Crosstalk between miRNA and protein expression profiles in nitrate exposed brain cells

doi:10.21203/rs.3.rs-2336998/v1

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Crosstalk between miRNA and protein expression profiles in nitrate exposed brain cells

https://doi.org/10.21203/rs.3.rs-2336998/v1

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published 27 Mar, 2023

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Growing evidence reported a strong association between the ingestion of nitrate and adverse health consequences in humans, including its detrimental impact on the developing brain. The present study identified miRNAs and proteins in SH-SY5Y human neuroblastoma cells and HMC3 human microglial cells using high throughput techniques in response to nitrate level most prevalent in the environment (mainly India) (X) and an exceptionally high nitrate level (5X) that can be reached in the near future. Cells were exposed to mixtures of nitrates for 72 h at doses of X and 5X, 320 mg/L and 1600 mg/L, respectively. OpenArray and LCMS analysis revealed that maximum deregulation in miRNAs and proteins was found in cells exposed to 5X dose. Top deregulated miRNAs include miR-34b, miR-34c, miR-155, miR-143, and miR-145. The proteomic profiles of both cell types include proteins that are potential targets of deregulated miRNAs. These miRNAs and their targeted proteins are involved in multiple functions, including cellular senescence, cell cycle, apoptosis, neuronal disorders, brain development, and homeostasis. Further, measuring mitochondrial bioenergetics in cells exposed to nitrate using a Seahorse XFp flux analyzer revealed that a 5X dose causes a significant reduction in oxygen consumption rate (OCR) and other bioenergetics parameters in both cell types. In summary, our studies have demonstrated that 5X dose of nitrate significantly alters cellular physiology and functions by deregulating several miRNAs and proteins. However, X dose of nitrate that is most prevalent in the environment has not caused any adverse effects on any cell type.

Nitrate

SH-SY5Y cells

HMC3 cells

miRNAs

proteomics

cellular bioenergetics

Neurodegeneration refers to a class of neurological diseases characterized by the slow and gradual deterioration of brain regions that are crucial for cognitive, psychological, behavioral, and motor functions [1]. These deliberating illnesses worsen with age [2] and can appear in hereditary or sporadic forms based on genetic and environmental variables. Protein misfolding/fibrillation, mitochondrial dysfunction, and oxidative stress are special features of various neurodegenerative diseases (NDDs), such as Alzheimer's disease (AD), Parkinson's disease (PD), Amyotrophic lateral sclerosis (ALS), Huntington's disease (HD) [1, 3]. Nitrates are a well-known water pollutant that can adversely affect groundwater systems. After pesticides, nitrates are the most prevalent anthropogenic pollutants of surface and groundwater. They are applied to the soil in the form of fertilizers during various agricultural practices. This has become a global concern and several international research communities have raised alarm to supply safe drinking water to the human population [4].

Numerous adverse outcomes have been reported in humans exposed to excess nitrates through drinking water, processed and preserved foods, and fertilizers [5–7]. According to Schwendimann et al., the brain development of the offspring born to pregnant mice who consumed water with high concentrations of nitrates (319 mg/L) and total dissolved salts (TDS) was altered. Brain from offsprings born to dams who had been given agricultural water (versus forest control water) were significantly smaller. Furthermore, the motor cortex of the brains of these offspring had significantly more microglia at postnatal day 21 (P21), significantly fewer white matter astrocytes, and a considerable increase in cell death, particularly in the dentate gyrus [8]. Nitrate exposure causes moderate anxiogenic-like behavior and alters the brain metabolomics profile in zebrafish [9]. These neuropathological effects suggest that nitrates may alter brain development. However, the underlying cellular and molecular mechanisms are still not well studied.

MicroRNAs (miRNA) are small non-coding RNAs that negatively control post-transcriptional gene expression. A huge number of miRNAs are expressed in the brain, and emerging research suggests that miRNAs are crucial for neuronal functions and brain development [10]. In recent decades, microRNA (miRNA) deregulation is emerging as a contributor to neurodegeneration by influencing most of the mechanisms responsible for NDDs [11]. MiR-34 family, miR-200 family, miR-143/145 cluster, and miR-155 are brain-specific miRNAs and have a crucial role in brain development [12–14]. The miR-34 family is involved in the regulation of neuronal maturation (proliferation and differentiation) and brain aging in neuronal or non-neuronal cells [15], and upregulation of miR-34 is associated with the pathogenesis of AD and metabolic syndrome [16]. Overexpression of miR-143/145 cluster has been reported in the brain following ischemic insult and multiple sclerosis [17, 18]. Increased miR-199a/b expression was strongly correlated with nitrate tolerance in humans [19]. A key research goal in miRNA biology is to understand the post-transcriptional/translational control of the targets and their effects on biological processes. MiRNA targets are typically identified computationally by seed sequence conservation, which yields numerous candidate targets with little overlap. This lack of linkage, together with the lack of correlation between mRNA and protein levels, encourages researchers to employ proteomic-based techniques for identifying, estimating, and confirming miRNA-induced protein alterations.

