Original Article A High-Throughput Screening Identifies MicroRNA Inhibitors That Influence Neuronal Maintenance and/or Response to Oxidative Stress Joan Pallarès-Albanell,1,7 M. Teresa Zomeño-Abellán,1,7 Georgia Escaramís,1,2,3,4 Lorena Pantano,5 Aroa Soriano,6 Miguel F. Segura,6 and Eulàlia Martí1,2,3,4 1Centre for Genomic Regulation (CRG), The Barcelona Institute for Science and Technology, Dr. Aiguader 88, Barcelona 08003, Spain; 2Departament de Biomedicina, Facultat de Medicina i Ciències de la Salut, Institut de Neurociències, Universitat de Barcelona, Barcelona 08036, Spain; 3Research Group on Statistics, Econometrics and Health, Universitat de Girona, 17003, Girona, Spain; 4Centro de Investigación Biomédica en Red de Epidemiología y Salud Pública (CIBERESP), Ministerio de Ciencia Innovación y Universidades, Madrid, Spain; 5Bioinformatics Core, Department of Biostatistics, Harvard T.H. Chan School of Public Health, Boston, MA 02115, USA; 6Group of Translational Research in Child and Adolescent Cancer, Vall d’Hebron Research Institute (VHIR), Universitat Autònoma de Barcelona (UAB), Passeig Vall d’Hebron 119, Barcelona 08035, Spain Small non-coding RNAs (sncRNAs), including microRNAs decay or inhibition of translation through incomplete base pairing (miRNAs) are important post-transcriptional gene expression with the 30 UTR of target genes.2 Each miRNA targets hundreds of regulators relevant in physiological and pathological processes. genes, and a gene can be targeted by several miRNAs.3 We have Here, we combined a high-throughput functional screening recently shown that the number of miRNAs is larger than antici- (HTFS) platform with a library of antisense oligonucleotides pated,4 with new miRNA candidates being evolutionarily young (ASOs) to systematically identify sncRNAs that affect neuronal and overrepresented in the human brain. cell survival in basal conditions and in response to oxidative stress (OS), a major hallmark in neurodegenerative diseases. Relative to other tissues, the nervous system is especially enriched in We considered hits commonly detected by two statistical diverse miRNAs and other types of ncRNAs of unknown function methods in three biological replicates. Forty-seven ASOs tar- that show specific spatial-temporal patterns of expression.5 Func- geting miRNAs (miRNA-ASOs) consistently decreased cell tional studies, with special emphasis on miRNAs, have revealed viability under basal conditions. A total of 60 miRNA-ASOs that their dynamic expression has essential roles in cell-type specifi- worsened cell viability impairment mediated by OS, with cation and differentiation during development,6 but also in the 36.6% commonly affecting cell viability under basal conditions. maintenance of adult post-mitotic cells. Conditional inactivation of In addition, 40 miRNA-ASOs significantly protected neuronal Dicer, the limiting enzyme of miRNA biogenesis, in specific areas cells from OS. In agreement with cell viability impairment, of the adult mouse brain consistently results in progressive loss of damaging miRNA-ASOs specifically induced increased free specific types of neurons.7–9 In addition, perturbation of selective radical biogenesis. miRNAs targeted by the detrimental ASOs miRNAs affects neuronal viability.10–12 Therefore, specific miRNAs are enriched in the fraction of miRNAs downregulated by and possibly other types of sncRNAs may be particularly relevant OS, suggesting that the miRNA expression pattern after OS in preserving neuronal viability. contributes to neuronal damage. The present HTFS high- lighted potentially druggable sncRNAs. However, future sncRNA expression is highly sensitive to physiological and patholog- studies are needed to define the pathways by which the identi- ical stimuli, with consequent modifications in gene expression leading fied ASOs regulate cell survival and OS response and to explore to a specific cell status. In neurodegenerative diseases, ncRNA alter- the potential of translating the current findings into clinical ations occurring at prodromal phases are especially relevant, as applications. they may determine initial gene expression changes with pathogenic impact. This scenario prompts the idea that the activity of specific INTRODUCTION sncRNAs could be targeted to overcome or delay pathogenesis. Different classes of small non-coding RNA (sncRNA) species have been uncovered with sequencing strategies, with many modulating Received 18 January 2019; accepted 11 June 2019; https://doi.org/10.1016/j.omtn.2019.06.007. gene expression through diverse mechanisms. MicroRNAs (miRNAs) 7 These authors contributed equally to this work. are the best known class of sncRNAs, transcribed as long precursors Correspondence: Eulàlia Martí, PhD, Departament de Biomedicina, Facultat de (pri-miRNAs) and processed to a mature, double-stranded, short Medicina i Ciències de la Salut, Institut de Neurociències, Universitat de Barcelona, RNA of 20–25 nt.1 The active strand of this duplex is loaded into C/Casanova, 143, 08036 Barcelona, Spain. the RNA-induced silencing complex (RISC), where it guides mRNA E-mail: eulalia.marti@ub.edu 374 Molecular Therapy: Nucleic Acids Vol. 17 September 2019 ª 2019 The Authors. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). www.moleculartherapy.org RESULTS HTFS Identifies Multiple miRNA-ASOs That Affect Cell Viability and Response to MPP+ Stress To identify sncRNAs with a role in neuronal survival, we set up an HTFS to evaluate cell viability after inhibiting the activity of miRNAs and other types of sncRNAs. We used a library of locked-nucleic-acid (LNA)-modified ASOs targeting 1,954 miRNAs and 50 sncRNAs not typified as miRNAs that our studies have described in deep-sequencing data analysis of human brain sam- ples20 (Table S1). The sncRNA-ASOs were individually transfected in three independent biological replicates into SH-SY5Y cells differ- entiated into a post-mitotic, dopaminergic phenotype, a cell model Figure 1. Scheme of the Experimental Design for the HTFS largely used to understand the molecular bases of neuronal dysfunc- tion.21,22 Two days after transfection, viability was determined with WST-1 tetrazolium reagent, whose bioreduction correlates with Stable, chemically modified antisense oligonucleotides comple- the number of metabolically active cells23 (Figure 1). In every plate, mentary to miRNAs (miRNA-ASOs) bind with high specificity we included a mock-transfected condition and a positive control of to the target sncRNAs and inhibit their activity by impeding the decreased cell viability, by transfecting a sncRNA-ASO that induces association of miRNAs with the complementary sites in the target cell death. mRNAs, which may restore expression levels of specific genes. miRNA-ASOs are therefore useful molecules for identifying miR- We used two statistical approaches to identify sncRNA-ASOs NAs with a relevant role in neuronal maintenance. Stabilized ASOs that affect neuronal cell