A Proteomics Approach to Investigate miR-153-3p and miR-205-5p Targets in Neuroblastoma Cells

MicroRNAs are key regulators associated with numerous diseases. In HEK293 cells, miR-153-3p and miR-205-5p down-regulate alpha-synuclein (SNCA) and Leucine-rich repeat kinase 2 (LRRK2), two key proteins involved in Parkinson’s disease (PD). We have used two-dimensional gel electrophoresis (2D-PAGE) coupled to mass spectrometry (MS) to identify a spectrum of miR-153-3p and miR-205-5p targets in neuronal SH-SY5Y cells. We overexpressed and inhibited both microRNAs in SH-SY5Y cells and through comparative proteomics profiling we quantified ~240 protein spots from each analysis. Combined, thirty-three protein spots were identified showing significant (p-value < 0.05) changes in abundance. Modulation of miR-153-3p resulted in seven up-regulated proteins and eight down-regulated proteins. miR-205 modulation resulted in twelve up-regulated proteins and six down-regulated proteins. Several of the proteins are associated with neuronal processes, including peroxiredoxin-2 and -4, cofilin-1, prefoldin 2, alpha-enolase, human nucleoside diphosphate kinase B (Nm23) and 14-3-3 protein epsilon. Many of the differentially expressed proteins are involved in diverse pathways including metabolism, neurotrophin signaling, actin cytoskeletal regulation, HIF-1 signaling and the proteasome indicating that miR-153-3p and miR-205-5p are involved in the regulation of a wide variety of biological processes in neuroblastoma cells.


Introduction
Parkinson's disease (PD) is the most common neurodegenerative movement disorder characterized by degeneration of dopaminergic neurons in the substantia nigra pars compacta [1]. Most PD cases are sporadic but genetic lesions in alpha-synuclein (SNCA) [2], Parkin [3], PINK1 [4], DJ-1 [5] and Leucine-rich repeat kinase 2 (LRRK2) [6] have been associated with both early-and late-onset PD. Despite extensive studies the molecular pathways leading to the onset and progression of PD are poorly understood.
Individual microRNAs can regulate several mRNAs [16]. Therefore, comparative proteomics profiling in cells with altered microRNA levels has the potential to reveal new microRNA target proteins. The aim of this study was to combine microRNA and proteomics technologies to identify new miR-153-3p and miR-205-5p targets in neuronal SH-SY5Y cells. We selected 2D-PAGE as opposed to LC-MS as although LC-MS analysis is more comprehensive 2D-PAGE offers the possibility of identifying more subtle changes in protein abundance. Several of the protein targets identified are associated with neuronal processes and key regulatory pathways, indicating that miR-153-3p and miR-205-5p are involved in a wide variety of biological processes.