In the present study, we have studied the effects of nitrate on human SH-SY5Y neuroblastoma cells and human HMC3 microglial cells at X nitrate level (320 mg/L) that is most prevalent in the environment (India) and at 5X (1600 mg/L) which is an exceptionally high nitrate level that appeared occationally, and might be reached in the future [8, 20]. By employing multi-omics approaches, this study identified miRNAs and proteins deregulated following nitrate exposure in cells. Additionally, the effect of nitrate on cellular bioenergetics has been investigated using the Seahorse extracellular flux analyzer.

Chemicals and Reagents

Sodium nitrate (Cat# 221341-550G), Potassium nitrate (Cat# P8291-500G), Magnesium nitrate (Cat# 63084-500G-F), Dithiothreitol (DTT) (Cat# 10,197,777), iodoacetamide (Cat# I1149), D-(þ)-glucose solution (Cat# G8769), and protease inhibitor (Cat# P-8340) were purchased from Sigma-Aldrich. SH-SY5Y neuroblastoma cells (Cat# SH-SY5Y CRL-2266) and HMC3 microglia cells (Cat# CRL 3304) were purchased from ATCC. Media and reagents including Minimum Essential Media (Cat# 61100–01), F-12 nutrient media (Cat# 21700–075), fetal bovine serum (FBS) (Cat# 10270106), antibiotic/antimycotic solution (Ab/Am) (Cat# 5240062), L-glutamine supplement (Cat# 25030), and Gibco sodium pyruvate (Cat# 11360–070) were procured from Thermo Fisher Scientific. Pierce™ Detergent Removal Spin Columns (Cat#87,776), Pierce™ C18 Spin Columns (Cat# 89,870), TaqMan Custom RT pool (Cat#A25630), TaqMan™ MicroRNA Reverse Transcription Kit (Cat# 4,366,597), TaqManR Custom openArray PreAmp Pool (Cat#2,874,647), TaqManR PreAmp Master Mix (Cat# 4,384,266), TaqManR openArray™ RT-PCR Plates 112 format (P/N # 4,470,813), TaqManR openArrayR Real-Time PCR Master Mix (Cat#4,462,159), High capacity cDNA RT kit (Cat# 4368813), mirVana miRNA isolation kit (Cat# AM1560), and many other reagents required for quantitative PCR were obtained from Applied Biosystems Thermo Fisher Scientific. Trypsin/Lys-C Mix, LCMS grade (Cat# V5073) was purchased from Promega, CA. XFp cell culture microplates (Part Number # 103023- 100), Seahorse XFp extracellular flux cartridge (Part Number # 103022–100), XF calibrant (Part Number # 103059–000), Extracellular flux assay kits (Part Number # 103010–100), and Agilent Seahorse XF base medium (Part Number # 102353–100) were procured from Agilent Technologies, India. RapiGest SF Surfactant (Cat#186,001,861) was procured from Waters, Milford, USA.

Cell culture and nitrate exposure

MEM/Ham F-12 nutrient media was used for SH-SY5Y cell culture with a 1% of the Ab/Am and 10% FBS whereas HMC3 cells were cultured in MEM medium with 1 % Ab/Am and with the supplementation of 10% FBS. Cells were incubated and maintained at 37 ℃ with 5% CO2 and 95% relative humidity. Nitrates were exposed to cells as mixtures of sodium nitrates, potassium nitrates and magnesium nitrates (320 mg/L nitrates mix named X dose and 1600 mg/L nitrates named 5X dose) for 72 h.

Cell viability assay

Cytotoxicity of nitrates on SH-SY5Y and HMC3 cells were identified using alamarBlue cell viability reagent (Cat# DAL1025). Equal numbers of cells were grown in each well of 96-well plates and exposed to various concentrations- X/10, X/5, X, 5X, 10X for 24, 48, 72, and 96 h. Fresh culture medium with doses were replaced every 24 h. AlamarBlue reagent was used one of the tenth ratio of medium in each well and incubated for 4 h and then fluorescence were measured at wavelength of 560 nm (excitation) and 590 nm (emission). Blank was used to normalize background florescence.

OpenArray technology based on real-time PCR of miRNA

Total RNA was isolated using the manufacturer’s instructions of the mirVana miRNA isolation kit from Thermo Fisher Scientific in Waltham, Massachusetts. Gel electrophoresis was used to confirm the integrity of the RNA samples. Selected miRNAs important in neural development and neurotoxicity were included in the unique “brain-specific miRNA” openArray panel (Part No. # 4,470,813) which was used for miRNA profiling. The openArray panel contains primers for 112 miRNAs. For the reverse transcription, a TaqMan Custom RT pool primer of 112 miRNAs was utilised. Thermo Fischer’s TaqMan miRNA Reverse Transcription Kit was used to perform reverse transcription on mature miRNAs. In a brief, the reaction mixture includes 100 ng of RNA, 75 units of MultiScribe reverse µtranscriptase, RT buffer, openArray Custom RT primers, Rnase inhibitor, MgCl₂, and dNTPs, with a maximum reaction volume of 7.5 µl. The reverse transcription process was thermally cycled a total of 40 times at 16 °C for 2 min, 42 °C for 1 min, and 50 °C for 1 s. Denaturation at 85 °C for 5 min was used to stop the process. Following that, cDNA was preamped using a pool of openArray preamp primers (Part No. # 4,485,255) and a TaqMan preamp master mixture. The ultimate volume for preamplification was 25µl, of which 2.5µl were custom preamp primer pools combined with 12.5 µl of preamp master mix, 2.5 µl cDNA, and additional nuclease-free water. Thermal cycling at 95 °C for 10 min, 55 °C for 2 min, 72 °C for 2 min, and 12 cycles of 95 °C for 15 s and 60 °C for 4 min were employed for preamplification. After cycling, preamplification products were incubated for 10 min at 99.9 °C to inactivate enzymes. Products from preamplification were diluted (1 to 40) in 0.1 TE buffer. The diluted preamplification products were combined with 2x TaqMan openArray Real-Time PCR Master Mix (Cat#4,462,159) and pipetted onto openArray plates using an openArray AccuFill System from Thermo Fisher Scientific. The preamplification product was then run as directed by the manufacturer on Thermo Fisher Scientific’s QuantStudio 12 K Flex RT-PCR machine. Using the Ct method and endogenous control, relative quantification of miRNA expression was calculated from the raw data using ExpressionSuite software. There were three biological replicates used in each experiment. The significant changes were determined by performing the Student’s t-test using the ExpressionSuite openArray analysis software. P value <0.05 Statistical significant.

