Quantitative Proteomics Reveals Diverse Roles of miR-148a from

Article pubs.acs.org/jpr

Quantitative Proteomics Reveals Diverse Roles of miR-148a from Gastric Cancer Progression to Neurological Development Chia-Wei Hu,†,# Chien-Wei Tseng,†,# Chih-Wei Chien,‡,# Hsuan-Cheng Huang,§ Wei-Chi Ku,∥ Shyh-Jye Lee,*,¶ Yu-Ju Chen,*,‡ and Hsueh-Fen Juan*,† †

Institute of Molecular and Cellular Biology and Department of Life Science, National Taiwan University, Taipei 106, Taiwan Department of Chemistry, National Tsing Hua University, Hsinchu 300, and Institute of Chemistry, Academia Sinica, Taipei 115, Taiwan § Institute of Biomedical Informatics and Center for Systems and Synthetic Biology, National Yang-Ming University, Taipei 112, Taiwan ∥ School of Medicine, Fu Jen Catholic University, New Taipei City 242, Taiwan ¶ Institute of Zoology, National Taiwan University, Taipei 106, Taiwan ‡

S Supporting Information *

ABSTRACT: MicroRNAs (miRNAs) are noncoding RNAs that control gene expression either by degradation of mRNAs or inhibition of protein translation. miR-148a has been reported to have the impacts on tumor progression. Here, a quantitative proteomics combined with stable isotope labeling was applied to identify the global profile of miR-148a-regulated downstream proteins. The data have been deposited to the ProteomeXchange with identifier PXD000190. A total of 2938 proteins were quantified, and 55 proteins were considered to be regulated by miR-148a. We found that not only proteins associated with cancer progression but also molecules involved in neural development were regulated by miR-148a. This study is the first to identify the function of miR148a in neural development by using a proteomic approach. Analysis of a public clinical database also showed that the patients with neural diseases could display abnormal expression of miR-148a. Moreover, silencing of miR-148a led to the abnormal morphology and decreased expression of neuron-related markers in the developing brain of zebrafish. These results provided important insight into the regulation of neurological development elicited by miR-148a. KEYWORDS: miR-148a, iTRAQ, proteome, neurological development, zebrafish

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INTRODUCTION MicroRNAs (miRNAs) are a class of small, endogenous, and highly conserved noncoding RNAs that are abundantly found in eukaryotic cells.1 They control gene expression either by degradation of target mRNAs or by inhibition of protein translation.2,3 There is emerging evidence to show that miRNAs are fundamental to various biological processes including cell development, proliferation, migration, and differentiation, with their deregulation causing abnormal gene expression in many diseases, such as cancer.4 For example, miR21 decreased expression of the tumor suppressor Pdcd4 and promoted invasion, intravasation, and metastasis in colorectal cancer.5 miR-155 targeted the tumor suppressor WEE1 homologue (S. pombe) (WEE1) and induced gene alterations required for cancer development and progression.6 Additionally, the microRNA let-7 decreased proliferation and migration of glioblastoma cells and reduced tumor size in a xenograft model.7 These reports indicate that miRNAs can function as either oncogenes or tumor suppressors in organisms. Using integrative network analysis, we previously reported that miR-148a was associated with metastasis-related biological processes in gastric cancer, such as integrin-mediated signaling, cell-matrix adhesion, and wound healing.8 Our findings © XXXX American Chemical Society

suggested that miR-148a was a potential tumor suppressor that inhibited gastric cancer metastasis. Furthermore, clinical data indicated that miR-148a could serve as a prognostic marker for gastric cancer. Other studies have also revealed the tumor suppressor function of miR-148a. Zhou et al. demonstrated that miR-148a was down-regulated in ovarian cancer cells and its overexpression reduced cell proliferation.9 Lujambio et al. found that miR-148a was hypermethylated in cancer cells, whereas tumor metastasis in xenograft models was reduced in response to miR-148a overexpression.10 Zheng et al. showed that miR-148a could inhibit cell invasion and metastasis in gastric cancer.11 Moreover, miR-148a induced apoptosis of colorectal cancer cells by down-regulating Bcl-2 expression.12 All the findings suggested a tumor suppressor role for miR-148a in the regulation of tumor progression. In addition to tumor progression, miR-148a was recently found to regulate myogenic differentiation by targeting ROCK1 expression.13 Besides, miR148a targeted HLA class I histocompatibility antigen, alpha chain G (HLA-G) expression, and consequently suppressed the leukocyte immunoglobulin-like receptor subfamily B member 1 Received: April 4, 2013

A

dx.doi.org/10.1021/pr400302w | J. Proteome Res. XXXX, XXX, XXX−XXX

Journal of Proteome Research

Article

Figure 1. Schematic representation of the experimental design used for quantitative proteomics analysis. (A) The quantitative protein profiles of miR-148a mimic-treated (pre-miR-148a) or negative control-treated (miR-CTL) AGS cells were generated. The relative protein expression ratio (pre-miR-148a/miR-CTL) was quantitated by iTRAQ, and the proteins of interest were further analyzed for functional annotation. The biological effects of miR-148a were verified using an in vivo zebrafish model. (B) For quantitative proteomic analysis, proteins were extracted, digested, and labeled with iTRAQ reagents. After SCX fractionation and LC−MS/MS analysis, peptides were identified and quantified. The detailed experimental procedure is described in the Materials and Methods.

Hospital. These cells were tested and authenticated in our laboratory on a monthly basis by morphology analysis using a microscope and mycoplasma detection using Hoechst 33258. AGS cells were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA). These cells were cultured at 37 °C in an atmosphere of 5% CO2.

(LILRB1)-mediated inhibition of natural killer (NK) cytotoxicity.14 These reports indicated that miR-148a might be a key regulator in cell physiology. However, its effects in other biological processes aside from cancer are rarely understood. Proteomics approaches have been widely used for the identification and quantification of proteins involved in biological functions regulated by miRNAs.15,16 For example, Khan et al. found that miR-128 could suppress prostate cancer invasion using isotope labeling quantitation.15 Taguchi et al. applied quantitative proteomics to show that miR-17−92 directly targeted hypoxia-inducible factor (HIF)-1α and affected cancer cell proliferation.16 A quantitation strategy based on the isobaric tags for relative and absolute quantitation (iTRAQ) reagents has been developed for protein quantitation, which is a relatively high-throughput approach due to sample multiplexing.17,18 Because of the isobaric nature of the iTRAQ reagents, the intensity of both the precursor ion and the resultant mass spectrometry (MS/MS) fragments is greatly enhanced from the sum of four sample sets.18 In addition, because of the 4-fold multiplexing, the N-terminus and the lysine side chains of tryptic peptides can be targeted, thus enabling the labeling of every peptide.18 Protein can be identified and quantified by the presence of more peptides, thereby enhancing the confidence of protein identification and quantitation. In the present study, we aimed to reveal the novel functions of miR-148a by using iTRAQ-based quantitative proteomics (Figure 1). The differentially expressed proteins of interest from the proteomic data were further analyzed and categorized by functional annotation. An in vivo animal model was used to elucidate the roles of miR-148a in cell physiology.

