Analysis of protein expression regulated by lumican in PANC‑1 cells using shotgun proteomics

  • Authors:
    • Tetsushi Yamamoto
    • Mitsuhiro Kudo
    • Wei-Xia Peng
    • Zenya Naito
  • View Affiliations

  • Published online on: July 11, 2013     https://doi.org/10.3892/or.2013.2612
  • Pages: 1609-1621
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Abstract

Lumican, a member of the class II small leucine-rich proteoglycan family, regulates the assembly and diameter of collagen fibers in the extracellular matrix of various tissues. We previously reported that lumican expression in the stromal tissues of pancreatic ductal adenocarcinoma (PDAC) correlates with tumor invasion, and tends to correlate with poor prognosis. Lumican stimulates growth and inhibits the invasion of a PDAC cell line. In the present study, we performed a global shotgun proteomic analysis using lumican-overexpressing PANC‑1 cells and lumican downregulated PANC‑1 cells to identify candidate proteins that are regulated by lumican and related to cell growth and invasion in PDAC cells. A total of 448 proteins were identified from lumican-overexpressing PANC‑1 and control cells. Additionally, 451 proteins were identified from lumican-downregulated PANC‑1 cells and control cells. As a result of semi-quantification based on spectral counting, 174 differentially expressed proteins were identified by lumican upregulation, and 143 differentially expressed proteins were identified by lumican downregulation. The expression levels of 24 proteins, including apoptosis- and invasion-related proteins correlated with lumican expression levels. It is likely that the expression of these proteins is regulated by lumican, and that they are involved in apoptosis and invasion in PDAC. These findings suggest that lumican may be involved in cell growth and invasion through the regulation of these 24 proteins expressed in PDAC.

Introduction

Lumican is a member of the class II small leucine-rich proteoglycan (SLRP) family. Members of this family have relatively small molecular sizes, with core proteins of approximately 40 kDa, and possess 6–10 leucine-rich repeat units in the core protein (1,2). Amino acid sequencing indicates that lumican has 4 potential sites for N-linked keratan sulfate (KS) or oligosaccharides (3,4). Therefore, lumican includes a core protein, glycoprotein and proteoglycan forms due to glycosylation (5). Lumican is a secreted collagen-binding extracellular matrix protein of the cornea, dermis and tendon stroma, arterial wall, and intestinal submucosa (69). Corneal opacity, as well as skin and tendon fragility due to disorganized and loosely packed collagen fibers in lumican-null mice suggest that lumican plays an important role in collagen fibrillogenesis (10,11).

Lumican was first reported as one of the major KS proteoglycans in the chicken cornea (12). In addition to the cornea, lumican expression has been reported in various human tissues, including malignant tumor tissues (5,1327). Among the clinicopathological characteristics of pancreatic ductal adenocarcinoma (PDAC), the localization of lumican in the stromal tissue adjacent to cancer cells correlates with advanced cancer stage, retroperitoneal and duodenal invasion, and residual tumor, and tends to correlate with shorter survival (21). These reports suggest that lumican localized in the stromal tissue is secreted from cancer cells and affects cancer cells through an autocrine and paracrine mechanism. We previously reported that PANC-1 cells, one of the PDAC cell lines, secrete only 70-kDa glycosylated lumican into the extracellular space. We also demonstrated that the secreted lumican stimulated cell growth through ERK activation and inhibited cell invasion and matrix metalloproteinase (MMP)-9 activation using lumican-overexpressing PANC-1 cells and lumican-downregulated PANC-1 cells (28). However, the mechanism of how lumican affects cell growth and invasion remains unclear.

In the present study, we performed shotgun liquid chromatography (LC)/mass spectrometry (MS)-based global proteomic analysis using protein from lumican-overexpressing PANC-1 cells and lumican-downregulated PANC-1 cells to examine how lumican regulates cell growth and invasion in PDAC cells. We identified 24 candidate proteins that may play an important role in cell growth and invasion and could be regulated by lumican.

Materials and methods

Materials

The following materials were purchased from Wako Pure Chemical Industries (Osaka, Japan): urea, 3-(3-cholamidopropyl) dimethylammonio-1-propanesulphonate (CHAPS), dithiothreitol (DTT), Tris (2-carboxyethyl) phosphine hydrochloride (TCEP) and iodoacetamide (IAA); Amicon Ultra 0.5-ml 3K was from Millipore (Tokyo, Japan), and thiourea from Nacalai Tesque, Inc. (Kyoto, Japan). All other chemicals and reagents were purchased from Sigma Chemical Corp. (St. Louis, MO, USA).