Label-Free Quantitative LC–MS/MS Proteomics

Label-Free quantification have been performed as described in our earlier lab studies [21-23]. In brief, protein was isolated from SH-SY5Y and HMC3 cells and quantified using Pierce bicinchoninic acid (BCA) protein assay kit (Catalogue 23,227). Samples were then cleaned by Pierce™ Detergent Removal Spin Columns (Cat#87,776). Total 100ug protein from each sample was mixed with 0.1% (V/V) RapiGest SF surfactant (Cat#186,001,861) for solubility and sample were reduced by adding 5mM dithiothreitol (DTT) and incubated for 30 min at 60 °C. Further, 15 mM iodoacetamide (IAA) added and incubated the samples for 30 min at room temperature (RT) in dark for alkylation. Finally protein samples were digested by using enzyme Trypsin/Lys-C (Cat# V5071) and incubated overnight at 37 °C in a water bath. The digestion reaction was quenched by mixing 0.5% formic acid (FA) and incubated for 60 min at 37 °C. Finally the digested samples were centrifuged at 20,000 g for 10 min. at 4 °C and supernatant containing peptides was transferred in fresh vial. The peptide samples were purified and concentrated by Pierce C18 Spin Columns (Cat# 89,870), as manufacturer protocol. The purified tryptic peptides were lyophilized and used for proteomic run.

The peptide identification was achieved by nanoLC (EASY-nLC 1200; Thermo Fisher) coupled with Q Exactive mass spectrometer (Q Exactive; Thermo Fisher) through a nano-electrospray ionisation source (Thermo Fisher Scientific). The lyophilized peptide samples were reconstituted in 0.1% FA and loaded on reversed phase column. The mobile phase used for the elution contain; solvents A (1% v/v acetonitrile and 0.1% v/v formic acid in water and 80% v/v acetonitrile ) and solvent B (1% v/v acetonitrile and 0.1% v/v formic acid in water and 80% v/v acetonitrile and 0.1% v/v FA). The loaded peptides in solvent A were eluted by applying a linear gradient of solvent B for 140 min at a flow rate of 300nl/min. The gradient began at 2% B to 5% B in 5 min and then increased linearly to 35% B in 120 min and finally increased to 90% for 15 min. A voltage of 2300 V was applied for electrospray ionisation and the heated capillary temperature was set at 300 °C. Data-dependent acquisition (DDA) was employed for peptide identification. Full MS had a resolution of 70,000, a scan range of 350 to 1700 m/z, and an automatic gain control (AGC) of 1e6 with a 60 ms maximum injection time. For dd-MS2, a resolution of 17,500 and AGC was 1e5 with a maximum injection duration of 100 ms. The MS data was obtained using the top 15 most abundant precursors and dynamic exclusion was set to 20 seconds.

Identification and Quantification of Proteins

The Proteome Discoverer software (version 2.4, Thermo Fisher, USA), all proteomics data were analysed. Peptide lists were then searched against the web-based Uniprot database of Homo sapiens (SwissProtTaxID 10,116) with the cysteine carbamidomethylation as fixed modification while methionine oxidation and N-terminal acetylation as static modification. A 1% false discovery rate was used for peptide identification (FDR).

Metabolic Flux Analysis using Seahorse XFp for Bioenergetics

Using the Seahorse XFp Analyzer, the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were assessed (Agilent Technologies, CA) with protocol that is described in our previous lab study [21,23]. SH-SY5Y and HMC3 cells were seeded and exposed to nitrates doses in each well of the Seahorse XFp cell culture mini plates and then real-time metabolic flux analysis was carried out. On the day of the test, cells were washed with unbuffered Seahorse XF base assay medium (10 mM glucose, 1 mM sodium pyruvate, 2 mM glutamine, pH 7.4). Prior to the start of the test, cells were cultured at 37 °C for 1 h in a CO2-free incubator. The impact of nitrate on mitochondrial respiration in SH-SY5Y cells and HMC3 was examined using the Seahorse XFp Analyzer. The effect of nitrate on the electron transport chain was assessed by adding oligomycin A (a complex V inhibitor), FCCP (an uncoupler), and rotenone (a complex I inhibitor)/Antimycin (a complex III inhibitor) in the order recommended by the manufacturer. The manufacturer’s instructions were followed when using the Cell Mito Stress Test kit, with ports A and B holding oligomycin, carbonyl cyanide-p-trifluoromethoxy phenylhydrazone (FCCP), and port C holding rotenone, a compound that inhibits complex I, and antimycin, a compound that inhibits complex III, respectively. The Seahorse XFp experiment was carried out using triplicate readings, and the outcomes were examined using Seahorse XFp Wave software.