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Isobaric Tag for Relative and Absolute Quantitation (iTRAQ)

AGS cells were transfected with a miR-148a mimics and its negative control. The transfection procedure of miR-148a was based on a previous study.8 At 48 h after transfection, cells were collected and lysed, and the protein concentration was determined by BCA assay (Pierce, Rockford, IL) for iTRAQ analysis. The reagents for iTRAQ were purchased from Applied Biosystems (Foster City, CA). Tris (2-carboxyethyl) phosphine hydrochloride (TCEP), triethylammonium bicarbonate (TEABC), methyl methanethiosulfonate (MMTS), trifluoroacetic acid (TFA), HPLC-grade acetonitrile (ACN), and 4-(2hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and rabbit antiactin polyclonal antibody were from Sigma-Aldrich (St. Louis, MO). Monomeric acrylamide/bisacrylamide solution (40%, 29:1), ammonium persulfate, and protein assay dye were from Bio-Rad (Hercules, CA). Iodoacetamide, N,N,N′,N′tetramethylethylethylenediamine (TEMED), and sodium dodecyl sulfate (SDS) were from Amersham Biosciences/GE Healthcare (Pittsburgh, PA). Trypsin (modified, sequencing grade) was from Promega (Madison, WI). Water was obtained from a Milli-Q Ultrapure water purification system (Millipore, Billerica, MA). All experiments were repeated three times. Gel-Assisted Digestion of Cell Lysates

MATERIALS AND METHODS

Cells were lysed in a lysis buffer (0.25 M Tris-HCl, pH 6.8, 1% SDS). The protein samples obtained from the cell lysates were subjected to gel-assisted digestion.18 Briefly, the samples were reduced with 5 mM TCEP, alkylated with 2 mM MMTS, and

Cell Culture and Authentication of Cell Lines

The human gastric cancer cell line, AGS, was obtained in 2008 from the cell line databank at the National Taiwan University B



Article

intense ions present in the full-scan mass spectrum. In this study, we performed two biological iTRAQ analyses, including two technical replicates in each experiment.

incorporated directly into a polyacrylamide gel contained in a microcentrifuge tube. For 100 μL of the protein sample, 37 μL of acrylamide/bisacrylamide solution (40%, v/v, 29:1), 3.7 μL of 10% (w/v) APS, and 1 μL of 100% TEMED were applied for gel formation. The gel was then cut into small pieces, which were washed for several times with 25 mM TEABC containing 50% (v/v) ACN, and further dehydrated with 100% ACN before it was completely dried using a SpeedVac (Thermo Savant SC210A, Holbrook, NY). Proteolytic digestion with trypsin was performed (10:1 protein-to-trypsin ratio, w/w) in 25 mM TEABC at 37 °C for overnight. The tryptic peptides were extracted from the gels sequentially with 25 mM TEABC, 0.1% (v/v) TFA in water, 0.1% (v/v) TFA in ACN, and finally 100% ACN. The peptide solutions were combined and vacuum-dried. The resultant peptides were resuspended in 0.5 M TEABC for downstream iTRAQ labeling.

Data Processing and Analyses

The peak lists in the resultant MS/MS spectra were generated by Mascot Distiller v2.1.1.0 (Matrix Science, London, United Kingdom) and searched using Mascot v2.2 (Matrix Science) against the International Protein Index (IPI) human database (v. 3.64, 84 032 sequences) from the European Bioinformatics Institute. Both the precursor peptide and fragment ion tolerances were set to ±0.1 Da. The search parameter settings were as follows: allowances for two missed cleavages from trypsin digestion and variable modifications of deamidation (NQ), oxidation (M), iTRAQ (N terminal), iTRAQ (K), and MMTS (C). For confident protein identification, we used only unique peptides with scores exceeding an expectation value (P < 0.05). To evaluate the FDR of protein identification, we also performed additional searches against a randomized decoy database created by Mascot with identical search parameters and validation criteria. The number of matches in the target database was true positive matches (TP) + false positive matches (FP) and the number of matches in the decoy database was FP. The quantity that was reported as the false discovery rate (FDR) = FP/(FP + TP). The MS/MS spectra and assignment for single-peptide based protein identification were included in Supporting Information Figure 1 and 2. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier PXD000190 and DOI 10.6019/PXD000190.19,20 For protein quantitation, the iTRAQ ratios were determined for all peptides and proteins using the Multi-Q software v1.6.5.4 as previously reported.18 The raw data files from the Q-TOF Premier were converted into files in mzXML format using Masswolf (MassLynx converter, Waters Corp.). The search results in Mascot were exported into files with commaseparated value (csv) format and eXtensive Markup Language (XML) data format. The converted files were then subjected to Multi-Q software for automated quantitation of the iTRAQlabeled peptides with confident MS/MS identification (Mascot score based on P < 0.05) by the intensities of signature ions (m/z 114 and 115). Only nondegenerate (unique) peptides were used for protein quantitation. We applied several selection filters during Multi-Q analyses. For the detector dynamic range filter, signature peaks with ion counts lower than 30 were filtered out. We also manually inspected every spectrum and kept the mass spectra with sufficiently good quality with at least 3 consecutive y- or b- ions. For peptide ratio normalization, Multi-Q performed a normalization procedure on the ratios with the Gaussian distribution. The reciprocal of the original mode in the peptide ratio distribution was set as the normalization factor to correct the systematic bias. To calculate average protein ratios, the ratios of the quantifiable and uniquely normalized iTRAQ peptides were weighted according to their peak intensities to minimize the standard deviation, as described below. For identification of differentially expressed proteins, the expression ratio of miR-148a mimic-treated and negative control-treated for each identified protein in AGS cells was calculated. The expression ratio (r) of the identified protein (X), was defined as the following:

iTRAQ Labeling and Strong Cation Exchange (SCX) Chromatography

To label the peptides with the iTRAQ reagent, one unit of iTRAQ reagent, which was sufficient to label 100 μg of peptides, was reconstituted in 70 μL of ethanol by vigorous vortexing for 1 min. The peptide solutions obtained from the cells containing the miR-148a precursor or precursor negative control were labeled with iTRAQ114 and iTRAQ115, respectively. The mixtures were incubated at room temperature for 1 h. The labeled peptides were acidified with buffer A (5 mM KH2PO4 and 25% can (v/v), pH 3.0) to a total volume of 1 mL and further fractionated by SCX chromatography. For peptide SCX fractionation, iTRAQ-labeled peptides were loaded onto a 2.1 × 200-mm polysulfethyl column containing 5-μm particles with 200-μm pore size (PolyLC, Columbia, MD). The peptides were eluted at a flow rate of 200 μL/min with a gradient of 0−25% buffer B (5 mM KH2PO4, 350 mM KCl, and 25% ACN (v/v), pH 3.0) for 30 min followed by a gradient of 25−100% buffer B for 20 min. The elution was monitored by measuring the absorbance at 214 nm, and fractions were collected every 1 min. Each fraction was vacuumdried and resuspended in 0.1% TFA for further desalting and concentration with ZipTips (Millipore, Bedford, CA). LC−MS/MS Analyses

Each iTRAQ-labeled SCX fraction was reconstituted in 6 μL of eluent buffer A (0.1% formic acid (FA) in H2O) and analyzed by online liquid chromatography tandem mass spectrometry (LC−MS/MS) using a Waters Quadrupole Time-of-Flight (QTOF) Premier (Waters Corp., Milford, MA). The peptide samples were injected into a 2-cm × 180-μm capillary trap column and separated on a 25-cm × 75-μm Waters ACQUITY 1.7-μm BEH C18 column using a nanoACQUITY Ultra Performance LC system (Waters Corp.). The column was maintained at 35 °C, and bound peptides were eluted for 120 min using a linear gradient of 0−80% eluent buffer B (0.1% FA in ACN). The mass spectrometer was operated in an electrospray-ionization (ESI)-positive V mode with a resolving power of 10 000. A NanoLockSpray source was used for accurate mass measurement, and the lock mass channel was sampled every 30 s. The mass spectrometer was calibrated with a synthetic human [Glu1]-fibrinopeptide B solution (1 pmol/ μL; Sigma Aldrich) delivered through the NanoLockSpray source. Data acquisition was performed using the data-directed analysis (DDA). The DDA method included one full MS scan (m/z 400−1600, 0.6 s) and three MS/MS scans (m/z 100− 1990, 1.2 s for each scan) sequentially on the three most C



rX =

Article

cells, the 2.3 nL of MOs were injected using an oil nanoliter injector (Nanoliter 2000, World Precision Instruments). Then, embryos were isolated from the injection trough and maintained in 0.3× Danieau’s buffer in an incubator set at 28.5 °C until further observation.

∑ Ti /∑ Ci i∈I

i∈I

where I represents the unique peptides identified as protein X, and Ti and Ci represent each normalized peptide signal in miR148a mimic-treated and negative control-treated AGS cells, respectively. We normalized the protein ratios by zeroing the mean of log2-transformed protein ratios. The normalized mean in the first and second biological iTRAQ results were 0.003 and −0.023, respectively. The significance thresholds for up- and down-regulated proteins were determined by mean ± 1.96 standard deviations (SD) of normalized log2-transformed protein ratios. The SD values in the first and second biological iTRAQ results were 0.251 and 0.215, respectively. Thus, the thresholds for up- and down-regulated proteins in the first iTRAQ results were 0.492 and −0.492, respectively; the thresholds for up- and down-regulated proteins in the second iTRAQ results were 0.422 and −0.422, respectively.

Whole-Mount in Situ Hybridization (WISH) and Confocal Microscopy

Embryos at 24 hpf were dechorionated manually and fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) at 4 °C overnight. The embryos were then hybridized with antisense digoxigenin (DIG)-labeled RNA riboprobes (Roche Applied Science, Penzberg, Germany) and maintained at 4 °C overnight. Hybridization with an alkaline phosphatase-coupled anti-DIG antibody (Roche Applied Science) was observed according to the method of Thisse et al.25 Briefly, the processed embryos were transferred to 100% methanol for 20 min and finally mounted in 100% glycerol and photographed using the Nikon CoolPIX 995 digital camera (Nikon, Tokyo, Japan). The GFP-labeled Huc embryos were observed by using the Zeiss LSM 780 confocal microscope (Zeiss, Jena, Germany).

Functional Enrichment Analysis

The significantly up- and down-regulated proteins in miR-148a overexpressed cell were used to analyze enriched functions using Ingenuity Pathway Analysis (IPA) software (Ingenuity Systems, Redwood City, CA) and gene ontology (GO) databases. IPA aids in the integration of complex “omics” data and provides insight into the regulatory mechanism and biological functions based on published studies. Thus, it was a useful tool in the analysis of miR-148a-regulated functions based on iTRAQ data.

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RESULTS

Quantitative Proteomic Analysis of miR-148a-Regulated Downstream Proteins

In this study, we used a quantitative proteomics approach to reveal novel functions of miR-148a. To identify downstream proteins regulated by miR-148a, we first used an iTRAQ approach to analyze the global changes in the expression profiles of miR-148a mimic-treated (Pre-miR-148a) and miR148a negative control-treated (miR-CTL) AGS cells. The workflow diagram is shown in Figure 1. The transfection efficiency of miR-148a mimics and negative control in AGS cells was confirmed. The nondegenerate peptides of each protein were selected and normalized, and these selected peptides were further used to calculate the expression ratio of each protein under the two conditions. To maximize the identification number and quantitation accuracy, we performed iTRAQ experiments with two biological replicates and two LC−MS/MS measurements in each biological replicate. Detailed information of the first and second iTRAQ results was shown in Tables S1 and S2 (Supporting Information). The overlap proteins identified in both biological replicates were listed in Table S3 (Supporting Information). The number of proteins with single identification was shown in Table S4 (Supporting Information). All MS/MS spectra for singlepeptide based quantification were summarized in Figures S1 and S2 (Supporting Information), respectively. From the two biological replicates of iTRAQ experiments, 2938 proteins were quantified with an expected value P < 0.05 in Mascot search. Under this criterion, the false discovery rate (FDR) in each biological replicate was 2.27% and 1.2%, respectively. When calculating the number of up- and downregulated proteins, we applied the cutoff threshold of mean ± 1.96 × standard deviation (SD) for each biological replicate. Under this threshold, we identified 130 proteins with 57 upregulated and 73 down-regulated proteins in the first iTRAQ result (cutoff threshold = ±0.492) as well as 122 proteins with 51 up-regulated and 71 down-regulated proteins in the second iTRAQ result (cutoff threshold= ±0.422) in response to miR148a overexpression. Among them, only the miR-148aregulated proteins showing the same tendency for up/down regulation in both iTRAQ results were used for further