viability. The first approach is based on the targeting other classes of sncRNA (sncRNA-ASOs) have the po- B score,19 a powerful method designed for plate-based assays that de- tential to similarly uncover the novel bioactive species implicated tects hits under the assumption that the vast majority of the screened in cell survival. sncRNA-ASOs are not active. Statistical B scores are constructed based on the observation that hits behave as outliers compared to High-throughput functional screenings (HTFSs) have used the majority of non-effective sncRNAs-ASOs. The normalized B miRNA-ASOs or RNA molecules designed to mimic endogenous scores follow a standardized Gaussian distribution, so that outliers, mature miRNA (miRNA-mimics), to identify species with an effect our potential hits, can be defined as values below 2. A positional in the expression of dosage-sensitive genes, in reporter assays.13,14 effect was detected, with B score values in close columns or rows In addition, most HTFS analyses have evaluated the effect of a clustering together (Figure S1). As indicated in the B score method, moderate number of miRNA-mimics or -ASOs in tumorigenic we applied an iterative median polished that robustly avoids posi- outcomes, including cell invasiveness,15 tumor cell death,16 or tion-related systematic errors.12 cell growth.17,18 HTFS studies to generally identify sncRNAs with a role in differentiated neuronal cell viability are missing. We consistently detected a decrease in cell viability in wells trans- In the present study, we developed a high-throughput screening fected with the positive control sncRNA-ASO (Figure 2). A signif- platform to evaluate the effect of 2,004 sncRNA-ASOs, targeting icant enrichment of hits was detected among the sncRNAs miRNAs (a total of 1,954) and non-miRNA sncRNAs (a total of expressed in our model system (Figure S2), which agrees with a 50) in neuronal cell viability, under both normal and basal specific effect of the ASOs on sncRNAs present in differentiated conditions and following oxidative stress (OS), a major hallmark neuronal cells, thus favoring the detection of true detrimental in neurodegenerative disorders. We used a statistical scoring effects. Among the sncRNAs (988 miRNAs and 9 sncRNAs) method developed for high-throughput screenings to target with mean normalized counts >1, according to sequencing analysis hits,19 a subgroup of which was confirmed by using an alternative (Table S2), a total of 76 sncRNA-ASOs targeting miRNAs statistical strategy that we developed, dealing with biological (miRNA-ASOs) significantly decreased viability in differentiated replicates. In addition, we used alternative functional approaches SH-SY5Y (B score <2), in at least two biological replicates to validate hits targeted by both statistical methods. Our (Table 1). approach has identified a reliable group of detrimental and protec- tive sncRNA-ASOs, which may reflect the involvement of the The second statistical approach is based on a linear mixed effects targeted species in neuronal maintenance and response to stress. model (LMM) that accounts for the three biological replicates as Finally, we analyzed sncRNA expression dynamics after OS, another source of variability in the experimental design. Similar to suggesting that deregulation of several sncRNA species may the B score method, the LMM uses the bulk of data as self-controls contribute to neuronal dysfunction. Our data provide a large cat- and accounts for systematic biases. The fact that the experimental alog of sncRNA-ASOs that may be selected for pre-clinical valida- design includes biological triplicates per sample, allows us to include tion in model organisms. sample random effects in the model so that outliers or candidate hits Molecular Therapy: Nucleic Acids Vol. 17 September 2019 375 Molecular Therapy: Nucleic Acids duced a stronger decrease in cell viability by the sncRNA-ASO used a positive control (70%), compared with basal, untreated con- ditions (60%) (Figure 2), and permitted the identification of other sncRNA-ASOs that worsen the response or protect it from MPP+ stress (Figure 1). A total of 60 miRNA-ASOs impaired the effect of MPP+ in cell viability according to both statistical methods (Table 2), of which 22 (36.6%) were common to those that decrease cell viability under basal conditions (Table 1). A miEAA analysis with the group of miRNAs whose ASOs worsened the response to MPP+ stress pointed to response to reactive oxygen species (ROS) and apoptosis pathways (Table S4). In addition, 40 miRNA-ASOs protected from the detrimental effect of MPP+ on cell viability (Table 2). Figure 2. Cell Viability Determinations in the HTFS under Basal Conditions and in Response to Oxidative Stress In summary, the HTFS identified 85 miRNA-ASOs with a detri- Cell viability determinations in every plate are shown in mock-transfected cells, mental effect in basal conditions and/or in response to MPP+, and cells transfected with a negative control sncRNA-ASO (C), and cells trans- fected with the positive control sncRNA-ASO (C+), under basal conditions and 40 miRNA-ASOs that protect from MPP+ stress, which may reflect following treatment with 3 mM MPP+. Each dot represents the percentage of the protective and detrimental roles of the targeted miRNAs, cell viability determinations in a well, with 100% considered to be the mock- respectively. 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 A Subset of Detrimental miRNA-ASOs Increases Free Radical conditions to be compared are included in the model as fixed affects, and plates Biogenesis and Impairs Mitochondrial Function are included as random effects to account for between-plate variability. A Bonferroni correction was applied to account for multiple comparisons Generation of ROS plays a critical role in damaging cellular com- (number of comparisons = 9). Significant differences are found between MPP+ ponents and initiating cell death. Therefore, we evaluated whether treatment and basal conditions, in each experimental condition (Mock, C, and the effect of specific miRNA-ASOs in cell viability is linked to C+; *p < 0.001), and in C+ versus mock and C under basal conditions and changes in ROS levels. We used CellROX Deep Red Reagent after exposure to MPP+ (+p < 0.001). (Molecular Probes) to quantify ROS levels that were expressed with respect to the total number of cells, measured with are summarized in a single value. This model provided a total of 87 CyQUANT Direct Cell Proliferation Assay (Invitrogen). This sncRNA-ASOs that decreased cell viability, of which 47 (54%) were strategy permitted the detection of increased ROS levels in cells coincidental with the B score-based approach (Table 1). These data transfected with the miRNA-ASO used as a positive control, and suggest that the 47 sncRNAs targeted by the correspondent ASOs a general increase in ROS levels in cells treated with 3 mM participate in the maintenance of cell viability. According to the