Statistical Analysis

There were three replicates of each experiment. Data are represented as mean ± standard error mean (SEM). GraphPad Prism 7.0 (GraphPad Software, CA) was used to statistically analyse the dataset using the student’s t test, and Proteome Discoverer 2.4 was used to evaluate the proteomic data. For statistical significance, a p value of less than 0.05 was selected.

Changes in cellular morphology of SH-SY5Y and HMC3 cells

Under normal conditions, SH-SY5Y cells grow in cluster form and possess a short branch neurite phenotype with mixed population of adherent and suspension cells, while HMC3 cells (human microglias) possess only adherent phenotype. Upon exposure to X dose of nitrate for 72 h, both SH-SY5Y & HMC3 cells showed no significant changes in their morphological aspect while on exposure to 5X dose only SH-SY5Y shows dramatic changes in their morphology as indicated in (Figure 1) with single-sided red arrow.

Assessment of Cell viability and Cytotoxicity

he toxic potency of nitrate was assessed using Alamar blue assay. SH-SY5Y and HMC3 cells were exposed to different concentrations of nitrate such as X/10, X/5, X, 5X and 10X with their respective controls. In SH-SY5Y cells, the cell viability assay has no significant changes up to X dose of nitrate following 24, 48, 72 and 96 h exposure to nitrate. However, the cell viability reached 70% at 5X dose of nitrate for 72 h in SH-SY5Y cells (Supplementary figure 1B). HMC3 cells showed a significant decrease in the percentage of viability at 5X and 10X with increasing concentration of nitrate after 72 h of exposure (Supplementary figure 1A). However, alterations in cell viability at X dose were non-significant.

Identification of nitrate induced miRNAs in neuronal and microglial cells using real-time PCR based openArray

SH-SY5Y and HMC3 cells were exposed to identified non-cytotoxic concentrations of X and 5X of nitrate for 72 h. Nitrate-regulated miRNAs were identified by a Real-Time system using a custom “brain-specific miRNA array” containing 112 miRNAs in an openArray plate. The expression profile of deregulated miRNAs was shown on a volcano plot drawn between log₂fold change with a minimum 1.5 fold change boundary and p-value less than or equal to 0.05. Results of customized 112 brain-specific openArray panel indicate the differential regulation of miRNAs in different groups following nitrate exposure as listed in Figure 2. The deregulation of miRNAs was more profound in the case of SH-SY5Y cells. In SH-SY5Y cells, hsa-miR-27b and Ipu-miR-155 have been found to be significantly increased and decreased, respectively at both X and 5X doses of nitrate. MiRNAs such as hsa-miR-34c (+7.664 fold), miR-34b (+4.017 fold), miR-194 (+2.7 fold), and miR-200c (+2.524 fold) are the topmost upregulated miRNAs identified in SH-SY5Y cells exposed to 5X dose of nitrate while hsa-miR-213 (+2.141) and hsa-miR-363(+2.03 fold) showed significant upregulation at X dose exposure. The miR-145/143 is commonly upregulated following exposure to both X and 5X doses of nitrate in HMC3 cells while hsa-miR-199a-3p, hsa-miR-213, hsa-miR-210, hsa-miR-34a, hsa-miR-9, and hsa-miR-339-5p were found to be significantly downregulated upon exposure with 5X dose of nitrate in HMC3 cells. The list of miRNA that are commonly deregulated in both group is shown in Figure 3 for both SH-SY5Y and HMC3 cells.

Changes in proteome profile due to nitrate exposure to SH-SY5Y and HMC3 cells

SH-SY5Y and HMC3 cells were exposed to nitrate for 72 h and identified the differentially expressed proteins (DEPs) by LC/MS-MS. A volcano plot of analysis of SH-SY5Y and HMC3 cells were performed to identify DEPs by setting fold change threshold boundary 2 and P value less than 0.05 (Figure 4A-D). In SH-SY5Y cells, we found total 6305 proteins in which 218 (117 down and 101 up-regulated) proteins are significantly deregulated in X dose group while in 5X dose group total 484 (229 down and 255 up-regulated) proteins found deregulated significantly. On the other hand, we identified total 5752 deregulated proteins in which 491 (219 down and 272 upregulated) proteins are found in X dose group while in 5X dose group total 623 (280 down and 343 up-regulated) proteins found deregulated significantly. Further, for more accuracy we followed strict selection criteria of unique peptide more than one. Finally, in SH-SY5Y cells, we found 47 significant deregulated proteins in X dose group while in 5X dose group we found 181 deregulated proteins. Moreover, venn diagram showed 7 (PURA, H2BC18, ADI1, ASAH1, LSM2, NAB2, MRPL54) significant proteins are common between X dose and 5X dose group of SH-SY5Y (Figure 5A). In HMC3 cells, 163 proteins in X dose group and 277 proteins in 5X dose group were significantly deregulated after applying criteria of unique peptide more than one.