miRNA Microarray Analysis

The miRNA expression profiles of patients with Alzheimer’s disease and Parkinson’s disease were retrieved from Gene Expression Omnibus (GEO) with the accession numbers GSE 16759 and GSE 16658, respectively.21,22 Levels of miR-148a expression in normal and diseased tissues were statistically analyzed using Student’s t-tests. P < 0.05 was considered to be statistically significant. Zebrafish Maintenance

All animal handling procedures were approved by the use of laboratory animal committee at National Taiwan University, Taipei, Taiwan (IACUC Approval ID: 97 Animal Use Document No. 55). The inbred wild-type zebrafish line was maintained at 28.5 °C under a 14-h light/10-h dark cycle. Embryos were collected by natural spawning and raised in 0.3× Danieau’s buffer (prepared by dilution of 1× Danieau’s buffer which included 58 mM NaCl, 0.7 mM KCl, 0.4 mM MgSO4, 0.6 mM Ca(NO3)2, and 5.0 mM HEPES buffer, pH 7.6, with double-distilled water) until observation or fixation.23 The embryo stage was defined as previously described, and different stages were indicated as hours postfertilization (hpf).24 Microinjection of Morpholino Oligonucleotides (MOs)

miR-148 (5′-ACAAAGTTCTGTAATGCACTGACTG-3′) antisense MOs were custom-made by Gene Tools, LLC (Philomath, OR). A standard MO sequence (5′-CCTCTTACCTCAGTTACAATTTATA-3′) not homologous to any known zebrafish sequences was used as a control. MOs were diluted to 10 ng using 1× Danieau’s buffer with 0.5% phenol red and kept at room temperature. Embryos at one-cell stage were immobilized in an injection trough on a 1% agar plate, and the desired MOs were prepared for loading into a pulled capillary. After a loaded capillary was inserted into the yolk D



Article

Figure 2. Validation of the regulation proteins of miR-148a by Western blotting. (A) Some regulated proteins shown in Table 1 were measured by Western blotting. Actin was used as an internal loading control. The expression levels of the regulated proteins were quantified. The band intensities were normalized to actin. (B) The comparison of the expression levels of the regulated proteins by mass spectrometry (MS) and by Western blotting (WB).

dysfunction, such as Alzheimer’s and Parkinson’s diseases. The information for the expression levels of miR-148a in patients was obtained from two miRNA expression profiles r e t r i e v e d f r o m G E O ( a cc e s s i o n G S E 1 6 7 5 9 a n d GSE16658).21,22 These data contain the miRNA expression profiles of 19 Parkinson’s patients and 13 controls (GSE16658), as well as 4 Alzheimer’s patients and 4 agematched controls (GSE16759). We found that miR-148a expression levels were lower in patients with Parkinson’s (P = 0.03) and Alzheimer’s (P = 0.09) diseases than those in controls (Table 3 and Figure 3B). The results support our findings that miR-148a might be involved in neurological development and functions.

investigation. The expression of some of these regulated proteins was validated by immunoblotting (Figure 2), and the proteins with single-peptide identification without validation were not shown. Therefore, 41 proteins were considered to be regulated by miR-148a (Table 1 and Figure 2). Functional Enrichment of miR-148a-Regulated Proteins

To further elucidate the biological processes affected by miR148a expression, the associated biological functions of these differentially expressed proteins listed in Table 1 were studied by IPA and GO analyses (Table 2 and Figure 3A). Many miR148a-regulated proteins, such as LLGL2 (Isoform C of Lethal(2) giant larvae protein homologue 2), EGFR (Isoform 1 of Epidermal growth factor receptor), LIG1 (DNA ligase 1), CYC1 (Cytochrome c1, heme protein, mitochondrial) were involved in cancer-related functions, including cell cycle, cell death, cell adhesion and migration. The results were consistent with our previous study and other reports showing the involvement of miR-148a in tumor progression.8,11,12 Moreover, miR-148a expression was also found to affect other important molecular mechanisms, including protein synthesis and degradation, DNA replication and repair, translation, and post-translational protein modification, suggesting a wide effect of miR-148a in cellular functions. Interestingly, a group of miR148a-regulated proteins, such as ENAH (Isoform 2 of Protein enabled homologue), STX3 (Isoform B of Syntaxin-3), and TMED2 (Transmembrane emp24 domain-containing protein 2) were found to associate with nervous system development and function, implying a possible role of miR-148a in neurological development.

miR-148a Affects Morphological Development in Zebrafish

Previous studies have reported that many miRNAs were identified with conserved seed regions in various vertebrate species, from fish to humans.1 Unsurprisingly, the human and zebrafish miR-148 also shared the same seed regions (nucleotides 2 to 8) (Figure 4). This characteristic and its transparent embryo enable zebrafish to be an ideal material for analyzing the effects of miR-148a in developmental processes. Here, an in vivo zebrafish model was applied to study the regulatory role of miR-148a in neurological development and functions. To determine the effects of miR-148 in the embryonic development of zebrafish, MOs specific for miR-148 were injected into wild-type 1-cell-stage embryos to abolish the expression of mature miR-148. We verified the expression of miR-148a in wild-type, standard MO-treated, and miR-148 MO-treated zebrafish at 24 hpf (Figure S3, Supporting Information). We found the expression of endogenous miR148 in miR-148 MO-treated zebrafish was decreased; however, its expression in standard MO-treated and wild type groups was not notably changed. Using the miR-148a-silencing embryo model, we further investigated whether the morphology of

miR-148a Expression in Patients with Neurological Diseases

To further evaluate the connection between miR-148a and neurological development and functions, we first examined the miR-148a expression under the condition of neuronal E