Materials and Methods
Cell culture and transient cell transfection SH-SY5Y cells (CRL-2266; ATCC) were cultured in a base medium mixture (Full medium: 1:1 DMEM/Ham's-F12) (Invitrogen) supplemented with 10% v/v fetal bovine serum (Atlanta biologics) and 2 mM GlutaMAX (Invitrogen) in 5% CO 2 atmosphere at 37°C. Transfections were performed, in triplicate, with scrambled control mimic, miR-153-3p mimic, miR-205-5p mimic, scrambled control hairpin inhibitor, miR-153-3p hairpin inhibitor and miR-205 hairpin inhibitor, all mirVana TM (Life Technologies), at a final concentration of 20 nM. Cells were seeded in 6-well plates at 5x10 5 cells/well. 2μl μRNA (20 μM), diluted with 100 μl of Opti-MEM, and 7 μl Lipofectamine RNAiMax (Invitrogen) diluted with 100 μl of Opti-MEM was incubated for 5 minutes (min) at room temperature (RT). The two solutions were mixed and incubated for 15 min at RT. The transfection mix was diluted to 2 ml with Opti-MEM, added to the wells and incubated at 37°C for 4-6 hours before replacing with full media. Cells were harvested after 24 hours for quantitative PCR (qRT-PCR) analysis and after 48 hours for Western blotting and 2D-PAGE analysis.
western blot analysis following published protocol [17]. Primary antibodies used are shown in S1 Table. The secondary antibodies used were goat anti-rabbit or a goat anti-mouse secondary antibody (Jackson Immunoresearch). The reported western blot results are representative of n = 3.
Scanning and analysis of the images. Gels were scanned using EPSON scan perfection V750 PRO software (Digital ICE technologies) at 600 dpi/16-bit grayscale. ImageMaster 2D platinum 7 (GE Healthcare) was used for spot detection, background subtraction, matching, and to identify statistically significant (ANOVA) differences between protein spots i.e. fold change over control. The experiments were performed in triplicate.
In-gel digestion. The differentially expressed protein spots were excised, cut into small pieces and placed in 0.6 ml Eppendorf tubes. The gel pieces were destained by incubating in 200 μl of 100 mM ammonium bicarbonate: acetonitrile (50:50 v/v) with shaking. When fully destained, the gel fragments were dehydrated with two washes of 100 μl of 100% acetonitrile (ACN) and were then dried in a vacuum centrifuge (Speed-Vac) for 5 min. The proteins were then cleaved enzymatically into peptides. For this, trypsin solution (2 μl of 0.02 μg/μl) was added to the wet the gel pieces and incubation was carried out for 4 hours at RT. Thirty μl of 50 mM ammonium bicarbonate was added to the gel pieces and left overnight at RT to allow for diffusion of the peptides from the gel. The digested proteins were stored at -80°C until further analysis.
Peptide mass fingerprinting. After digestion, POROS 20 R2 resin (Applied Biosystems) was added to the digested gel samples with 5% formic acid and 0.2% trifluoroacetic acid for extraction at 4°C for 4 hours on a shaker. Prior to MALDI-MS analysis, the peptide digests were further desalted using ZipTip C 18 (Millipore). The ZipTips were conditioned with 10 μl of 0.1% TFA twice, 70% ACN/0.1% TFA twice, and 10 μl of 0.1% TFA twice. Samples containing the digest and bead mixture were transferred to the ZipTips and bound to the C18 resin. The loaded tips were then washed with 10 μl of 0.1% TFA. The peptide digests were eluted by placing 2 μl of 10 mg/mL CHCA matrix solution in 0.003% TFA, 13% ethanol, and 84% ACN onto the top of the ZipTips and slowly dispensing onto the MALDI plate. Mass spectrometric analysis was performed using a Thermo LTQ XL linear ion trap mass spectrometer (Thermo Scientific) equipped with a vacuum MALDI source, after the solvent evaporated at RT and the CHCA matrix was crystallized with peptides on the MALDI plate. A data-dependent acquisition was performed using Xcalibur software, in which the top 40 of the most abundant miR153-3p and miR205-5p Protein Targets in SH-SY5Y Cells precursor ions from the survey scan (mass range 700-3500 Da) were chosen and MS/MS acquisition was triggered to fragment them by CID (collision-induced dissociation). The normalized collision energy was 50%, and the isolation width was 3 Da. The raw-files from the LTQ mass spectrometer were analyzed by using Mascot Distiller 2.3.2 (Matrix Science, Boston, MA) for protein identification. Peptide masses were matched against the taxonomy Homo sapiens in the National Centre for Biotechnology Information non-redundant (NCBInr) database. One missed trypsin cleavage per peptide was allowed and an initial mass tolerance of 0.3 Da was used in all searches. Complete carboxyamidation of cysteine sulfhydryls and partial oxidation of methionine were assumed [19].

Cell viability and reactive oxygen species measurements
Cell viability was measured using the neutral red uptake assay 48 hours post-transfection. Cells were washed with PBS, 100μl of neutral red working solution (40μg/ml) added to each well and plates were incubated for 2 hours. Cells were then washed with PBS, neutral red extracted using 150μl of destain solution (50% ethanol, 1% glacial acetic acid, 49% deionized water) per well and the plates were subjected to shaking for 10 min. Absorbance was measured at 540nm using an Epoch microplate spectrophotometer (BioTeck).
Cellular reactive oxygen species (ROS) were measured using 2', 7'-dichlorofluoresceine diacetate (DCF-DA) (Sigma-Aldrich). Cells were plated on solid black opaque plates at 5x10 4 cells per well and after 48 hours incubated with 100μl of DCF-DA (25μM) for 45 min. Fluorescence was measured using a GLoMas1-Multi Detection System fluorescence plate reader (Promega) at 485nm (excitation) and 528nm (emission). The assays were performed in triplicate.

Image analysis, statistical analysis and contextual analysis
Western blot images were analyzed using IQTL software (GE Healthcare). Microsoft excel tools was used for two-tailed Student's t-test. The standard error was used to display variation. The targets of miR-153-3p and miR-205-5p were used as input queries for the Partek Genomics Suite software, version 6.6 (Partek) to perform Gene ontology (GO) analysis and generate interactive maps and pathways.
Altered levels of miR-153-3p and miR-205-5p results in protein changes associated with a spectrum of biological processes We next sought to identify additional targets of miR-153-3p and miR-205-5p in SH-SY5Y cells using 2D-PAGE analysis. However, before performing 2D-PAGE analysis we showed that miR-153-3p and miR-205-5p transfections had no significant effect on SH-SY5Y cell viability ensuring that any observed protein changes were due to changes in miR-153-3p and miR-205-5p levels ( Fig 1D).
We identified thirty-three protein spots that showed significant abundance changes (fold change > 1.4, n = 3, p-value < 0.05) between control transfected and miR-153-3p/miR-205-5ptransfected SH-SY5Y cells. In response to altered levels of miR-153-3p seven protein spots were up-regulated whilst eight protein spots were down-regulated (Fig 2, Table 1). In response to miR-205-5p perturbations twelve protein spots were up-regulated whilst six protein spots were downregulated (Fig 3, Table 1). The protein spots were subjected to MS and the fragment spectra were searched against the NCBInr database (taxonomy Homo sapiens) using Mascot Distiller revealing the identity of the differentially expressed proteins (S1 Fig; Table 1, S2 Table).