Additionally, venn diagram analysis showed 11.1% (44) proteins, named in table 1, common between X dose and 5x dose group of HMC3 cells (Figure 5B). Detailed list of significantly deregulated proteins in SH-SY5Y and HMC3 group provided in supplementary tables 2-9.

Table-1: Common deregulated proteins found in SH-SY5Y & HMC3 cells compared with X and 5X dose

Common proteins in both doses of SH-SY5Y cells	Common proteins in both doses of HMC3 cells
PURA, H2BC18, ADI1, ASAH1, LSM2, NAB2, MRPL54	ATP6AP2, SAFB2, ISCA2, CXADR, BGN, ALD, SSR4, MGARP, HIST1H4J, FAM120A, STAM2, MX1, MIF, H1-5, TRIM22, POLA1, EIF2A, C6orf139, SCAF1, GOLGA1, C1orf122, CDK5, BABAM2, LENG1, H1-2, RPL14, HIST1H1E, SMURF2, CHMP4A, ACSS2, TIMMDC1, MED8, H1-3, ERLIN1, TPI, ZYX, EFNB1, KIAA0319L, FLOT1, TBCEL, STAT2, APIP, DNASE2, SLU7

Functional enrichment and Protein-protein Interactions (PPIs) network of nitrate regulated proteins in SH-SY5Y and HMC3 cells

Most significantly deregulated proteins obtained with unique peptide more than one were uploaded on DAVID online pathway search engine tool (https://david.ncifcrf.gov). For KEGG and Gene Ontology (GO) analysis. KEGG pathway analysis in SH-SY5Y cells, indicated the involvement of significantly deregulated proteins in various biological pathways such as pathways of neurodegeneration- multiple diseases, Amyotrophic lateral sclerosis (ALS), focal adhesion, autophagy, sphingolipid metabolism (Figure 6A) in 5X dose group while in no significant pathway found in X dose group. In HMC3 cells, major significant pathway identified as pathways of neurodegeneration-multiple disease ALS, Parkinson’s Disease (PD), Alzheimer Disease (AD), Prion Disease, Huntington Disease (HD), protein processing in endoplasmic reticulum, ribosome, oxidative phosphorylation, arginine and proline metabolism, proteasome, arginine biosynthesis, tyrosine metabolism, 2-oxocarboxylic acid metabolism, phenyl alanine metabolism and various types of N-glycan biosynthesis in 5X dose group and Endocytosis, Epstein-Barr virus infection, Ubiquitin mediated proteolysis, Pyrimidine metabolism, Viral myocarditis, p53 signalling pathway, Nucleotide metabolism in X dose group. The detailed list of pathways provided in the supplementary table 14- 15. Further, GO analysis was performed to classify the significantly deregulated proteins in biological processes, cellular components and molecular functions (Supplementary table 10-13).

The STRING algorithm (https://string-db.org) was used to identify the Protein-protein interactions (PPIs) amongst significantly deregulated proteins in SH-SY5Y and HMC3 cells exposed to nitrate that revealed strong and complex interactions. Further, STRING analysis showed that several deregulated proteins formed complex clusters in both SH-SY5Y and HMC3 cells such as mitochondrial functions and nitrogen compound metabolic processes (Figure 7 A-B and 8 A-B). Several other interaction complexes exclusively found in SH-SY5Y (Cellular nitrogen compound metabolic process, cellular response to stress, Ras signalling, DNA ligation, ALS, spliceosomal complex, disease of metabolism and cellular component disassembly) and HMC3 cells (Nitrogen compound transport, organonitrogen compound metabolic process, cellular response to cytokine stimulus, PD, negative regulation of cell cycle G2-M phase transition, negative regulation of protein silencing, programmed cell death, Wnt signaling pathway and cell cycle) provided in supplementary figure 2-3.

Differentially miRNAs and proteins correlation studies

MiRNA-protein interaction studies were carried out to identify the post transcriptional gene regulation by miRNAs using TargetScan software (https://www.targetscan.org). In general, miRNAs negatively regulates the expression of target genes. Therefore, upregulated miRNAs reduces the level of their target proteins and vice-versa. The downstream target analysis of common deregulated miRNAs; X and 5X dose of SH-SY5Y cells (miR-27b and miR-155), HMC3 cells (miR-143 and miR-145) and 5X doses of HMC3 and SH-SY5Y cells (miR-34a, -34c, -143, -200c, -210, -339-5p) having binding site to the significantly deregulated proteins identified in SH-SY5Y and HMC3 cells displayed in Figure 9. In SH-SY5Y cells, the upregulated miR-27b showed interaction with maximum number of downregulated proteins (6) such as QKI, KCMF1, ZEB2, AK4, KMT2A, and PURA. Interestingly, PURA protein is commonly downregulated in both X and 5X group of SH-SY5Y cells while remaining proteins (QKI, KCMF1, ZEB2, AK4, KMT2A) found only in 5X dose group. In addition, miR-155 was also showed the interaction with RAPH1 and KANSL1 in X dose group and SCG2 protein in 5X dose group of SH-SY5Y cells. In HMC3 cells, upregulated miR-145 have shown the binding site to the maximum number of downregulated proteins (13) in which ERLIN1 and KIAA0319L protein commonly found in X and 5X dose group. Remaining 11 proteins targeted by miR-145; 6 proteins (RAB5C, DPYSL2, NEDD4L, STAM, MBD2, STX16) found in X group and 5 proteins (EPN1, CAMK2D, PKN2, IRS1, STK25) found in 5X group. A protein SLU7, commonly downregulated in X and 5X group have shown the binding site for the miR-143 in HMC3 cells