F

IPI00022536 IPI00032038

Posttranslational protein modification

Fatty acid metabolism

IPI00012048

IPI00550746 IPI00219604

IPI00477355 IPI00018274

IPI00013744

IPI00026182 IPI00219111

Cell signaling; posttranslational protein modification Translation

Cell junction, adhesion and migration; cell morphology

IPI00030919

Cell signaling

IPI00029264

IPI00012833

Cell signaling

IPI00299116 IPI00167572 IPI00102685

IPI00023673 IPI00021794 IPI00023101

Cell junction, adhesion and migration Cell signaling Cell signaling

Unknown Unknown Unknown Up-Regulation Nervous system development and function Cell cycle and cell proliferation; cell junction, adhesion and migration; cell death Cell cycle and cell proliferation; organelle localization Cell cycle and cell proliferation; cell morphology; cellular function and maintenance Cell cycle and cell proliferation; cell junction, adhesion and migration; DNA replication and repair; cell death Cell junction, adhesion and migration

IPI00218918 IPI00172656 IPI00011996

Cell cycle and cell proliferation; cell junction, adhesion and migration Response to unfolded protein Cell death; protein synthesis and degradation

IPI00063827

IPI00028160 IPI00220099 IPI00465050

Nervous system development and function Nervous system development and function Cell cycle and cell proliferation

Hydrolase activity

IPI00374054 IPI00016608

accession no.

Down-Regulation Nervous system development and function Nervous system development and function

biological function

Table 1. Differentially Regulated Proteins in miR-148a-Treated Cells

KIF1-binding protein Isoform 1 of Epidermal growth factor receptor Nuclear migration protein nudC Dual specificity mitogen-activated protein kinase kinase 1 Isoform 1 of Nucleoside diphosphate kinase A Cytochrome c1, heme protein, mitochondrial Integrin alpha-2

Isoform 2 of protein enabled homologue Transmembrane emp24 domain-containing protein 2 Isoform 1 of Porphobilinogen deaminase Isoform B of Syntaxin-3 Isoform C of Lethal(2) giant larvae protein homologue 2 Annexin A1 FAS-associated factor 2 Isoform 1 of Ubiquitin-conjugating enzyme E2 Z Galectin-3-binding protein Lysosomal protective protein Cell differentiation protein RCD1 homologue Serine/threonine-protein phosphatase 4 catalytic subunit Mitogen-activated protein kinase scaffold protein 1 F-actin-capping protein subunit alpha-2 Translocating chain-associated membrane protein 1 Isoform 1 of Ribosomal protein S6 kinase alpha-4 Isoform 1 of Carnitine Opalmitoyltransferase 1, liver isoform Isoform 1 of Abhydrolase domaincontaining protein 14B Podocalyxin-like protein 1 precursor Protein FAM98B Myeloid-associated differentiation marker

protein name

ITGA2

CYC1

NME1

NUDC MAP2K1

KIAA1279 EGFR

PODXL FAM98B MYADM

ABHD14B

CPT1A

RPS6KA4

CAPZA2 TRAM1

MAPKSP1

PPP4C

LGALS3BP CTSA RQCD1

ANXA1 FAF2 UBE2Z

HMBS STX3 LLGL2

ENAH TMED2

gene symbol

129214

35367

17138

38219 43411

71768 134190

55536 37167 35250

22332

88311

85552

32929 43044

43411

35057

65289 54431 33610

35885 52591 38186

39306 28842 113377

63886 22746

mass (Da)

509

238

1123

998 192

164 195

208 98 98

159

141

80

483 159

129

142

280 183 100

2065 373 64

319 84 199

209 373

protein scorea

8

2

7

8 3

3 4

3 3 2

2

4

2

4 3

2

2

4 2 3

11 3 2

4 1 2

4 2

no. of peptidesb

1.02 ± 0.05 1.47 ± N/A 1.14 ± 0.1

2.27 ± 0.25 2.04 ± 0.68 1.46 ± 0.33

0.66 ± N/A

0.93 ± 0.31

1.36 ± 0.24 1.39 ± 0.47

0.72 ± N/A

0.91 ± 0.02

1.05 ± 0.23 1.03 ± 0.2

0.94 ± 0.09 0.67 ± N/A

0.58 ± N/A 0.72 ± 0.34

1.1 ± 0.15 1.25 ± 0.06

0.72 ± 0.09

0.99 ± 0.38

1.54 ± N/A 1.53 ± 0.31

0.72 ± N/A

0.97 ± N/A

0.85 ± N/A 0.66 ± 0.04 0.73 ± N/A

0.87 ± 0.13 0.51 ± 0.24 0.7 ± N/A

0.71 ± 0.21 0.75 ± 0.31 0.86 ± 0.09

0.69 ± 0.12 0.84 ± 0.59 0.84 ± 0.09

0.71 ± 0.14 0.84 ± 0.12 0.99 ± 0.3

0.87 ± 0.27 0.67 ± 0.22 0.27 ± N/A

0.92 ± N/A

0.71 ± N/A 0.63 ± N/A 0.63 ± 0.05

0.93 ± 0.13 0.69 ± 0.17 0.59 ± N/A

0.67 ± 0.17

0.81 ± 0.09 0.67 ± 0.07

replicate 2 (FDR: 1.2%)

0.67 ± N/A 0.84 ± 0.27


normalized ratio ± STD (pre-miR148a/miR-CTL)

Journal of Proteome Research Article


IPI00017895

Gluconeogenesis

G

a

DNA ligase 1 Stromal interaction molecule 1 Isoform 1 of Prolyl 4-hydroxylase subunit alpha-1 Isoform A of Ras GTPase-activating protein-binding protein 2 Lamina-associated polypeptide 2, isoform alpha Isoform 1 of E3 ubiquitin-protein ligase UHRF2 Epsilon subunit of coatomer protein complex isoform b Phosphomannomutase 2 Serine/threonine-protein kinase VRK1 Acidic leucine-rich nuclear phosphoprotein 32 family member E Isoform 1 of Glycerol-3-phosphate dehydrogenase, mitochondrial Coproporphyrinogen-III oxidase, mitochondrial WD repeat-containing protein 75

protein name

WDR75

CPOX

GPD2

PMM2 VRK1 ANP32E

COPE

UHRF2

TMPO

G3BP2

LIG1 STIM1 P4HA1

gene symbol

94438

50120

80802

28064 45447 30674

28779

89928

75446

54088

101673 77375 61011

mass (Da)

The best Mascot score of protein. bThe maximum number of nondegenerate (unique) peptides used for quantitation in two biological replicates.