MPP+ (Figure S3). miRNA enrichment analysis and annotation (miEAA) tool,24 the pathways targeted by the group of miRNAs whose ASOs decrease We chose a subset of miRNA-ASOs with a negative effect on cell cell viability include nervous system development, apoptosis, and viability under basal conditions and/or in response to MPP+, a DNA damage response and pathways associated with diverse neurode- group of miRNA-ASOs that protect against MPP+, and a subset generative conditions, including Parkinson’s and Huntington’s of miRNA-ASOs with no effect on cell viability. The subsets of diseases (Table S3). detrimental and protective miRNA-ASOs are based on the altered expression of the targeted miRNAs in neurodegenerative condi- An independent HTFS was performed to evaluate the effect of tions (Table S5). We particularly focused on miRNAs perturbed sncRNA-ASOs in cell viability after a stressful stimulus (Figure 1). in PD paradigms, since differentiated SH-SY5Y cells are widely Differentiated SH-SY5Y cells were transfected with the sncRNA- used to understand neuronal death mechanisms in this disease.21 ASOs, and 24 h later, they were subjected to 1-methyl-4-phenylpyr- After transfection of the chosen miRNA-ASOs we evaluated idinium (MPP+) stress for an additional 24 h, after which cell ROS levels, both under basal conditions and in response to viability was determined. MPP+ impairs oxidative phosphorylation MPP+, as an alternative readout of cell dysfunction (Figure 3). in mitochondria by inhibiting complex I, leading to depletion of We calculated Pearson’s correlation coefficient to evaluate the ATP and cell death, and has been used largely to understand degree of association between the cell viability (B score) and OS dopaminergic neuronal death in Parkinson’s disease (PD).21 We (ROS levels) measures. The statistically significant negative corre- used a concentration of MPP+ (3 mM) that produced a significant lation coefficient (Figure S4) suggests that both measures are but moderate decrease (35%) in cell viability. MPP+ (3 mM) pro- inversely associated. 376 Molecular Therapy: Nucleic Acids Vol. 17 September 2019 www.moleculartherapy.org Table 1. sncRNA-ASOs Decreasing Cell Viability Table 1. Continued sncRNA ID Replicate 1 Replicate 2 Replicate 3 Average sncRNA ID Replicate 1 Replicate 2 Replicate 3 Average hsa-miR-1236-3pa 15.43 3.66 5.84 8.31 hsa-miR-18a-5pa 2.70 3.34 2.97 3.00 hsa-miR-4758-3pa 8.84 5.99 9.63 8.15 hsa-miR-152-5p 3.01 3.25 2.70 2.99 hsa-miR-1913a 9.25 8.26 6.50 8.00 hsa-miR-766-3pa 2.37 0.80 5.65 2.94 hsa-miR-5006-3p a 12.47 5.29 5.83 7.86 hsa-miR-5699-5p 5.14 1.55 2.12 2.94 hsa-miR-3677-5pa 6.53 5.42 8.23 6.73 hsa-miR-767-5p 2.19 4.43 2.16 2.93 hsa-miR-204-5pa 4.99 2.48 12.39 6.62 hsa-miR-7974a 2.35 4.66 1.67 2.89 hsa-miR-4640-3pa 4.56 6.80 8.14 6.50 hsa-miR-642a-5p 1.15 4.95 2.58 2.89 hsa-miR-496a 3.48 10.49 4.55 6.18 hsa-miR-362-5p 2.97 3.39 2.27 2.88 hsa-miR-5579-3p a 6.94 7.57 3.89 6.14 hsa-miR-3116 3.25 3.10 2.25 2.87 hsa-miR-6817-3pa 5.11 8.36 4.89 6.12 hsa-miR-885-5pa 2.43 4.03 2.05 2.84 hsa-miR-3171a 7.03 6.81 3.88 5.91 hsa-miR-2116-3pa 4.35 0.76 3.39 2.83 hsa-miR-6840-5p a 9.52 4.47 2.48 5.49 hsa-miR-636 2.76 1.39 3.96 2.70 hsa-miR-6511a-3pa 3.70 3.80 8.34 5.28 hsa-miR-330-3p 2.97 3.20 1.90 2.69 hsa-miR-2355-5pa 1.80 7.51 6.33 5.21 hsa-miR-6803-3p 1.00 2.87 4.02 2.63 hsa-miR-1224-3pa 2.68 4.31 8.14 5.04 hsa-miR-106b-3p 2.02 0.75 5.09 2.62 hsa-miR-24-1-5p a 0.21 7.64 6.88 4.91 hsa-miR-543a 2.08 3.87 1.90 2.62 hsa-miR-133a-3p a 0.47 4.26 9.79 4.84 hsa-miR-654-3p a 0.64 2.40 4.79 2.61 hsa-miR-346a 5.00 4.97 4.55 4.84 hsa-miR-151a-5p 1.96 2.18 3.45 2.53 hsa-miR-3940-3pa 7.26 2.62 4.64 4.84 hsa-miR-4685-3pa 0.08 3.24 4.19 2.50 hsa-miR-454-5p a 1.55 4.50 8.32 4.79 hsa-miR-1298-5p 1.66 2.84 2.68 2.39 hsa-miR-7976a 6.17 3.79 4.34 4.77 hsa-miR-1234-3pa 3.30 2.10 1.75 2.38 hsa-miR-574-3p a 2.62 8.13 3.47 4.74 hsa-miR-758-5p 2.70 2.76 1.68 2.38 hsa-miR-361-3pa 6.68 2.09 4.39 hsa-miR-4638-3p 1.39 2.76 2.92 2.35 hsa-miR-5587-3p 4.67 4.73 3.36 4.25 hsa-miR-2355-3p 2.18 0.90 3.94 2.34 hsa-miR-7-1-3p 4.81 3.13 3.97 hsa-miR-939-3pa 3.70 2.86 0.45 2.34 hsa-miR-3200-3p a 3.05 2.10 6.76 3.97 hsa-miR-1305 1.72 2.12 3.02 2.29 hsa-miR-374b-5pa 3.00 1.92 6.93 3.95 hsa-miR-616-5pa 2.53 2.84 1.45 2.27 hsa-miR-6511b-3p 3.38 1.08 7.11 3.86 hsa-miR-590-3p 0.10 3.63 3.05 2.26 hsa-miR-4484a 2.52 4.60 4.15 3.76 hsa-miR-3130-5pa 2.49 0.02 4.17 2.22 hsa-miR-532-3p a 4.42 3.76 2.60 3.59 hsa-miR-551a 2.56 3.79 0.11 2.15 hsa-miR-3176 3.30 2.62 4.85 3.59 hsa-miR-128-3p 3.24 1.99 1.19 2.14 hsa-miR-6770-5p 2.66 3.98 4.08 3.57 hsa-miR-3944-3p 2.39 0.45 3.27 2.03 hsa-miR-500a-3p a 4.79 2.52 3.30 3.54 Hits show a median polished B score <2, consistent in at least two biological replicates. Only ASOs targeting sncRNAs with mean normalized counts >1 in differentiated hsa-miR-6800-3pa 2.09 4.44 4.06 3.53 SHSY5Y cells are shown. hsa-miR-1301-3p 4.18 2.91 3.31 3.47 a Hits validated with a LMM. hsa-miR-2114-5p 2.68 8.04 0.37 3.45 hsa-miR-4647 2.13 3.55 4.60 3.43 miRNA-ASOs impairing cell viability induced a significant increase hsa-miR-4793-3p 1.59 4.78 3.84 3.40 in ROS levels under basal conditions and in response to MPP+ hsa-miR-324-3pa 1.33 4.21 4.11 3.22 (Exp 1; Figure 3C). The miRNAs-ASOs impairing cell viability, in hsa-miR-767-3pa 4.18 3.75 1.50 3.14 both basal conditions and response to OS, strongly contributed to hsa-miR-210-5p 0.93 2.28 6.08 3.10 increased ROS levels (Exp 2a versus Exp 2b, upper panel; Figure 3C). miRNA-ASOs with no effect on cell viability induced a slight increase hsa-miR-1914-5pa 3.14 5.55 0.59 3.09 in ROS levels compared with mock-transfected cells, suggesting that hsa-miR-6738-3pa 2.40 3.90 2.93 3.08 LNA-ASOs may produce minor OS (Exp 4; Figure 3C). Compared hsa-miR-551b-3pa 1.95 1.64 5.46 3.02 with this group, protective miRNA-ASOs tended to decrease ROS (Continued) levels (Exp 3; Figure 3C) in agreement with their protective outcome Molecular Therapy: Nucleic Acids Vol. 17 September 2019 377 Molecular Therapy: Nucleic Acids Table 2. sncRNA-ASOs That Modify the Cell Response to Oxidative Stress sncRNA-ASO Replicate 1 Replicate 2 Replicate 3 Average sncRNA-ASO Replicate 1 Replicate 2 Replicate 3 Average hsa-miR-636a 11.08 8.06 7.50 8.88 hsa-miR-363-5p 1.93 3.91 0.97 2.27 hsa-miR-6840-5pa -7.63 -8.46 -5.76 -7.28 hsa-miR-146b-3pa 2.60 2.69 1.52 2.27 hsa-miR-204-5pa -6.19 -7.23 -7.66 -7.03 hsa-miR-410-3pa 2.42 2.92 1.46 2.27 hsa-miR-1236-3p a -8.41 -7.02 -4.92 -6.78 hsa-miR-330-3p 2.34 2.43 2.02 2.26 hsa-miR-532-3pa -8.45 -5.70 -5.86 -6.67 hsa-miR-1253 3.69 2.01 1.07 2.26 hsa-miR-5006-3pa -4.23 -8.42 -5.45 -6.03 hsa-miR-6806-3p 2.13 3.79 0.84 2.25 hsa-miR-6803-3p 9.90 3.96 3.26 5.71 hsa-miR-6857-3p 2.14 3.98 0.50 2.21 a a hsa-miR-6817-3p -3.66 -4.75 -7.49 -5.30 hsa-miR-574-3p -2.55 -1.41 -2.66 -2.21 hsa-miR-362-5pa 4.45 4.87 6.14 5.16 hsa-miR-6511a-3pa -2.67 -2.14 -1.80 -2.20 hsa-miR-1271-5pa 2.38 4.76 8.01 5.05 hsa-miR-129-2-3pa 1.54 2.32 2.74 2.20 hsa-miR-2355-5pa -3.03 -6.88 -4.29 -4.74 hsa-miR-3176a 2.60 2.87 1.10 2.19 hsa-miR-4522 4.50 6.35 3.15 4.67 hsa-miR-99b-5p 2.89 1.49 2.15 2.18 hsa-miR-1913a -5.73 -2.95 -5.31 -4.66 hsa-miR-6797-3pa 2.62 2.89 0.81 2.11 hsa-miR-454-5pa -7.71 -3.05 -3.00 -4.59 hsa-miR-342-3p 2.68 0.59 2.95 2.07 hsa-miR-6511b-3pa 5.59 3.85 4.26 4.57 hsa-miR-548s 2.83 1.01 2.33 2.06 hsa-miR-4479 3.90 4.18 5.39 4.49 hsa-miR-4767 a 2.08 2.71 1.36 2.05 hsa-miR-7977a 6.04 4.55 2.62 4.40 hsa-miR-615-3p 2.25 3.29 0.55 2.03 hsa-miR-133a-3pa -3.20 -5.02 -4.84 -4.35 hsa-miR-143-5p 3.48 2.65 0.08 2.01 hsa-miR-654-5p 6.43 3.00 3.23 4.22 hsa-miR-182-5p 2.73 2.37 0.61 1.50 hsa-miR-767-5p a 2.02 4.82 5.80 4.21 hsa-miR-548b-5p a 0.83 3.30 3.57 2.01 hsa-miR-24-2-5pa 4.44 2.25 5.86 4.18 hsa-miR-7854-3pa 0.41 2.89 2.89 2.06 hsa-miR-6750-3p 5.93 4.33 1.98 4.08 hsa-miR-4745-5pa 1.15 2.56 2.49 2.07 hsa-miR-615-5pa 2.41 4.51 5.31 4.07 hsa-miR-197-5pa 2.19 2.79 1.37 2.12 hsa-miR-642a-5pa 1.02 4.78 6.32 4.04 hsa-miR-6129a 2.01 3.18 1.19 2.12 hsa-miR-766-3p a -4.95 -3.95 -2.80 -3.90 hsa-miR-548l 4.41 0.52 2.77 2.22 hsa-miR-4685-3pa -2.77 -4.40 -4.32 -3.83 hsa-miR-548b-3p 2.54 2.15 2.11 2.27 hsa-miR-346a -4.33 -3.04 -3.69 hsa-miR-3126-5pa 0.26 4.11 2.65 2.34 hsa-miR-6798-3pa 3.26 3.42 4.11 3.59 hsa-miR-101-3pa 0.57 2.34 4.15 2.35 hsa-miR-4642 a 7.34 3.31 0.06 3.57 hsa-miR-656-3p 0.95 2.94 3.26 2.38 hsa-miR-3619-5p 4.64 3.02 2.95 3.54 hsa-miR-4443a 1.04 2.03 4.10 2.39 hsa-miR-605-5pa 3.41 4.68 2.50 3.53 hsa-miR-5695a 3.73 1.95 1.82 2.50 hsa-miR-1226-5p 3.29 1.28 5.77 3.44 hsa-miR-505-5pa 5.36 0.81 3.05 2.53 hsa-miR-4758-3pa -3.91 -1.23 -5.18 -3.44 hsa-miR-9-3p 3.59 2.62 1.54 2.58 hsa-miR-1914-5p 3.48 2.52 4.32 3.44 hsa-miR-548ka 3.22 2.72 1.97 2.64 hsa-miR-5587-3p -2.10 -3.78 -4.38 -3.42 hsa-miR-651-5pa 3.25 2.50 2.21 2.65 hsa-miR-152-5p 6.01 1.46 2.69 3.39 hsa-miR-876-5pa 0.00 5.17 2.97 2.71 hsa-miR-4800-3p 5.11 2.29 2.63 3.34 hsa-miR-425-3p 1.94 4.33 1.87 2.71 hsa-miR-339-5pa 3.82 2.36 3.78 3.32 hsa-miR-574-5pa 2.79 2.94 2.71 2.81 hsa-miR-767-3pa -2.00 -5.01 -2.93 -3.31 hsa-miR-548e-3pa 5.30 2.28 0.94 2.84 hsa-miR-3938 3.73 3.39 2.79 3.30 hsa-miR-200c-3pa 1.36 3.20 3.98 2.85 hsa-miR-99a-5p a 4.30 2.27 3.28 hsa-miR-629-5p a 2.70 1.49 4.48 2.89 hsa-miR-455-3pa 3.94 3.50 2.38 3.27 hsa-miR-766-5p 4.21 3.50 1.26 2.99 hsa-miR-26a-1-3pa 4.39 3.52 1.74 3.22 hsa-miR-1303a 0.67 4.57 3.76 3.00 (Continued on next page) 378 Molecular Therapy: Nucleic Acids Vol. 17 September 2019 www.moleculartherapy.org Table 2. Continued sncRNA-ASO Replicate 1 Replicate 2 Replicate 3 Average sncRNA-ASO Replicate 1 Replicate 2 Replicate 3 Average hsa-miR-6738-3pa -3.19 -3.97 -2.47 -3.21 hsa-miR-3163a 2.23 6.21 0.74 3.06 hsa-miR-874-5pa 4.32 1.80 3.34 3.16 hsa-miR-105-3p 1.44 3.84 3.91 3.07 hsa-miR-331-3pa 2.94 3.32 3.13 hsa-miR-10b-3pa 5.86 0.92 2.67 3.15 hsa-miR-2277-3p 2.64 2.68 4.00 3.11 hsa-miR-215-5pa 3.49 2.96 3.23 hsa-miR-152-3p a 2.32 3.73 3.03 hsa-miR-1185-5p a 3.30 1.95 4.92 3.39 hsa-miR-361-3pa -1.00 -4.74 -3.33 -3.03 hsa-miR-3141a 0.14 2.70 7.48 3.44 hsa-miR-329-3pa 3.24 2.43 3.40 3.02 hsa-miR-1268aa 0.94 5.76 3.64 3.45 hsa-miR-4516 3.59 3.68 1.68 2.98 hsa-miR-193b-5pa 1.48 3.86 5.04 3.46 hsa-miR-3130-5p 5.37 3.71 0.15 2.98 hsa-miR-4705 a 3.57 4.43 2.41 3.47 hsa-miR-370-5p 2.46 3.19 3.21 2.96 hsa-miR-3609a 2.18 2.82 6.28 3.76 hsa-miR-6800-3pa -4.01 -2.06 -2.79 -2.95 hsa-miR-320a 4.71 2.91 3.81 hsa-miR-105-5pa 4.71 3.74 0.28 2.91 hsa-miR-221-5pa 6.00 1.98 3.52 3.83 hsa-miR-106b-3p 1.71 3.65 3.30 2.89 hsa-miR-219a-1-3p a 6.93 3.64 1.00 3.86 hsa-miR-6726-3pa 4.97 2.07 1.49 2.84 hsa-miR-29b-1-5pa 2.71 4.73 4.21 3.88 hsa-miR-4746-5pa 2.24 3.82 2.35 2.80 hsa-miR-1277-3pa 5.78 3.07 3.06 3.97 hsa-miR-151a-5p 2.58 2.88 2.86 2.77 hsa-miR-134-5pa 3.63 4.96 3.34 3.98 hsa-miR-15a-3p 3.22 3.81 1.16 2.73 hsa-miR-1269p a 6.33 3.70 1.99 4.01 hsa-miR-3187-3pa 3.30 1.91 2.84 2.69 hsa-miR-627-5p 1.43 7.54 3.40 4.12 hsa-miR-129-1-3pa 0.67 3.15 5.52 2.67 hsa-miR-590-5p 5.24 3.92 3.44 4.20 hsa-miR-496a -2.42 -1.26 -4.29 -2.65 hsa-miR-99a-3pa 6.49 2.23 4.41 4.38 hsa-miR-23a-3pa 4.22 2.13 1.55 2.63 hsa-miR-147pa 5.29 4.50 4.11 4.63 hsa-miR-1296-3p a 0.81 2.87 4.11 2.60 hsa-miR-585-3pa 3.89 5.75 4.51 4.72 hsa-miR-1538 3.61 2.28 1.79 2.56 hsa-miR-186-3pa 5.11 4.76 4.29 4.72 hsa-miR-148a-5pa 1.35 3.27 2.78 2.47 hsa-miR-664a-5pa 7.02 2.83 4.92 4.92 hsa-miR-550a-3pa 4.37 0.79 2.14 2.43 hsa-miR-6847-5pa 7.62 3.90 3.65 5.06 hsa-let-7d-3p a 1.21 3.76 2.12 2.36 hsa-miR-1268b a 12.43 9.03 7.61 9.69 Hits show a median polished B score <2; worsening cell viability in response to MPP+; or B score >2, which protects against the detrimental effect of MPP+, consistent between at least two biological replicates. Only ASOs targeting sncRNAs with mean normalized counts >1 in differentiated SHSY5Y cells treated with 3 mM MPP+ for 24 h, are shown. ASOs high- lighted in bold are common to those found in basal conditions (Table 1). a Hits validated with a LMM. in viability determinations. Overall, these data indicate that ROS the activity of these miRNAs is important for neuronal survival and levels confirm the previously selected hits, especially those producing response to MPP+ stress. a detrimental effect on cell viability. miRNA Expression Dynamics in Response to MPP+ May To test whether the detrimental effect of miRNA-ASOs was linked Contribute to Cell Dysfunction to mitochondrial dysfunction, we measured the mitochondrial Inhibition of sncRNAs activity may reflect, at least to some extent, the membrane potential by using Mitotracker fluorescent dye consequences of the downregulation of the targeted sncRNAs that (Molecular Probes). We selected ASOs targeting miR-204-5p, occurs in response to specific stimuli. We analyzed the sncRNA miR-532-3p, and miR-324-3p, which are reliably expressed in expression profiles in response to MPP+ to understand if sncRNA the human brain, perturbed in several neurodegenerative and dynamics contribute to cell viability outcome. SH-SY5Y cells were neurological disorders,20,25–31 and involved in neuronal differenti- differentiated, and sncRNA expression profiles were determined by ation.32–34 deep sequencing 24 h after exposure to MPP+ (3 mM). A total of 128 miRNAs were significantly deregulated after exposure to MPP+ Confirming the effect of three miRNA-ASOs in decreasing cell (Table 3). viability (Tables 1 and 2) and inducing ROS, miR-204-5p-, miR-324-3p-, and miR-532-3p-ASO significantly impaired mito- Among the miRNAs whose ASOs worsened cell viability in basal con- chondrial membrane potential (Figure 4). These results suggest that ditions and/or response to MPP+ (according to both statistical tests), Molecular Therapy: Nucleic Acids Vol. 17 September 2019 379 Molecular Therapy: Nucleic Acids A 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 B 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. C oxidative stress response, p53, and DNA damage response. In addition, MPP+-upregulated miRNAs were enriched in analogous pathways. Furthermore, MPP+-deregulated miRNAs