The target analysis of 6 common deregulated miRNAs in 5X group of SH-SY5Y and HMC3 cells showed only 4 miRNAs (miR-34a, miR-34c, miR-143, miR-339-5p). VPS37B is the target protein of miR-34a identified only in HMC3 cells. Target of miR-34c are ABR (5X dose of SH-SY5Y cell), ANP32B and AXL (5X dose of HMC3 cell) illustrated in Figure 9. PDF is the target protein of miR-339-5p commonly found in both cells exposed to 5X dose while NEDD4L is identified only in SH-SY5Y cell. In SH-SY5Y, miR-143 targets QKI, CBL, KCMF1, BLMH, SLC35A4 while in HMC3 only SLU7 is found.

Effect of nitrate exposure on cellular bioenergetics and mitochondrial function in HMC3 and SH-SY5Y cells

We found that nitrate dramatically affects cellular bioenergetics in both cell types. In both SH-SY5Y and HMC3 cells, we found that exposure to 5X dose of nitrates showed a dramatic reduction in oxygen consumption rate (OCR), a sign of dysfunctional oxidative phosphorylation (OXPHOS), however, X dose could not induce such significant changes in cells. (Figure 10 A). Furthermore, the extracellular acidification rate (ECAR) was also found to be considerably decreased at 5X dose, but changes in ECAR at X dose were not so prominent (Figure 10 B).

In SH-SY5Y and HMC3 cells, the level of changes in basal respiration at X dose was found to be non-significant. However, at 5X dose exposure, SH-SY5Y shows a dramatic reduction in basal respiration rate with respect to control, while no changes in basal respiration was observed in HMC3 cells. Similar pattern of changes in maximal respiration and proton leak in both cell lines was observed where results indicated no signification alteration at X dose whereas a notable decrease in maximal respiration and proton leak was observed in both HMC3 and SH-SY5Y cells exposed to 5X dose of nitrate. A distinct pattern in the alterations in ATP synthesis in response to nitrate exposure was seen in SH-SY5Y cells compared to HMC3 cells. A marked decrease in ATP production was observed in SH-SY5Y cells following exposure to 5X dose of nitrate whereas the level of ATP production showed no significant changes in HMC3 cells at both X and 5X doses of nitrate. There was no significant change in spare respiratory capacity in both cell types exposed to X dose of nitrates. The change in spare respiratory capacity in SH-SY5Y cells at 5X dose was non-significant (Figure 10 C-G). However, Spare respiratory capacity was found to be significantly reduced at 5X dose in HMC3 cells.

Environmental factors have been identified as the major contributors to neural dysfunction-associated disorders. The majority of neurological illnesses are mostly caused by prenatal or postnatal exposure to environmental toxins [24]. Some experimental and clinical studies have also reported the adverse effect of nitrates on health by targeting various physiological and metabolic functions in the body [8, 25–27]. Epidemiological studies have shown that high levels of nitrate can be toxic and its chronic exposure may affect brain development [8, 27]. Studies from our lab and elsewhere have provided evidence that miRNA plays an important role in neuronal development and neurodegeneration [15, 12, 28–31]. Indeed, increasing literature has also suggested the involvement of miRNAs and proteins in regulating nitrate-induced toxicity [32, 19]. However, the role of miRNAs and proteins in regulating nitrate-induced toxicity of the brain is yet to be elucidated. The present study aimed to identify critical miRNAs and associated proteins regulating nitrate-induced toxicity in neuronal and microglial cells employing high-throughput technology. Furthermore, the effect of nitrate on the bioenergetic function of SH-SY5Y and HMC-3 cells have also been explored. The doses of nitrate used in the study are 320 mg/L designated as X dose (the most common and studied dose) and 1600 mg/L designated as 5X dose (five times higher dose found in few sources of water)[8, 33]. Our results demonstrate that nitrate exposure has an adverse effect on brain function by affecting the neurons and microglia at a molecular level, as indicated by alterations in the morphological features of cells. Earlier studies reported that offsprings born to pregnant and lactating mice fed with nitrate-contaminated water from heavily farmed terrain exhibit neurodevelopmental issues as indicated by the loss of neurons and astrocytes, rise in microglial cells, and cell death [8]. Several studies demonstrated the adverse effect of nitrate/nitrite on the pathophysiology of the brain leading to anxiety-like behavior whereas other studies revealed no significant changes in the level of epinephrine, nor-epinephrine, or dopamine as well as no alteration in electrical activity following infusion of sodium nitrite/nitrate [9, 34]. Various types of cancers have been caused by exposure to nitrate/nitrite in drinking water, however, brain tumors showed no significant association with nitrate from well water [35].