IPI00217240

IPI00006092 IPI00019640 IPI00165393

Posttranslational protein modification Posttranslational protein modification Phosphatase inhibitor activity

Unknown

IPI00399318

Protein transport

IPI00093057

IPI00044681

Protein synthesis and degradation

Heme biosynthetic process

IPI00216230

Cell morphology

IPI00219841 IPI00299063 IPI00009923

accession no.

IPI00009057

biological function

Up-Regulation Cell death; DNA replication and repair Cellular function and maintenance Extracellular matrix organization; cellular amino acid derivative metabolic process Cell signaling

Table 1. continued

438

345

356

136 235 943

695

91

1110

213

170 125 111

protein scorea

7

6

4

3 3 3

5

2

8

2

3 3 3

no. of peptidesb

1.11 ± 0.17 1.42 ± 0.2 1.09 ± 0.11 1.34 ± 0.37 1.61 ± 0.31

1.7 ± 0.13 1.17 ± 0.54 1.42 ± 0.14 1.21 ± 0.24 1.11 ± 0.45

1.01 ± 0.24

1.18 ± 0.02

1.64 ± N/A

1.44 ± N/A

1.14 ± 0.13

1.51 ± 0.43

1.26 ± 0.17

1.07 ± 0.09

1.69 ± 0.31

1.47 ± 0.18

1.17 ± 0.13 1.47 ± N/A 1.79 ± N/A


1.43 ± 0.17 1.09 ± N/A 1.03 ± 0.18


normalized ratio ± STD (pre-miR148a/miR-CTL)

Journal of Proteome Research Article



Article

Table 2. Functional Categories of the Regulated Proteins in miR-148a-Treated Cells biological functionsa Nervous system development and function Cell cycle and cell proliferation Cell death Cell junction, adhesion and migration Cell morphology Cellular function and maintenance Extracellular matrix organization Cell signaling DNA replication and repair Translation Protein synthesis and degradation Response to unfolded protein Protein transport Posttranslational protein modification Phosphatase inhibitor activity Organelle localization Fatty acid metabolism Gluconeogenesis Heme biosynthetic process Hydrolase activity Cellular amino acid derivative metabolic process Unknown

Table 3. Expression of miR-148a in Patients with Neurological Diseases

gene names

samplea

ENAH, STX3, TMED2, HMBS, KIAA1279 LLGL2, ANXA1, EGFR, NME1, MAP2K1, NUDC EGFR, LIG1, NME1, UBE2Z EGFR, CYC1, NME1, LGALS3BP, ANXA1, ITGA2 MAP2K1, ITGA2, TMPO SNX1, MAP2K1, STIM1 P4HA1 CTSA, CAPZA2, RQCD1, PPP4C, MAPKSP1, G3BP2 LIG1, NME1, SMARCA5 TRAM1 UHRF2, UBE2Z FAF2 COPE, CAPZA2, RPS6KA4, PMM2, VRK1

normalized signal intensities

Parkison’s Disease disease 0.786 control 1.219 Alzheimer’s Disease disease 5.8 control 32.47

no. of samples

fold change (disease/control)

p-valueb

19 13

0.645

0.03

4 4

0.180

0.09

a The miRNA expression profiles were retrieved from GEO (accession number GSE16658 and GSE16759). bp-value was calculated by t-test.

Figure 4. Comparison of mature sequences of miR-148a in human and miR-148 in zebrafish. The mature sequences of miR-148a in human (has-mir-148a) and miR-148 in zebrafish (dre-mir-148) were shown according to Targetscan 6.2 and miRbase databases. The gray color indicates the seed region of has-miR-148a/dre-miR-148.

ANP32E NUDC CPT1A GPD2 CPOX ABHD14B P4HA1

brain, which is an important structure for neuronal system, was influenced after miR-148 MO treatment. Interestingly, during brain development, the boundary between the midbrain and hindbrain as well as the brain size of embryo were abnormally developed in miR-148 knock-downs (Figure 5C and F), and these changes were notably prominent compared to wild-type embryos at 24 and 48 hpf after microinjection (Figure 5A and D). Moreover, the phenotypes of embryos treated with standard MOs (Figure 5B and E) were indistinguishable from those of the wild-type embryos. All these results indicate that

PODXL, FAM98B, MYADM

a

The biological functions were assigned by IPA and Gene Ontology database.

Figure 3. Functional categories of the proteins regulated by miR-148a and the fold changes of miR-148a in neurological diseases. (A) The functional distribution of miR-148a-regulated proteins. The percentage indicates the proportion of proteins relative to the total number of combinations in the diagram. (B) Comparison of expression levels of miR-148a in patients with Parkinson’s and Alzheimer’s diseases and their controls, respectively. Fold change of miR-148a expression shown was obtained by dividing the average intensity of control group by the average intensity of disease group. H



Article

Figure 5. Reduction in miR-148 levels affected the morphological development of zebrafish. The effects of altered miR-148 expression on the morphological development of zebrafish were observed at 24 hpf (A−C) and 48 hpf (D−F). The brain morphology (black triangle) and the midbrain−hindbrain boundary (arrow) were missing in miR-148-silenced zebrafish (C and F) compared with wild-type (A and D) and standard MO control (B and E) groups.

Figure 6. Reduction in miR-148 levels affected the expression of Zic1 in brain and spinal cord of zebrafish. The expression of Zic1 at 24 hpf in wildtype (A), standard MO control (B), and miR-148 MO-treated zebrafish (C) was detected by in situ hybridization. Silencing of miR-148 affected Zic1 expression in telencephalon (black triangle), diencephalon (arrow) and dorsal spinal cord (asterisk) compared with control groups.

suggesting a regulatory function of miR-148a in neuronal development.

miR-148a might be a regulatory molecule during neurological development.