were significantly perturbed in Alzheimer’s-, Parkinson’s-, and Huntington’s-disease-related pathways. Overall, these data suggest that the miRNA expression dynamics in response to MPP+ contributes to detrimental processes. DISCUSSION The evidence shows that perturbed activity of spe- cific sncRNAs influence neuronal function under both normal and pathological conditions. In the hsa-miR-7974, miR-3619-5p, miR-455-3p, miR-3176, miR-3187-3p, present study we developed an HTFS to generally identify sncRNAs miR-636, and miR-454-5p were downregulated after exposure to involved in neuronal cell viability, both under basal conditions and in MPP+ (Table 3), which suggests that their deregulation pattern response to OS, a major player in neurodegenerative processes. may contribute to impairment of cell viability. For four downregu- lated miRNAs—miR-4745-5p, miR-574-5p, miR-1303, and miR- Here, we used LNA-modified AOSs that bind to target sequences and 6847-5p—the correspondent ASOs protected from MPP+ stress. decrease the availability and activity of each particular sncRNA. Among the MPP+-downregulated miRNAs, the increased number Although LNA modifications in ASOs increase stability and speci- of detrimental ASOs versus protective ones is in contrast with the ficity for the target sequence,35 binding to unintended targets could few hits found among the MPP+-upregulated miRNAs (only one account for nonspecific effects in cell viability. However, the signifi- protective and one detrimental miRNA-ASO). cant enrichment of hits among SH-SY5Y-expressed sncRNAs favors the idea that LNA-ASOs induce a detrimental effect by specifically We used the miEAA tool to identify the pathways targeted by targeting sncRNAs expressed in our model system. MPP+-deregulated miRNAs (Tables S6 and S7). The analysis showed that MPP+-downregulated miRNAs were significantly To identify reliable sncRNA-ASOs affecting cell viability, we used the depleted in signaling pathways related to stress, including apoptosis, B score method, which is designed to detect hits in HTFS.19 We 380 Molecular Therapy: Nucleic Acids Vol. 17 September 2019 www.moleculartherapy.org have validated the toxic effect of miRNA-ASOs tested in other HTFSs, including miR-204; miR-210 inhibitors in HeLa cells;36 and miR- 133a-3p, miR-361-3p, and miR-346 inhibitors in lung cancer cells.37 In addition, in line with the detrimental effect of the miR-7-1-3p-ASO observed in the present study, miR-7-1 activity is involved in motor neuron protection.38 miR-134-5p expression induces neuronal death in the brain,39 which agrees with the protective effect of miR-134- 5p-ASO in front of the stressful conditions observed here. Further- more, reduction of miR-23a following traumatic brain injury induces neuronal death,40 which matches with the worsened effect of the correspondent ASO observed in the present study. Although our results validate many of the miRNAs reported to have a role in cell viability, there are some exceptions. The present study did Figure 4. Mitochondrial Activity after Transfection with Specific not reveal the neuroprotective activity of miR-7-5p and miR-153.41 In miRNA-ASOs addition, we did not reproduce the neuroprotective effect of miR-181 Mock, cells treated with Lipofectamine; negative control (C), cells transfected with inhibition42 or the detrimental effect of miR-29b inhibition43 in front an ASO that does not produce any effect in cell viability; positive control (C+), cells of cerebral ischemia. Whether the lack of confirmation is because the transfected with a control sncRNA-ASO inducing cell death; positive control activity of these miRNAs is context dependent or involves alternative (CCCP), cells treated with CCCP were used as a positive control for mitochondrial functional loss. The boxplot shows absorbance determinations after incubation with experimental conditions remains to be determined. Validation of the MitoTracker Deep Red reagent. *p < 0.001, by the Kruskal-Wallis test, followed by present results in diverse cell types, including primary neurons, is the Mann-Whitney U test with Bonferroni’s correction as a post hoc test (n = 5). needed to understand how general and consistent the effects of the miRNA-ASOs reported herein are. Furthermore, evaluation of the applied an iterative median polish that robustly avoids row- and effect of miRNA-ASOs in the brain in model organisms will answer column-position-related systematic errors, which we detected in the whether validation in simpler neuronal cell cultures can be extended different HTFSs. Z scores are also based on the raw sample values; to a complex structure with heterogeneous neuronal and non- however, as Brideau and colleagues19 discussed, these scores suffer neuronal (glial) cells. from the fact that they do not take into account positional effects, and their performance is compromised by outliers, which are the The temporal and spatial miRNA dynamics regulate gene expression key hits. However, when no positional effects and changes occur in programs in normal conditions and in response to stressful stimuli. a plate, Z scores are slightly more efficient. That is why the combina- The effect of miRNA-ASOs mimic the consequences of miRNA tion of different methods may help to choose the best top hits. Under downregulation, and our data suggest that the overall miRNA expres- this premise, we proposed another statistical approach that is based sion patterns in response to MPP+ contribute to MPP+-mediated on Z scores, but improved in a way similar to the B score. The damage. Analogous assumptions considering the miRNA expression approach is based on a LMM, where the positional effects are taken patterns in human brains in neurodegenerative conditions are diffi- into account, and, in addition, it considers the three independent rep- cult to determine. Neurodegenerative diseases involve continuous licates as a source of variability in the experimental design. This and long-lasting molecular changes that shape disease evolution. method detected from 55% to 70% of the B score hits, and additional For instance, according to our data, miR-204-5p and miR-532-3p functional validations were performed for a subset of sncRNA-ASOs are protective miRNAs (their inhibition impairs cell viability). In commonly identified in both strategies. the substantia nigra of PD patients, miR-204-5p is upregulated, and miR-532-5p is downregulated;27 however, both miRNAs are downre- Only sncRNA-ASOs targeting canonical miRNAs produced signifi- gulated in the amygdala of PD patients.20,27 It is difficult to anticipate cant effects in cell viability in agreement with both statistical methods. the significance of these changes, since neuropathological alterations According to the miEAA (Tables S3 and S4), the pathways targeted strongly differ in these two brain areas. Furthermore, the expression by the group of miRNAs whose ASOs decrease cell viability favor pattern of miR-128-3p, another miRNA that