The present study identified several miRNAs and associated proteins deregulated following both X dose and 5X dose in both SH-SY5Y neuroblastomas and HMC3 cells. The high dose of nitrate caused a more profound deregulation in miRNA expression in both neuronal and microglial cells, with more miRNAs deregulated in neuronal cells. Top significant deregulated miRNAs identified on exposure to nitrate include miR-213, miR-363, miR-27b, miR-146a, and miR-203 at X dose and miR-34c, miR-34b, miR-194, miR-200c, miR-34a, miR-55, miR-7 and others at 5X dose in SH-SY5Y cells. Significant deregulation was observed in miR-143 and miR-145 at X dose and miR-145, miR-200c, miR-34c, miR-296 at 5X dose in HMC3 cells. Several experimental studies have reported the role of miR-34 family in various biological functions such as cellular senescence, apoptosis, cell cycle, immune homeostasis, stress response, signal transduction, and organ development [36, 37]. ANP32B an important protein involved in the extra cellular vesicle secretion in neuronal cell and neurodegenerative disease [38]. Target analysis of miR-34c have shown the binding site on ANP32B and may be downregulated by the induced miR-34c. LCMS analysis revealed that 47 and 181 proteins from SH-SY5Y cells exposed to X and 5X doses and 163 and 277 proteins in the HMC3 cells, respectively, were deregulated following nitrate exposure. KEGG enrichment analysis showed that proteins deregulated in both cells following exposure to a 5X dose of nitrate are involved in neurodegeneration and associated disorders. Furthermore, deregulated proteins identified in SH-SY5Y cells at X dose of nitrate showed no association with relevant pathways. However, proteins altered at X dose in HMC3 cells are found to be involved in pathways such as endocytosis, ubiquitin mediated proteolysis, pyrimidine metabolism, p53 signalling pathway, and nucleotide metabolism. Functional protein-protein interaction network analysis revealed that deregulated proteins were found to be involved in pathways such as mitochondrial functions, nitrogen compound metabolic processes in both SH-SY5Y and HMC3 cells. Proteins regulating cellular response to stress cellular nitrogen compound metabolic process, spliceosomal complex, disease of metabolism, Ras signaling pathway, ALS, cellular component disassembly and DNA ligation have been significantly deregulated in SH-SY5Y cells. However, proteins regulating processes such as cellular response to cytokine stimulus, nitrogen compound transport, organonitrogen compound metabolic processing, oxidoreductase complex, electron transport system, Wnt signalling, programmed cell death, cellular component disassembly, negative regulation of protein silencing, negative of cell cycle G2-M phase transition and PD were significantly deregulated in HMC3 cells. Overexpression of miR-27b can lower the expression of some mitochondrial genes that are essential for promoting mitochondrial biogenesis and decrease the numbers of mitochondria [39]. In line with the study, we have also found upregulation of miR-27b at both dosages (X & 5X) in SH-SY5Y cells. Interestingly, target analysis showed the maximum downstream proteins interacted with the miR-27b.As per Targetscan analysis, PURA was commonly deregulated in SH-SY5Y at both X and 5X doses. PURA has been reported to be involved in various neuronal disorders and transcriptional control mechanisms [40]. The level of PURA protein was increased at the high dose group in the 5X dose group and down-regulated in the X dose group in SH-SY5Y cells. Consistent upregulation of PURA was also reported in Alzheimer’s disease (AD) [41], thus indicating a high dose of nitrate may have neurotoxic effects on brain. Finger E-Box Binding Homeobox 2 (ZEB2) is a substantial contributor to astrogliosis and, by inducing astrogliosis, can enhance neuroprotection and neurological function recovery in stroke and spinal cord injury [42, 43].The expression of ZEB2 was found downregulated in SH-SY5Y cells at high dose and could be responsible for the neuronal death. TargetScan analysis revealed that ZEB2 have the binding site for the miR-27b and could be involved in the downregulation of ZEB2. MiR-155 is known as the master regulator of inflammation and can be regulated by multiple signaling pathways [44]. Regulatory cytokines such as TGF-β, anti-inflammatory proteins, natural and synthetic glucocorticoids, and IL-10 induce or inhibit the expression of miR-155 [44]. TargetScan analysis revealed that SCG2 is a direct target of miR-155, which was downregulated in nitrate-exposed SH-SY5Y cells. Earlier research has indicated that SCG2 overexpression in plasma is a biomarker for early neurodevelopmental disorders [45]. SLU7, regulates the process of alternate splicing [46]. Alternative splicing is a frequent occurrence in brain tissues and plays a role in all stages of brain development, including cell fate determination, axon guidance, neuronal migration, and synapse formation [47]. Downregulation of SLU7 can affect various processes of brain development. TargetScan analysis of SLU7 have shown the binding site for the miR-143; which was found upregulated in both the cell lines after nitrate exposure. So, higher expression of miR-143 could be responsible for the downregulation of the SLU7 and leads to the neurodegeneration. Gostic et al. in their study found that KIAA03119L, which is a dyslexia susceptible gene, is involved in the development of brain and neuronal migration [48]. A membrane protein called ERLIN 1 is associated with a number of neurological diseases and may be the root cause of the early onset of ALS [49, 50]. The precise role of identified proteins such as KIAA03119L, ERLIN1, and SLU7 which are putative targets of miR-143/145 is not well understood but these are found to be involved in the developmental process of brain and altered expression of these proteins may affect various developmental processes. Bleomycin hydrolase (BLMH) is also an important protein involved in ageing, brain plasticity, learning, and neurodegenerative disorders such as Alzheimer’s disease [51]. The expression of BLMH was downregulated in SH-SY5Y cells after higher dose exposure of nitrate. CBL is an E3-ubiquitin ligase involved in the inhibition of microglial-mediated neuroinflammation via PI3K/AKT/NFKB pathway [52]. In the present study the CBL protein was downregulated that may be lead to the increase inflammation in the neuronal SH-SY5Y cells. Interestingly, both the proteins BLMH & CBL showed the interaction with the miR-143 by TargetScan analysis. So the higher expression of the miR-143 caused the downregulation of BLMH and CBL and involved in the neurodegeneration.