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miR-148a Affects Neuron-Related Marker Expression in Zebrafish

DISCUSSION miRNAs has been shown to play an indispensable role of biological regulation in various fields.2−4 High-throughput proteomics is a useful and convenient way to investigate the complexity of miRNA regulation.15,16,30 In this study, a quantitative proteomic analysis was successful to reveal the global change of protein expression directly or indirectly affected by miR-148a, providing a new insight for uncovering the effect of miR-148a in cell physiology and identifying new targets of miR-148a. Previous studies have shown that miR148a could induce changes in cell proliferation, apoptosis, cell invasion and metastasis.8−12 Consistently, our study showed that many miR-148a-regulated proteins were involved in the same functional categories, such as LLGL2, ANXA1, LGALS3BP, and LIG1 (Table 2 and Figure 3A). To date, most studies of miR-148a mainly focused on discovering its role in tumor progression. However, in this study, the global changes of protein expression in miR-148aexpressing cells revealed another group of molecules involved in nervous system development might also have connection with miR-148a. Among them, the expression of ENAH, STX3, TMED2, and HMBS were decreased in miR-148a expressing cells, albeit they might not be the direct targets of miR-148a. ENAH, known as a mammalian ortholog of Drosophila

To further confirm the regulatory function of miR-148 in the neurological development and functions in zebrafish model, the expression of two neuron-related markers, Huc and Zic1, were detected in miR-148 MO-treated groups. The zebrafish zic1 gene expressed in brain and somites is known to control the forebrain patterning and midline formation.26,27 In this study, we used in situ hybridization to evaluate the expression of zic1 in miR-148 MO-treated zebrafish. Our findings showed that the expression level of zic1 was lower in the telencephalon and diencephalon as well as the dorsal spinal cord of miR-148 MOtreated zebrafish (Figure 6C), compared to wild-type (Figure 6A) and standard MO-treated control (Figure 6B). We further detected the expression of green fluorescent protein (GFP)labeled huc gene in miR-148a MO-treated zebrafish. Huc is a homologue of Drosophila elav. Its expression in the neural plate at the early stages of neurogenesis plays a role in neuronal determination and differentiation.28,29 We found that the expression of huc in brain and spinal cord was also reduced in miR-148 MO-treated zebrafish at 24 and 48 hpf compared with wild-type and MO standard control groups (Figure 7). Thus, these results showed that the reduced expression of miR148 caused abnormal development of brain and spinal cord, I



Article

In this study, we performed a quantitative proteomic analysis to reveal the novel functions of miR-148a. Moreover, the regulatory role of miR-148a in neurological development was further validated using a zebrafish model. It should be noted that some miRNAs with conserved sequences also display conserved expression patterns, providing further evidence for the conservation of their functions.44 For example, musclespecific miR-1 and nervous system-specific miR-124 are conserved in mammals, flies, and fish.45 In our study, we found that miR-148a in both human and zebrafish shared the same seed regions, including nucleotides 2−8 (Figure 4). Thus, we proposed that the zebrafish is a suitable model for analyzing the regulatory roles of miR-148a in neurological development and functions. Our in situ hybridization data showed that the expression of Zic1 gene was lower in the telencephalon and diencephalon as well as the dorsal spinal cord of miR-148 MOtreated zebrafish. Moreover, the expression of GFP-labeled Huc in the brain and spinal cord was also reduced in miR-148 MOtreated zebrafish at 24 and 48 hpf. The in vivo experiments further supported the hypothesis that miR-148 plays an important role in the neurological development and functions.

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Figure 7. Reduction in miR-148 levels affected the expression of Huc in brain and spinal cord of zebrafish. The expression of Huc gene expression was observed at 24 hpf (A−C) and 48 hpf (D−F) in GFPlabeled wild type (A and D), standard MO control (B and E), and miR-148 MO groups (C and F). The expression level of Huc was decreased in the brain and spinal cord of miR-148 MO-treated zebrafish compared with control groups.

CONCLUSION miR-148a is known to function in suppressing gastric cancer metastasis and can serve as a prognostic marker for gastric cancer. Here, we used an iTRAQ-based quantitative proteomic approach to reveal another role of miR-148a in neurological development and function. Clinical data showed that abnormal expression levels of miR-148a were associated with neurological disorders. Using an in vivo animal model (zebrafish), we showed that miR-148 knock-down affected the morphology of developing brain and the expression of the neuron-related markers in brain and dorsal spinal cord. These results demonstrated that miR-148a might be able to regulate neurological development and function.

Enabled (Ena), is required for neural development.31 The disruption of ENAH can induce abnormal development of forebrain.31 Moreover, STX3 is identified as a plasma membrane protein and plays an important role in the growth of neuritis.33 STX3-silenced neurons derived from PC12 cells are unable to grow neuritis even after stimulation with nerve growth factor (NGF).32 Mutation in TMED2 can also lead to the abnormalities of neural tube in embryo.34 On the other side, deregulation in these neuronal system-related genes, including ENAH, TMED2, and HMBS are associated with neuronal dysfunction and diseases.33−37 Our results showed that the miR-148a might regulate the expression of these neural function-related proteins, suggesting a possible effect of miR148a in neural development and diseases. There is emerging evidence to indicate that miRNAs have diverse functions in organisms, and their deregulation has been implicated in various diseases. For example, reduced miR-133b expression levels are associated with poor patient survival and metastasis in colorectal cancer.38 Moreover, miR-133b has been found to be deficient in the midbrain of mouse models and patients with Parkinson’s disease and is involved in the spinal cord regeneration of adult zebrafish.39,40 On the other hand, miR-107 might suppress the tumor invasion and proliferation of gastric cancer cells by targeting cyclin-dependent kinase 6.41 Besides, miR-107 is also expressed at low levels in Alzheimer’s disease and is implicated in neurodegenerative diseases through the regulation of granulin expression.42,43 Our proteomic results also uncovered diverse functions regulated by miR148a, particularly in neurological development. We also found that the expression level of miR-148a was clinically related to neurological diseases, including Alzheimer’s and Parkinson’s diseases (Figure 3B). All these results suggest that miRNA might be a potential candidate for targeting therapy in neurological diseases.

