Oxidative Stress and Its Modulation by Ladostigil Alter the Expression of Abundant Long Non-Coding RNAs in SH-SY5Y Cells

Abstract

Neurodegenerative disorders, brain injury, and the decline in cognitive function with aging are accompanied by a reduced capacity of cells in the brain to cope with oxidative stress and inflammation. In this study, we focused on the response to oxidative stress in SH-SY5Y, a human neuroblastoma cell line. We monitored the viability of the cells in the presence of oxidative stress. Such stress was induced by hydrogen peroxide or by Sin1 (3-morpholinosydnonimine) that generates reactive oxygen and nitrogen species (ROS and RNS). Both stressors caused significant cell death. Our results from the RNA-seq experiments show that SH-SY5Y cells treated with Sin1 for 24 h resulted in 94 differently expressed long non-coding RNAs (lncRNAs), including many abundant ones. Among the abundant lncRNAs that were upregulated by exposing the cells to Sin1 were those implicated in redox homeostasis, energy metabolism, and neurodegenerative diseases (e.g., MALAT1, MIAT, GABPB1-AS1, NEAT1, MIAT, GABPB1-AS1, and HAND2-AS1). Another group of abundant lncRNAs that were significantly altered under oxidative stress included cancer-related SNHG family members. We tested the impact of ladostigil, a bifunctional reagent with antioxidant and anti-inflammatory properties, on the lncRNA expression levels. Ladostigil was previously shown to enhance learning and memory in the brains of elderly rats. In SH-SY5Y cells, several lncRNAs involved in transcription regulation and the chromatin structure were significantly induced by ladostigil. We anticipate that these poorly studied lncRNAs may act as enhancers (eRNA), regulating transcription and splicing, and in competition for miRNA binding (ceRNA). We found that the induction of abundant lncRNAs, such as MALAT1, NEAT-1, MIAT, and SHNG12, by the Sin1 oxidative stress paradigm specifies only the undifferentiated cell state. We conclude that a global alteration in the lncRNA profiles upon stress in SH-SY5Y may shift cell homeostasis and is an attractive in vitro system to characterize drugs that impact the redox state of the cells and their viability.

Keywords: MALAT1; Nrf2 signaling; RNA-seq; UPR; ceRNA; neurodegenerative disease.

Figure 1

Flow cytometry analysis in cells under oxidative stress conditions in response to treatment with ladostigil. (A) Results from FACS analysis with 50k cells per experiment. The fraction of live and dead cells upon 40 μM H₂O₂ and ladostigil treatment was tested at 5 h following stimulus. Asterisks indicate the statistical significance of the results. The attenuation in the fraction of PI-positive cells by ladostigil (5.4 μM) was substantial (p-value = 0.02). (B) Cell viability upon increasing the concentration of Sin1 with and without the presence of ladostigil (5.4 μM, with mean and standard error; s.e.). Each data point is based on 8 replicates. (C) Results from 3 repeated experiments with mean and standard deviation (s.d.) from the flow cytometry run of SY-SY5Y cells treated with Sin1 for 5 h. All measured data were from FACS that was repeated in triplicate. The total FACS cells was considered as 100%, and the percentage of them stained with PI is indicated as a fraction. (D) Results from triplicate experiments with the mean and standard deviation (s.d.) from the flow cytometry run for Annexin-V of SY-SY5Y cells treated with Sin1 for 5 h. NT indicates cells non-treated with Sin1. Asterisks indicate the t-test statistical results with p-values of <0.005 (***), <0.01 (**), and <0.05 (*).

Figure 2

Partition of ncRNA biotypes in SH-SY5Y cells. (A) Partition of the RNA-seq results of untreated SH-SY5Y cells according to the major molecular biotypes. Altogether, there were 19,475 identified transcripts. Blue, coding genes and purple, ncRNAs portioned by biotype classes. Mito, mitochondrial transcript; TEC, to be experimentally confirmed; and PG, pseudogene. Combined related biotypes (e.g., transcribed unprocessed and unitary pseudogenes) are marked as multi. (B) Partition of 780 expressed ncRNA transcripts partitioned by major ncRNA classes. The ncRNAs were based on transcripts with an average TMM of >4. There were 11,588 aligned transcripts that met this threshold. (C) Top expressing ncRNAs (TMM > 100) colored by broad-sense functional annotations (inset). MT, mitochondria; snRNA, small nucleus RNA; and XCI, X-chromosome inactivation.

Figure 3

Differentially expressed (DE) ncRNAs following 24 h of exposure to Sin1 relative to untreated cells. (A) Transcripts associated with upregulation (Up) and downregulation (Down) relative to naïve, untreated cells. The number of genes associated with each expression trend is shown in parentheses. The rest of the genes (9719 genes) were unchanged. See the Materials and Methods for the expression trend definition. (B) Histogram showing the DE ncRNAs ratio for downregulated and upregulated transcripts. The dashed line marks the expression ratio (1.25 and 0.8) vs. naïve cells for Up and Down, respectively. The pie diagram applies to 94 genes that satisfy the threshold.

Figure 4

Expression of members of the small nucleolar RNA host genes (SNHGs). (A) Mean expressions (by log₁₀TMM) of the 17 SNHG members identified by DE lncRNA analysis. (B) Fold changes (FCs) following treatment with 50 μM Sin1 are shown for each of the SNHGs. FCs below and above zero show downregulated and upregulation of the indicated genes. Note that the values are logFC. Despite the small effect of the differential expression genes, the filled bar shows statistically significant genes, and the striped bar indicates a gene belonging to the SNHG group, but failed to reach FDR (p-value < 0.05).

Figure 5

Sample of the lncRNAs by the measured expression levels in naïve untreated cells (NT), cells exposed to Sin1 (24 h) with or without ladostigil. (A) The transcripts shown are those with maximal induction by Sin 1 relative to NT. The average TMM and the standard deviations from biological triplicates are shown for each experimental group. (B) The transcripts shown are those with a maximal effect by ladostigil in the presence of Sin1. The average TMM and the standard deviations from biological triplicates are shown for each experimental group. The statistical significance with the p-value FDR ranging from 0.05–0.005 is depicted by * and <0.005 by **. (C) The genomic segments of AC105339.2 and RPARP-AS1 cover 10 and 12k nucleotides, respectively. AC105339.2 and RPARP-AS1 show the overlap between the lncRNA transcript and the gene in the vicinity. AC105339.2 acts as antisense to FSD2 (704 aa) and RPARP-AS1 is the antisense for C10orf95 (213 aa). Numerous transcripts of RPARP-AS1 are shown above the ENCODE annotations for enhancers (red) and proximal promotors (yellow).

Figure 6

Differentially expressed (DE) ncRNAs following 24 h of exposure to Sin1 relative to untreated cells. (A) RT-PCR results on Agarose gel. The RT-PCR product of β-actin was used as an internal control. Each condition is shown for two biological duplicates. The indicated ncRNA results are for the untreated culture (N.T.) and cells in the presence of Sin1, and Sin1 and ladostigil (5.4 μM). (B) Quantification of the results in (A) by a heatmap visualization. Orange, comparison of the baseline of the untreated cells. Blue, changes in cells by Sin1, and Sin1 and ladostigil (5.4 μM).