, ncRNA-ASOs with no effect on cell viability, including a scrambled sequence used as a negative control (C−); red, an ASO with a known toxic effect used as a positive control (C+). (C) Plot showing the fold change (FC) of ROS levels in cells transfected with miRNA-ASOs versus mock-transfected cells, under basal conditions (upper panel) or in response to MPP+ (lower panel). Exp1: FC considering the miRNA-ASOs in blue; Exp2a, FC considering miRNA-ASOs in green and those in blue marked with an asterisk; Exp2b, FC considering miRNA-ASOs in green; Exp3, FC considering miRNA-ASOs in orange; and Exp4, FC considering miRNA-ASOs in black.

Figure 4

Mitochondrial Activity after Transfection with Specific miRNA-ASOs Mock, cells treated with Lipofectamine; negative control (C−), cells transfected with an ASO that does not produce any effect in cell viability; positive control (C+), cells transfected with a control sncRNA-ASO inducing cell death; positive control (CCCP), cells treated with CCCP were used as a positive control for mitochondrial functional loss. The boxplot shows absorbance determinations after incubation with MitoTracker Deep Red reagent. *p < 0.001, by the Kruskal-Wallis test, followed by the Mann-Whitney U test with Bonferroni’s correction as a post hoc test (n = 5).