PDAC cell line

PANC-1 cells were obtained from the Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer, Tohoku University (Sendai, Japan).

Protein extraction of lumican-regulated PANC-1 cells

The lumican-overexpressing PANC-1 cells, lumican-downregulated PANC-1 cells, and control cell lines (Mock and NC, respectively) were prepared as previously described (28). The lumican-regulated PANC-1 cells were cultured at a density of 5×105 cells in a 100-mm dish in RPMI-1640 medium with 10% fetal bovine serum (FBS) for 72 h. Then, cells were solubilized in urea lysis buffer (7 M urea, 2 M thiourea, 5% CHAPS, 1% Triton X-100). Protein concentration was measured using the Bradford method.

In-solution trypsin digestion

A gel-free digestion approach was performed in accordance with the protocol described by Bluemlein and Ralser (29). In brief, 10 μg of protein extract from each sample was reduced by addition of 45 mM DTT and 20 mM TCEP and was then alkylated using 100 mM IAA. Following alkylation, samples were digested with Proteomics Grade Trypsin (Agilent Technologies, Inc., Santa Clara, CA, USA) at 37°C for 24 h. Next, digests were evaporated in a vacuum concentrator centrifuge and the residue was resuspended in 0.1% trifluoroacetic acid/5% acetonitrile. The digests were filtered through Amicon Ultra 0.5-ml 3K to remove undigested proteins and the flow-through was used in the following analyses.

LC-MS/MS analysis of protein identification

Approximately 2-μg peptide samples were injected into a peptide L-trap column (Chemicals Evaluation and Research Institute, Tokyo, Japan) using an HTC PAL autosampler (CTC Analytics, Zwingen, Switzerland) and further separated through an Advance-nano UHPLC using a Reverse-Phase C18-column (Zaplous column α, 3-μm diameter gel particles and 100 Å pore size, 0.1×150 mm; both from AMR, Inc., Tokyo, Japan). The mobile phase consisted of solution A (0.1% formic acid in water) and solution B (acetonitrile). The column was developed at a flow rate of 500 nl/min with a concentration gradient of acetonitrile from 5 to 45% B over 120 min. Gradient-eluted peptides were analyzed using an amaZon ETD ion-trap mass spectrometer (Bruker Daltonics, Billerica, MA, USA). The results were acquired in a data-dependent manner in which MS/MS fragmentation was performed on the 10 most intense peaks of every full MS scan.

All MS/MS spectra data were searched against the SwissProt Homo sapiens database using Mascot (v2.3.01; Matrix Science, London, UK). The search criteria was set as follows: enzyme, trypsin; allowance of up to two missed cleavage peptides; mass tolerance ± 0.5 Da and MS/MS tolerance ± 0.5 Da; and modifications of cysteine carbamidomethylation and methionine oxidation.

Semi-quantitative analysis of identified proteins

The fold-changes in expressed proteins on the base 2 logarithmic scale were calculated using Rsc based upon spectral counting (30). Relative amounts of identified proteins were also calculated using the normalized spectral abundance factor (NSAF) (31). Differentially expressed proteins were chosen so that their Rsc satisfy >1 or <−1, which correspond to fold-changes of >2 or <0.5.

Bioinformatics

Functional annotations for the identified proteins whose expression level was regulated by lumican were processed using the Database for Annotation, Visualization and Integrated Discovery (DAVID), v6.7 (http://david.abcc.ncifcrf.gov/home.jsp) (3234).