we have identified as the idea that the detrimental effects of the miRNA-ASOs are not neuroprotective, differs in the amygdala of PD patients, showing nonspecific but are caused by the selective blockage of miRNAs downregulation at pre-clinical stages and upregulation at clinical that participate in neuronal survival. stages.20 miRNA expression dynamics may reflect complex reactive responses with functional consequences, either protecting from stress miRNA-ASOs that impair cell viability in basal conditions and/or in or contributing to dysfunction, depending on the brain regions and response to MPP+ may prevent the activity of miRNAs involved in disease evolutionary stages. Experimentally induced alterations in neuronal maintenance or in neuronal protection in front of OS. the activity of miRNAs in vivo should highlight the relevance that Conversely, miRNA-ASOs protecting from MPP+ stress may target changes in the expression levels of particular miRNA (or groups of miRNAs contributing to MPP+-mediated cell death. Our results miRNAs) undergo in a pathogenic process. Molecular Therapy: Nucleic Acids Vol. 17 September 2019 381 Molecular Therapy: Nucleic Acids Table 3. miRNAs Significantly Deregulated, 24 h after Treatment with 3 mM MPP Log2 Fold Average Average Log2 Fold Average Average Mean Change B Score B Score Mean Change 24 h B Score B Score Normalized 24 h MPP+ Basal MPP+ Normalized MPP+ Basal MPP+ miRNA Counts versus C padj Assay Assay miRNA Counts versus C padj Assay Assay hsa-let-7a-5p 212,828.53 0.46 0.01 3.00 0.77 hsa-miR-335-5p 33.84 0.73 0.02 1.29 0.30 hsa-let-7c-5p 9,910.12 0.47 0.03 0.65 1.51 hsa-miR-34a-3p 65.64 1.09 0.00 1.18 1.46 hsa-let-7f-1-3p 37.86 1.00 0.00 0.48 0.09 hsa-miR-34a-5p 1,366.74 0.98 0.00 1.50 0.58 hsa-let-7i-5p 23,119.45 0.34 0.00 1.18 0.45 hsa-miR-34b-3p 17.96 1.54 0.00 0.27 0.06 hsa-miR-10a-5p 628.77 0.53 0.04 3.57 0.95 hsa-miR-34b-5p 62.77 1.88 0.00 1.16 1.58 hsa-miR-1180-3p 1,366.77 0.60 0.00 1.57 1.06 hsa-miR-34c-5p 1,062.35 1.91 0.00 0.60 0.81 hsa-miR-1224-5p 83.98 0.60 0.01 1.72 0.90 hsa-miR-3615 1,227.71 0.40 0.02 0.02 0.14 hsa-miR-1247-5p 78.77 0.60 0.00 1.73 1.92 hsa-miR-3619-5p 15.30 1.73 0.00 0.54 3.54 hsa-miR-125b-1-3p 1,030.31 0.47 0.01 2.16 0.35 hsa-miR-3679-5p 20.00 0.97 0.01 0.44 0.70 hsa-miR-126-3p 1,021.38 0.21 0.03 0.28 0.92 hsa-miR-378a-3p 3,805.50 0.41 0.00 0.30 1.17 hsa-miR-126-5p 5,597.90 0.39 0.00 0.94 0.01 hsa-miR-378i 14.00 0.92 0.01 1.29 1.45 hsa-miR-1260a 3,628.71 0.81 0.00 3.77 0.74 hsa-miR-381-3p 1,565.58 0.36 0.00 0.45 1.67 hsa-miR-1260b 3,920.23 0.79 0.00 1.47 0.33 hsa-miR-3909 142.17 0.62 0.00 2.21 0.58 hsa-miR-1272 13.23 2.00 0.00 0.95 0.97 hsa-miR-3929 44.66 1.10 0.00 0.80 1.60 hsa-miR-1276 240.10 0.56 0.00 1.67 1.00 hsa-miR-3939 17.64 0.78 0.02 0.08 1.33 hsa-miR-1298-3p 14.61 1.45 0.00 1.30 0.26 hsa-miR-409-3p 1,080.93 0.36 0.01 1.63 1.60 hsa-miR-1298-5p 289.63 1.68 0.00 2.39 1.76 hsa-miR-424-5p 312.73 0.64 0.00 0.13 0.04 hsa-miR-1303 152.76 1.30 0.00 0.89 3.00 hsa-miR-425-3p 924.49 0.33 0.00 0.15 2.71 hsa-miR-130b-5p 2,949.55 0.41 0.00 0.75 0.19 hsa-miR-4284 28.38 2.82 0.00 0.41 0.42 hsa-miR-132-3p 6,025.80 0.67 0.02 1.22 0.25 hsa-miR-4301 53.24 1.15 0.00 1.34 0.50 hsa-miR-135a-3p 73.40 0.88 0.00 0.77 0.25 hsa-miR-4455 455.18 0.90 0.00 2.71 1.17 hsa-miR-135a-5p 143.13 0.51 0.03 0.15 1.36 hsa-miR-448 79.61 1.00 0.00 0.24 2.77 hsa-miR-138-1-3p 547.79 0.86 0.00 2.14 0.95 hsa-miR-449a 47.36 0.57 0.03 0.42 0.47 hsa-miR-140-3p 7,556.14 0.25 0.02 0.13 1.61 hsa-miR-4516 15.07 0.96 0.01 0.41 2.98 hsa-miR-146a-5p 77.95 0.51 0.04 1.97 0.42 hsa-miR-454-5p 459.79 0.42 0.01 4.79 4.59 hsa-miR-149-5p 3,328.89 0.49 0.00 1.38 0.60 hsa-miR-455-3p 65.94 0.80 0.03 0.20 3.27 hsa-miR-152-5p 367.41 0.29 0.03 3.41 3.39 hsa-miR-4638-3p 24.56 0.70 0.04 2.35 1.40 hsa-miR-17-5p 13,154.08 0.22 0.01 1.15 1.85 hsa-miR-4741 79.93 0.72 0.01 0.24 0.13 hsa-miR-181a-3p 1,831.47 0.50 0.00 0.30 0.37 hsa-miR-4745-5p 60.91 0.79 0.03 0.80 2.07 hsa-miR-182-5p 46,002.45 0.39 0.04 0.03 1.50 hsa-miR-4792 680.59 1.29 0.00 0.58 1.29 hsa-miR-183-3p 148.31 0.62 0.00 0.10 1.35 hsa-miR-483-3p 69.91 1.22 0.00 0.56 2.00 hsa-miR-183-5p 5,824.28 0.38 0.01 0.46 0.42 hsa-miR-486-3p 81.75 0.82 0.03 0.34 0.38 hsa-miR-1908-5p 48.76 0.72 0.01 2.47 1.93 hsa-miR-486-5p 5,471.97 0.55 0.01 1.76 1.36 hsa-miR-192-5p 5,963.58 0.79 0.00 0.19 0.55 hsa-miR-489-3p 709.95 1.86 0.00 0.33 1.31 hsa-miR-193a-5p 62.50 0.75 0.02 1.44 1.57 hsa-miR-490-5p 37.79 0.99 0.00 0.28 0.37 hsa-miR-193b-3p 235.11 0.32 0.04 0.43 1.42 hsa-miR-497-5p 2,210.67 0.40 0.04 4.12 0.67 hsa-miR-194-5p 429.58 0.49 0.00 0.15 0.24 hsa-miR-499a-5p 25.96 1.20 0.00 0.30 1.77 hsa-miR-195-3p 154.61 0.54 0.00 1.92 1.10 hsa-miR-501-3p 5,352.55 0.38 0.04 0.56 0.45 hsa-miR-195-5p 1,398.68 0.55 0.01 1.09 0.51 hsa-miR-532-5p 12,026.27 0.23 0.05 0.08 1.33 hsa-miR-197-3p 4,746.12 0.47 0.00 1.20 1.18 hsa-miR-548h-5p 53.59 0.52 0.03 2.32 0.54 hsa-miR-200b-3p 20.49 0.64 0.03 0.36 0.71 hsa-miR-550a-3p 258.48 0.42 0.01 0.10 2.43 hsa-miR-20b-5p 584.11 0.50 0.00 0.95 1.24 hsa-miR-550a-5p 316.40 0.45 0.00 1.19 1.03 (Continued on next page) 382 Molecular Therapy: Nucleic Acids Vol. 17 September 2019 www.moleculartherapy.org Table 3. Continued Log2 Fold Average Average Log2 Fold Average Average Mean Change B Score B Score Mean Change 24 h B Score B Score Normalized 24 h MPP+ Basal MPP+ Normalized MPP+ Basal MPP+ miRNA Counts versus C padj Assay Assay miRNA Counts versus C padj Assay Assay hsa-miR-210-5p 73.28 0.46 0.04 3.10 0.42 hsa-miR-574-5p 752.87 0.90 0.00 1.27 2.81 hsa-miR-212-3p 1,057.58 0.77 0.02 1.72 0.09 hsa-miR-576-5p 202.17 0.51 0.00 1.29 1.47 hsa-miR-212-5p 423.95 0.75 0.01 1.03 0.47 hsa-miR-584-5p 1,617.69 0.62 0.00 0.25 1.63 hsa-miR-219a-1-3p 35.29 0.54 0.03 0.02 3.86 hsa-miR-627-5p 70.33 0.78 0.00 1.48 4.12 hsa-miR-22-3p 183,682.84 0.16 0.03 0.15 0.09 hsa-miR-636 57.03 0.82 0.00 2.70 8.88 hsa-miR-222-3p 734.01 0.32 0.02 0.24 0.67 hsa-miR-6516-5p 27.95 0.72 0.01 1.00 0.41 hsa-miR-23a-5p 126.01 0.89 0.00 0.62 0.30 hsa-miR-653-3p 60.24 1.85 0.00 1.38 2.09 hsa-miR-25-5p 984.00 0.51 0.00 0.60 0.70 hsa-miR-653-5p 60.58 1.39 0.00 2.44 0.06 hsa-miR-26b-3p 273.41 0.38 0.04 2.73 1.31 hsa-miR-671-3p 2,130.38 0.44 0.02 0.00 0.83 hsa-miR-26b-5p 23,380.92 0.41 0.00 2.77 1.16 hsa-miR-6716-3p 47.82 0.57 0.04 1.72 2.98 hsa-miR-27a-5p 477.16 0.56 0.00 2.51 0.38 hsa-miR-6847-5p 15.45 0.82 0.03 3.04 5.06 hsa-miR-27b-5p 2,323.36 0.34 0.04 0.19 0.91 hsa-miR-760 345.77 0.53 0.02 1.92 2.44 hsa-miR-301a-5p 125.21 0.49 0.04 0.31 0.52 hsa-miR-769-5p 14,172.66 0.33 0.00 1.18 0.45 hsa-miR-30a-3p 1,131.05 0.34 0.00 0.47 0.19 hsa-miR-7974 370.02 2.32 0.00 2.89 0.21 hsa-miR-30b-3p 46.79 0.63 0.04 0.05 1.84 hsa-miR-873-3p 1,154.19 0.53 0.00 0.20 0.99 hsa-miR-3158-3p 118.66 0.68 0.00 0.25 1.71 hsa-miR-887-3p 5,009.15 0.27 0.00 0.52 1.03 hsa-miR-3174 23.19 0.78 0.01 0.68 1.61 hsa-miR-887-5p 207.56 0.65 0.00 2.86 0.28 hsa-miR-3176 216.31 0.68 0.00 3.59 2.19 hsa-miR-92a-1-5p 180.98 0.81 0.00 1.61 0.55 hsa-miR-3187-3p 57.82 0.98 0.00 1.07 2.69 hsa-miR-92b-5p 