Regulation of key neuronal processes by miR-153-3p and miR-205-5p
Overexpression of miR-153-3p resulted in up-regulation of proteasome subunit alpha type-1 isoform 2 (PSMA1) (  21). Efficient proteasome activity is vital in neurons as inappropriate degradation of misfolded proteins, such as amyloids and SNCA, results in aggregate formation, a hallmark of AD and PD [12,20].
miR-153-3p overexpression also resulted in increase abundance of Prefolding subunit 2 (PFDN2) (Table 1, spot 7), which transfers misfolded proteins to chaperonin ensuring proper folding [21]. This indicates that miR-153-3p may up-regulate PFDN2 in response to increased levels of misfolded proteins as a neuroprotective mechanism.
We also found that cathepsin Z (CTSZ) ( Table 1, spot 13) is down-regulated in response to miR-153-3p inhibition. In aging mouse brains cathepsin is upregulated, impairing neuronal survival and neuritogenesis, indicating that miR-153-3p may regulate cathepsin levels to maintain a healthy neuronal population [22].
Inhibition of miR-153-3p also results in the up-regulation of the calcium activated chloride channel family member 4 (CLCA4) (Table 1, spot 9). Calcium activated chloride channels are highly expressed in microglia and activated microglia and a reduction in toxicity is seen in response to CLCA inhibitors [23]. CLCA4 inhibition by miR-153-3p may contribute to neuroprotection.
Our data indicates that both miR-205-5p and miR-153-3p influence direct and peripheral processes associated with neurodegenerative disorders, providing clues towards the possible regulation of key pathways (S4 Table, Fig 4).

miR-153-3p and miR-205-5p alter known cell cycle regulators
Numerous microRNAs are involved in the cell cycle, cancer proliferation and metastasis [37]. In response to miR-153-3p inhibition we identified Nucleoside diphosphate Kinase B (Nm23) ( Table 1, spot 10) and tumor suppressor alpha-enolase (Table 1, spot 11), two cell cycle x, y, z -Single peptides identified from individual protein spots in two ( x ), three ( y ) or seven ( z ) independent MALDI-MS detections. All the protein spots were picked and analyzed from at-least two independent experiments. Fold change ± error are calculated with respect to control mimic and control inhibitor by ImageMaster 2D platinum 7 (GE) software. The significance was calculated using two-tailed t-test.
Combined these results indicate that miR-153-3p and miR-205-5p may play a role in cell proliferation and migration involving various target proteins. Glucose stimulation increases miR-153 expression and miR-153 expression is reduced in PTPRN2 (Protein tyrosine phosphatase receptor type N polypeptide 2) mouse knockout models [46]. We found that the expression of adipokine Serpin A12 (SERPINA12) ( Table 1, spot 5) is down-regulated in response to miR-153-3p overexpression whilst the pyruvate dehydrogenase complex beta subunit (PDHB) ( Table 1, spot 2), a key enzyme linking the glycolytic pathway to the TCA cycle, is up-regulated (Table 1) [47].
As microRNAs are most commonly involved in translational regulation, the up-regulation of eukaryotic translation initiation factor 5A-1 isoform B (EIF5A) (

Concluding remarks
MicroRNA biology is complex and we have shown that miR-153-3p and miR-205-5p influences the abundance of numerous proteins integral to many biological processes in neuroblastoma cells (Fig 4, S3 Table). Interestingly, we observed that some proteins (cofilin-1 and HSFD48) show reciprocal regulatory effects in response to miRNA mimic and antagomir whilst other proteins did not show this reciprocal regulation. This suggests that the proteins identified in this study represent a combination of direct and indirect targets of miR-153-3p and miR-205-5p.
Some of these processes associated with the identified proteins are fundamental in nature whilst others are specifically associated with cell survival, cell proliferation and neuroprotection. Although we acknowledge that the altered abundance of a small number of proteins in a pathway may not necessarily indicate that the entire pathway is affected, our study highlights that to fully understand microRNA-mediated processes a holistic approach is needed, which will pave the way for further insight into neuronal processes associated with normal development and disease.  Table. Cellular processes and pathway analysis of the differentially regulated proteins in response to mimics and antagomirs of miR-153-3p and miR-205-5p. (DOCX)