Results

Protein identification and profile in lumican-regulated PANC-1 cells

To examine the effect of lumican on cell growth and invasion of PDAC cells, we created two types of PANC-1 cells whose lumican expression level was regulated: lumican-overexpressing PANC-1 cells and lumican-downregulated PANC-1 cells (28). We then investigated the molecular profile of proteins whose expression level was regulated by lumican using shotgun proteomics. Fig. 1 shows the Venn map for the identified proteins in lumican-regulated PANC-1 cells. In lumican-overexpressing PANC-1 cells (Lum), 321 proteins were identified, and 347 were identified in control cells (Mock) under the search parameter settings used (Fig. 1A). On the other hand, 388 proteins were identified in lumican-downregulated PANC-1 cells (siLum) and 287 in control cells (NC) (Fig. 1B). Among the 448 proteins identified from lumican upregulated cells, 220 (49.1%) proteins were identified in both cell lines, while 101 (22.6%) and 127 (28.3%) proteins were unique to Lum and Mock, respectively (Fig. 1A). Of the 451 total proteins identified from lumican downregulated cells, 224 (49.7%) proteins were identified in both cell lines, whereas 164 (36.3%) and 63 (14.0%) proteins were unique to siLum and NC, respectively (Fig. 1B).

Semi-quantitative comparison of identified proteins in lumican-regulated PANC-1 cells

Next, we performed a label-free semi-quantitative method based on spectral counting, as described in the Materials and methods section, to find proteins whose expression levels were regulated by lumican. The Rsc value was plotted against the corresponding protein (X-axis) from left to right for proteins identified in the Lum and Mock groups (Fig. 2A). The positive and negative Rsc values indicate increased and decreased expression, respectively, in the Lum group. The NSAF value (bar) plotted against the corresponding protein (X-axis), NSAF of Lum (black bar) and Mock (gray bar) proteins are indicated above and below the X-axis, respectively (Fig. 2A). Proteins with either a high positive or negative Rsc value were considered candidate proteins whose expression level was regulated by lumican. Fig. 2B shows the Rsc and NSAF values of the siLum and NC groups as described above. In the lumican upregulated cells, the Rsc of metallothionein (MT)-1X was positive, and the Rsc of galectin-1 was negative (Fig. 2A). In the lumican downregulated cells, the Rsc of Annexin A5 was positive and the Rsc of vinculin was negative (Fig. 2B). Housekeeping proteins such as β-actin, histone H4 and GAPDH were located near the center of the X-axis (Fig. 2).

As a result of semi-quantification, 174 differentially expressed proteins were identified in lumican upregulated PANC-1 cells (Table I), and a total of 143 differentially expressed proteins were identified in lumican downregulated PANC-1 cells (Table II). However, the expression level of housekeeping proteins such as β-actin, GAPDH and histone H4 was not changed in lumican upregulated cells or lumican downregulated cells. In order to identify proteins whose expression levels were regulated by lumican, we selected proteins whose Rsc value was inversely associated between lumican upregulated cells and lumican downregulated cells. Twenty four proteins were identified as candidates (Table III).

Table I

Differentially expressed proteins in lumican upregulated PANC-1 cells.

Table I

Differentially expressed proteins in lumican upregulated PANC-1 cells.