The mitochondria undergo some of the most significant changes during development and differentiation. The mitochondria are essential for cellular functions such as energy metabolism, ageing, formation of reactive oxygen species, calcium homeostasis, and differentiation [53]. In our study, we have measured OCR in live cells using Seahorse extracellular flux analyser and observed no significant alteration in bioenergetics parameters when cells were exposed to X dose of nitrate. However, OCR as well as other bioenergetics parameters were dramatically deregulated in both neuronal and microglial cells at 5X dose. Proliferative cells have been reported to engage in various metabolic activities and thus demand high ATP consumption [54]. During the conversion of ADP to ATP, active mitochondria require oxygen. To satisfy the demands, cells may speed up glycolysis or respiration rate [55]. Therefore, we evaluated the major indices of mitochondrial respiration including OCR, ATP synthesis, extracellular acidification rate (ECAR), and their balance.

Our results showed that SH-SY5Y cells exposed to a high nitrate dose (5X) possess lower OCR as well as ECAR compared to control cells. Furthermore, significant down-regulation in basal respiration, maximal respiration, proton leak and ATP production have been observed in SH-SY5Y cells exposed to a 5X dose of nitrate whereas a slight reduction in OCR and ECAR was observed with X dose of nitrate. In HMC3 cells, changes observed in the X dose group were non-significant. However, both OCR and ECAR were dramatically reduced in HMC3 cells following exposure to a 5X dose of nitrate. Bioenergetic parameters such as maximal respiration, spare respiratory capacity, and proton leak were significantly reduced in HMC3 cells exposed to 5X dose of nitrate. Mitochondrial bioenergetics analysis suggests that nitrate exposure at higher doses targets mitochondria and drastically alters its functions, which may be linked to various pathological mechanisms underlying nitrate-induced toxicity. However, the most prevalent X dose did not cause any prominent impact on mitochondrial bioenergetics.

In summary, this study is the first to identify miRNAs and associated proteins at lower and higher doses of nitrate in both neuronal and microglial cells. We found that a higher nitrate dose (5X) deregulated more miRNAs and proteins in both cell types, with higher changes observed in SH-SY5Y cells. Dysfunction of cellular bioenergetics indicated that nitrate has adverse effects on neuronal and microglial cells, targeting mitochondrial functions with more pronounced effects at a higher dose of nitrate. Thus, X dose, which has been highly prevalent in many locations, did not lead to any adverse repercussions. However, the 5X dose, which has been detected in very few locations or could be reached in the near future, has the potential to cause adverse health outcomes in the brain.

Ethical approval- Not applicable

Consent to participate- Not applicable

Consent for publication- All the authors have read the contents of the manuscript and have provided their conscent to publish the data.

Competing interests- The authors declare no competing interests.

Acknowledgements

Ms Saumya Mishra is grateful to University Grant Commission (UGC), New Delhi, for providing fellowship. Ms Deepshikha Srivastava is acknowledged for extending the technical support in the Central Instrmentation Facility for LC-MS/MS during proteomics studies. Authors are also gretful to Science and Engineering Research Board (DST-SERB), New Delhi, India for providing the financial support thorough an extramural resrach grant (Grant Sanction No. EMR/2017/000889).

Author Contribution

AB Pant has conceptualized the program, design the protocols and monitored throughout the course of experimentations. Saumya Mishra performed the experiments, data analysis and prepared the manuscript. Sanjeev Kumar Yadav assisted in carrying out the openArray experiments. Anuj Pandey assisted in carrying out the cellular bioenergetics experiments, and data analysis. Sana Sarkar has helped in the data analysis and editing of the manuscript. Renu Negi assisted in the in vitro experiments. Sanjay Yadav provided the intellectual inputs and reviewed the manuscript.

Availability of data and materials: Not applicable

Funding

The research was funded by DST-Science and Engineering Research Board (DST-SERB), New Delhi (Grant Sanction no. EMR/2017/000889).

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Crosstalk between miRNA and protein expression profiles in nitrate exposed brain cells

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