173.91 0.49 0.00 0.75 1.29 hsa-miR-328-3p 528.91 0.45 0.00 1.20 0.95 hsa-miR-93-3p 1,166.74 0.33 0.02 3.47 0.92 hsa-miR-335-3p 29.44 0.84 0.00 0.77 0.82 hsa-miR-935 980.02 0.45 0.00 1.10 0.05 hsa-miR-98-5p 7,822.48 0.67 0.00 0.95 1.18 The B score under basal conditions and following MPP+ treatment is shown for each ASO targeting the corresponding deregulated miRNA. padj., adjusted p value. In summary, the present study provides a catalog of protective and Transfection of LNA-Modified ASOs detrimental miRNAs with activity that affects neuronal viability, The library consisted of 1,954 miRNA-ASOs plus 50 custom both in resting conditions and/or in response to oxidative stress. sncRNA-ASOs (Human miRCURY LNA microRNA Inhibitor Our approach is a first step in defining druggable miRNAs in preclin- Library 190103; Exiqon). Transfection was performed using a Sci- ical studies aiming at validating the manipulation of miRNA activity clone AHL 3000 (Caliper). Briefly, 50 mL of transfection mix was as a therapeutic strategy in neurological and neurodegenerative added to differentiated cells grown in 100 mL of differentiation me- disorders. dium. The transfection mix consisted of 43.5 mL of Opti-MEM (Gibco), 0.5 mL of Lipofectamine 2000, and 6 mL of miRNA- or MATERIALS AND METHODS sncRNA-ASO. The final concentration of the ASO was 70 nM. Cell Cultures Then, 100 mL of the transfection medium was removed 24 h later SH-SY5Y cells were grown in DMEM with L-glutamine with a Platewasher ELx405 (BioTek Instruments) and replaced with (Biowest), supplemented with 10% inactivated fetal bovine serum 100 mL of fresh differentiation medium, with a Multidrop 348. (FBSi; Gibco), 1% of penicillin-streptomycin (P/S; Gibco), and 250 ng/mL of amphotericin B (Sigma-Aldrich). SH-SY5Y Each plate was transfected with 58 unique miRNA- or sncRNA-ASO, cells were differentiated for 4 days with retinoic acid (RA; Merck) a negative mock-transfected control, and a positive control, consisting at 10 mM and for 3 days with 12-O-tetradecanoylphorbol- of a sncRNA-ASO with known detrimental activity. The peripheral 13-acetate (TPA; Merck) at 80 nM. This protocol was adapted wells were not included in the analysis and contained only medium. from Presgraves et al.44 Approximately 5,000 cells were seeded The 2,004 sncRNA-ASOs were distributed in 37 plates, and transfec- per well in 96-well plates (Thermo Fisher Scientific), using a Mul- tions were performed in triplicate. An additional plate was included tidrop 348 (Thermo Fisher Scientific) or in 100,000 cells in 6-well in triplicate containing mock-transfected cells (treated only with plates. Lipofectamine 2000; Thermo Fisher Scientific); cells transfected Molecular Therapy: Nucleic Acids Vol. 17 September 2019 383 Molecular Therapy: Nucleic Acids with a sncRNA-ASO that does not alter cell viability, used as the nega- denominator were adjusted to account for systematic biases. A row- tive control (miRCURY LNA Inhibitor Negative Control A; Exiqon); column-additive model was added to the plate using the two-way and cells transfected with a sncRNA-ASO, inducing cell death, used as median polish procedure xrcp = mp + Rrp + Ccp + ercp , where xrcp a positive control (Custom miRCURY LNA Inhibitor Positive Con- is the raw value of a sample in row r, column c, and plate p. mp is trol A; Exiqon). Taking into account all conditions and the replicates, the plate median, Rrp is the row effect, and Ccp is the column effect. the HTFS included 114 plates. Under this model, ercp is the plate-specific row-and-column-normal- ized value of xrcp . Taking all that into account, the final B score was Two independent HTFSs were performed in triplicate to evaluate the obtained by: effect of the miRNA- or sncRNA-ASOs in basal conditions and in ercp b’i = : response to MPP+ stress. In the HTFS under basal conditions, cell madp ðeÞ viability was determined 24 h after the addition of the fresh medium (48 h after transfection). In the HTFS aiming to evaluate the response As this positional-effect-normalized B score behaves as a standard- to MPP+, the fresh medium contained MPP+ at a final concentration ized Gaussian distribution, the hits can be defined as values below of 3 mM. In this case, viability was determined 24 h after treatment 2 (two SDs) in the screening looking at neuronal survival. In the with MPP+ and 48 h after transfection. screening related to compounds affecting viability under OS, values below 2 are considered to worsen viability in an OS context. Conversely, values above 2 in that screening indicate that the effect MPP+ Treatment of the ASO is protective. To evaluate the involvement of miRNA- or sncRNA-ASOs in in the neuronal response to oxidative stress, cells were treated with MPP+. Hits Detection Based on a LMM After differentiation and 24 h following transfection in 96-well plates, The previous approach was not able to deal with the variance produced the cells were treated with a dose of MPP+ (3 mM, final concentra- by the fact that each plate was run in triplicate. Therefore, we comple- tion; Sigma-Aldrich), inducing a moderate effect on cell viability mented the B score approach with another one based on a LMM: (30%–40% decrease), which was determined 24 h later. xrcpik = m + Rr + Cc + gp + ai + ercpik : MPP+ (3 mM) treatment was also performed in six-well plates after In this equation, xrcpik is the measurement value of the k-th biological SHSY-5Y seeding (100,000 cells per well) and differentiation, to evaluate replicate of the i-th compound sample in row r, column c, and plate p. sncRNA perturbations occurring as a consequence of MPP+ stress. m is the overall average across all measurements. Rr and Cc are row and column fixed effects, respectively. ai is the random effect for Cell Viability Determinations the i-th sample, which accounts for inter-sample variation. gp is the Cell viability was assessed with the WST-1 reagent45 (Roche) 48 h random effect for the p-th plate, which accounts for inter-plate vari- after transfection. WST-1 was added to the cell medium at a ratio ation. ercpik is a random-error variable, that accounts for the vari- of 1:10. WST-1 was dispensed using a Sciclone AHL 3000. Cell plates ability inherent to the sample. The model assumes that ai , gp and were incubated with WST-1 at 37 C for 2 h. Then, each plate was read ercpik are Gaussian variables with mean 0 and s2a ; s2g s2e variances, twice: at 440 nm and at 690 nm, using an Infinite M200 instrument respectively. Therefore, the model accounts for positional effects (Tecan). The final absorbance value was the result of subtracting through the inclusion of row and column fixed effects and adds in- the 690 nm absorption measure from that at 440 nm and from that ter-plate variability. The model can easily be extended to include of a blank plate. This blank plate was also measured at 440 and specific plate positional biases by the inclusion of a random effect 690 nm and contained growth medium without