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ASSOCIATED CONTENT

S Supporting Information *

Figures S1−S3 and Tables S1−S4. This material is available free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*(S.-J.L.) Tel: +886-2-3366-5902. Fax: +886-2-3366-2457. Email: jeffl[email protected]. (Y.-J.C.) Tel: +886-2-2789-8660. Fax: +886-2-2783-1237. E-mail: [email protected]. (H.F.J.) Tel: +886-2-3366-4536. Fax: +886-2-23673374. E-mail: [email protected]. Author Contributions #

Chia-Wei Hu, Chien-Wei Tseng, and Chih-Wei Chien equally contributed to this work. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the Institute of Chemistry, Academic Sinica, and Department of Chemistry, National Taiwan University, for mass spectrometric analysis, Technology Commons, College of Life Science, National Taiwan University, for providing technical assistance, and Kuan-Hao Hsu for the miR-148atreated cell experiment. The deposition of mass spectrometry J



Article

mediated control of HLA-G expression and function. PLoS One 2012, 7, e33395. (15) Khan, A. P.; Poisson, L. M.; Bhat, V. B.; Fermin, D.; Zhao, R.; Kalyana-Sundaram, S.; Michailidis, G.; Nesvizhskii, A. I.; Omenn, G. S.; Chinnaiyan, A. M.; Sreekumar, A. Quantitative proteomic profiling of prostate cancer reveals a role for miR-128 in prostate cancer. Mol. Cell. Proteomics 2010, 9, 298−312. (16) Taguchi, A.; Yanagisawa, K.; Tanaka, M.; Cao, K.; Matsuyama, Y.; Goto, H.; Takahashi, T. Identification of hypoxia-inducible factor-1 alpha as a novel target for miR-17-92 microRNA cluster. Cancer Res. 2008, 68, 5540−5545. (17) Ross, P. L.; Huang, Y. N.; Marchese, J. N.; Williamson, B.; Parker, K.; Hattan, S.; Khainovski, N.; Pillai, S.; Dey, S.; Daniels, S.; Purkayastha, S.; Juhasz, P.; Martin, S.; Bartlet-Jones, M.; He, F.; Jacobson, A.; Pappin, D. J. Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents. Mol. Cell. Proteomics 2004, 3, 1154−1169. (18) Han, C. L.; Chien, C. W.; Chen, W. C.; Chen, Y. R.; Wu, C. P.; Li, H.; Chen, Y. J. A multiplexed quantitative strategy for membrane proteomics: opportunities for mining therapeutic targets for autosomal dominant polycystic kidney disease. Mol. Cell. Proteomics 2008, 7, 1983−1997. (19) Vizcaino, J. A.; Cote, R.; Reisinger, F.; Barsnes, H.; Foster, J. M.; Rameseder, J.; Hermjakob, H.; Martens, L. The Proteomics Identifications database: 2010 update. Nucleic Acids Res. 2010, 38, D736−742. (20) Wang, R.; Fabregat, A.; Rios, D.; Ovelleiro, D.; Foster, J. M.; Cote, R. G.; Griss, J.; Csordas, A.; Perez-Riverol, Y.; Reisinger, F.; Hermjakob, H.; Martens, L.; Vizcaino, J. A. PRIDE Inspector: a tool to visualize and validate MS proteomics data. Nat. Biotechnol. 2012, 30, 135−137. (21) Zhou, X. J.; Finch, C. E.; Nunez-Iglesias, J.; Liu, C.; Morgan, T. E. Joint genome-wide profiling of miRNA and mRNA expression in Alzheimer’s disease cortex reveals altered miRNA regulation. PLoS One 2010, 5, e8898. (22) Martins, M.; Rosa, A.; Guedes, L. C.; Fonseca, B. V.; Gotovac, K.; et al. Convergence of miRNA expression profiling, alpha-synuclein interacton and GWAS in Parkinson’s disease. PLoS One 2011, 6, e25443. (23) Yeh, C. M.; Liu, Y. C.; Chang, C. J.; Lai, S. L.; Hsiao, C. D.; Lee, S. J. Ptenb mediates gastrulation cell movements via Cdc42/AKT1 in zebrafish. PLoS One 2011, 6, e18702. (24) Kimmel, C. B.; Ballard, W. W.; Kimmel, S. R.; Ullmann, B.; Schilling, T. F. Stages of embryonic development of the zebrafish. Dev. Dyn. 1995, 203, 253−310. (25) Thisse, C.; Thisse, B.; Schilling, T. F.; Postlethwait, J. H. Structure of the zebrafish snail1 gene and its expression in wild-type, spadetail and no tail mutant embryos. Development 1993, 119, 1203− 1215. (26) Rohr, K. B.; Schulte-Merker, S.; Tautz, D. Zebrafish zic1 expression in brain and somites is affected by BMP and hedgehog signalling. Mech. Dev. 1999, 85, 147−159. (27) Maurus, D.; Harris, W. A. Zic-associated holoprosencephaly: zebrafish Zic1 controls midline formation and forebrain patterning by regulating Nodal, Hedgehog, and retinoic acid signaling. Genes Dev. 2009, 23, 1461−1473. (28) Kim, C. H.; Ueshima, E.; Muraoka, O.; Tanaka, H.; Yeo, S. Y.; Huh, T. L.; Miki, N. Zebrafish elav/HuC homologue as a very early neuronal marker. Neurosci. Lett. 1996, 216, 109−112. (29) Kim, C. H.; Bae, Y. K.; Yamanaka, Y.; Yamashita, S.; Shimizu, T.; Fujii, R.; Park, H. C.; Yeo, S. Y.; Huh, T. L.; Hibi, M.; Hirano, T. Overexpression of neurogenin induces ectopic expression of HuC in zebrafish. Neurosci. Lett. 1997, 239, 113−116. (30) Iliopoulos, D.; Malizos, K. N.; Oikonomou, P.; Tsezou, A. Integrative microRNA and proteomic approaches identify novel osteoarthritis genes and their collaborative metabolic and inflammatory networks. PLoS ONE 2008, 3, e3740.

proteomics data to ProteomeXchange Consortium was supported by PRIDE team, EBI. This work was supported by the National Science Council, Taiwan (NSC 99-2621-B-002005-MY3, NSC 99-2621-B-010-001-MY3) and National Taiwan University Cutting-Edge Steering Research Project (NTU-CESRP-102R7602C3).

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ABBREVIATIONS iTRAQ, isobaric tags for relative and absolute quantitation; TCEP, tris (2-carboxyethyl) phosphine hydrochloride; TEABC, triethylammonium bicarbonate; MMTS, methyl methanethiosulfonate; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; TEMED, N,N,N′,N′-tetramethylethylethylenediamine; ESI, electrospray ionization; IPA, ingenuity pathway analysis; GO, gene ontology; GEO, gene expression omnibus; WISH, whole-mount in situ hybridization; hpf, hours postfertilization; FDR, false discovery rate; MOs, morpholino oligonucleotides

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L


Quantitative Proteomics Reveals Diverse Roles of miR-148a from

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