cells and WST-1 at that interacts with row and column fixed effects. a ratio of 1:10. As in this approach ai is the element of interest. The BLUP46 (the best In the HTFS to evaluate the response to MPP+, a triplicate plate was linear unbiased predictor of these random effects) of ai can be used to added to measure the effect of MPP+ in cell viability in mock-trans- catch samples that significantly deviate from the overall adjusted fected cells and in cells transfected with the sncRNA-ASO used as a average. Because the experimental design includes biological tripli- positive control. cates, the model takes advantage of this by including all triplicates, instead of summarizing them, and therefore gains statistical power. Hits Detection Based on B Score In this approach, the sample outliers, the putative hits, are summa- The B score method19 is based on the median, which is not distorted rized in a single value given by the BLUPs. by outliers (the hits or ASOs affecting cell viability): ROS Quantification xi  medðxÞ bi = : Subsets of miRNA-ASOs were selected to analyze their effect on the madðxÞ ROS production that normally accompanies neuronal damage. In this equation, medðxÞ is the median of all sample values on a plate, miRNA-ASOs were transfected in triplicate, in six independent bio- whereas madðxÞ is the median absolute deviation. The numerator and logical replicates. After 24 h, half of them were treated with 3 mM 384 Molecular Therapy: Nucleic Acids Vol. 17 September 2019 www.moleculartherapy.org MPP+. CellROX Deep Red Reagent (Molecular Probes) was used to RNA integrity was evaluated with Bioanalyzer 2100 (Applied Bio- measure ROS production, and CyQUANT Direct Cell Proliferation systems), with RNA integrity number (RIN) > 9 in all samples. Small Assay (Invitrogen) was used to normalize ROS levels by the RNA (sRNA) indexed libraries for sequencing were prepared from number of cells. After the transfection (48 h), with or without treat- 1 mg of total RNA, using the Trueseq kit (Illumina), according to ment with MPP+ (24 h), cells were treated with CellROX Deep Red the manufacturer’s specifications. Library preparation for control Reagent at 5 mM and incubated for 30 minutes at 37 C, and and MPP+-treated cells was performed in parallel, and indexed sam- fluorescence was measured at 640/665 nm. Afterward, the same cells ples were sequenced in a single lane in a HiSeq2000 (Illumina). The were treated with CyQUANT Direct Cell Proliferation Assay. reads generated were 50 nt long. CyQUANT (15 mL; 10 solution) was added to each well, the cells were incubated for 2 h at 37 C, and fluorescence was measured at sRNA Profiling 485/535 nm. Reads were trimmed to 36 nt and ligation adapters removed using the adrec.jar program from the seqBuster suite.47 Sequences were map- Fold change measures were used in order to assess whether the sub- ped to the hg19 genome. sRNA processing, mapping, and annotation sets of miRNA-ASOs had collective effects in the production of ROS, were performed using the SeqBuster and SeqCluster tools, as by comparing each subset of miRNA-ASOs affecting cell viability described in prior studies.47,48 We used DESeq2 for differential with the mock-transfected condition. The approach used was again expression analysis and log2 transformation of the count data. We a LMM, as it allowed accounting for the different sources of variability followed the standard steps for the analysis: load count matrix, esti- that the experiment involved. It also allowed the attainment of a mation of dispersion, and negative binomial Wald test, with outlier normalized fold change (FC). The formula used was: removal using DESeq2 functions.49  log xjpik = m + bj + gp + ai + ercpik : SUPPLEMENTAL INFORMATION In this equation, xjpik is the measurement value of the k-th technical Supplemental Information can be found online at https://doi.org/10. replicate of the i-th miRNA-ASO in the p-th plate and the j-th group. 1016/j.omtn.2019.06.007. If the miRNA-ASO affects cell viability, j = 1; if it does not, j = 0. m is the overall average across all measurements, bj is the group fixed ef- AUTHOR CONTRIBUTIONS fect, ai is the random effect for the i-th miRNA-ASO (accounting for J.P.-A. and M.T.Z.-A. conducted the experiments. G.E. performed the inter-miRNA-ASO variation), gp is the random effect for the p-th statistical analyses and helped write the manuscript. L.P. performed plate (accounting for inter-plate variation), and ejpik is a random-error bioinformatic analyses of sRNA sequencing data. A.S. and M.F.S. variable (accounting for the intra-sample variability). The model as- helped in designing the high-throughput experimental approaches. sumes that ai , gp ; and ercpik are Gaussian variables with mean 0 and E.M. designed the study, organized the presentation of the results, s2a ; s2g ; and s2e variances, respectively. Under the assumptions of and wrote the manuscript. this LMM, the adjusted FC is obtained as FC = expðbÞ. CONFLICTS OF INTEREST Determination of Mitochondrial Functionality The authors declare no competing interests. To determine mitochondrial activity, we used the MitoTracker Deep Red probe (Invitrogen), which passively diffuses across plasma ACKNOWLEDGMENTS membranes and accumulates in active mitochondria. After the This work was supported by the Spanish Ministry of Economy and transfection (48 h), the cells were treated with MitoTracker Deep Competitiveness and FEDER funds (SAF2014-60551-R and Red reagent at 0.1 mM and incubated for 1 h at 37 C, and fluores- SAF2017-88452-R). We acknowledge the support of the Spanish cence was measured at 579/599 nm. Carbonylcyanide chlorophenyl- Ministry of Economy, Industry and Competitiveness (MEIC) to the hydrazone (CCCP) 5 mM was used as the positive control for the EMBL partnership and the Centro de Excelencia Severo Ochoa mitochondria depolarization. The Kruskal-Wallis test was used to 2013–2017 (SEV-2012-0208). We acknowledge the support of the compare all conditions, followed by the Mann-Whitney U test Spanish Ministry of Science Innovation and Universities, Maria with Bonferroni’s post hoc correction. p < 0.05 was considered sta- Maeztu Unit of Excellence Programme. We thank the staff of the Ge- tistically significant. nomics Unit for the preparation of sRNA libraries and sequencing and the staff of the Biomolecular Screening and Protein Technologies RNA Extraction and Small RNA Sequencing Unit for their help in the setting up the high-throughput screening. Total RNA was isolated from cells, 24 h after treatment with MPP+ or a control solution, in four independent biological replicates. After the REFERENCES medium was removed from cells treated with MPP+ for 24 h, the 1. Treiber, T., Treiber, N., and Meister, G. (2019). Regulation of microRNA biogenesis plates were washed once in PBS and immediately frozen at 80 C. and its crosstalk with other cellular pathways. Nat. Rev. Mol. 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