Spectral counting

No.IDAccession no. and descriptionNo. of amino acidsMockLumFold-change (Rsc)
1ACTH_HUMAN(P63267) Actin, γ-enteric smooth muscle376350−4.69652
2ACTC_HUMAN(P68032) Actin, α cardiac muscle 1377180−3.7706617
3TBB2A_HUMAN(Q13885) Tubulin β-2A chain445100−2.9897736
4H2B1H_HUMAN(Q93079) Histone H2B type 1-H12690−2.8547299
5HS904_HUMAN(Q58FG1) Putative heat shock protein HSP 90-α A441880−2.7058891
6TBA4A_HUMAN(P68366) Tubulin α-4A chain44880−2.7058891
7H2AJ_HUMAN(Q9BTM1) Histone H2A.J12970−2.540088
8H2B3B_HUMAN(Q8N257) Histone H2B type 3-B12670−2.540088
9RLA0_HUMAN(P05388) 60S acidic ribosomal protein P031770−2.540088
10H2B1O_HUMAN(P23527) Histone H2B type 1-O12640−1.885788
11K2C71_HUMAN(Q3SY84) Keratin, type II cytoskeletal 7152340−1.885788
12NDKB_HUMAN(P22392) Nucleoside diphosphate kinase B15240−1.885788
13RA1L2_HUMAN(Q32P51) Heterogeneous nuclear ribonucleoprotein A1-like 232040−1.885788
14CH10_HUMAN(P61604) 10 kDa heat shock protein, mitochondrial10240−1.885788
15H2A1H_HUMAN(Q96KK5) Histone H2A type 1-H12830−1.5801931
16HNRPK_HUMAN(P61978) Heterogeneous nuclear ribonucleoprotein K46330−1.5801931
17K1C25_HUMAN(Q7Z3Z0) Keratin, type I cytoskeletal 2545030−1.5801931
18K22O_HUMAN(Q01546) Keratin, type II cytoskeletal 2 oral63830−1.5801931
19MYL6_HUMAN(P60660) Myosin light polypeptide 615130−1.5801931
20NFH_HUMAN(P12036) Neurofilament heavy polypeptide102630−1.5801931
21TAGL3_HUMAN(Q9UI15) Transgelin-319930−1.5801931
22YI016_HUMAN(A6NKZ8) Putative tubulin β chain-like protein37230−1.5801931
23ANXA5_HUMAN(P08758) Annexin A532030−1.5801931
24MT1G_HUMAN(P13640) Metallothionein-1G6230−1.5801931
25K1C14_HUMAN(P02533) Keratin, type I cytoskeletal 1447261−1.5040946
26K2C80_HUMAN(Q6KB66) Keratin, type II cytoskeletal 8045251−1.2892288
27ACTBL_HUMAN(Q562R1) β-actin-like protein 2376134−1.259281
281433B_HUMAN(P31946) 14-3-3 protein β/α24620−1.1924301
29AINX_HUMAN(Q16352) α-internexin49920−1.1924301
30H2AZ_HUMAN(H2AZ_HUMAN) Histone H2A.Z12820−1.1924301
31H2B1D_HUMAN(P58876) Histone H2B type 1-D12620−1.1924301
32H2B1J_HUMAN(P06899) Histone H2B type 1-J12620−1.1924301
33K1C13_HUMAN(P13646) Keratin, type I cytoskeletal 1345820−1.1924301
34K2C6C_HUMAN(P48668) Keratin, type II cytoskeletal 6C56420−1.1924301
35KRT85_HUMAN(P78386) Keratin, type II cuticular Hb550720−1.1924301
36MLL3_HUMAN(Q8NEZ4) Histone-lysine N-methyltransferase MLL3491120−1.1924301
37NDK8_HUMAN(O60361) Putative nucleoside diphosphate kinase13720−1.1924301
38NFM_HUMAN(P07197) Neurofilament medium polypeptide91620−1.1924301
39SERPH_HUMAN(P50454) Serpin H141820−1.1924301
40U17L5_HUMAN(A8MUK1) Ubiquitin carboxyl-terminal hydrolase 17-like protein 553020−1.1924301
41URFB1_HUMAN(Q6BDS2) UHRF1-binding protein 1144020−1.1924301
42MDHM_HUMAN(P40926) Malate dehydrogenase, mitochondrial33820−1.1924301
43IF5A1_HUMAN(P63241) Eukaryotic translation initiation factor 5A-115420−1.1924301
44SFPQ_HUMAN(P23246) Splicing factor, proline- and glutamine-rich70720−1.1924301
45NACA_HUMAN(Q13765) Nascent polypeptide-associated complex subunit α21520−1.1924301
46EF1B_HUMAN(P24534) Elongation factor 1-β22520−1.1924301
47RS4X_HUMAN(P62701) 40S ribosomal protein S4, X isoform26320−1.1924301
48IF4A2_HUMAN(Q14240) Eukaryotic initiation factor 4A-II40793−1.0866682
49EF2_HUMAN(P13639) Elongation factor 2858166−1.0697973
50HSP72_HUMAN(P54652) Heat shock-related 70 kDa protein 2639114−1.0396137
51DESM_HUMAN(P17661) Desmin470135−1.0068984
52LEG1_HUMAN(P09382) Galectin-1135135−1.0068984
53CC113_HUMAN(Q9H0I3) Coiled-coil domain-containing protein 113377011.03639903
54H11_HUMAN(Q02539) Histone H1.1215011.03639903
55UBA1_HUMAN(P22314) Ubiquitin-like modifier-activating enzyme 11058011.03639903
56TCPH_HUMAN(Q99832) T-complex protein 1 subunit eta543011.03639903
57RL31_HUMAN(P62899) 60S ribosomal protein L31125011.03639903
58ADT4_HUMAN(Q9H0C2) ADP/ATP translocase 4315011.03639903
59BRE1B_HUMAN(O75150) E3 ubiquitin-protein ligase BRE1B1001011.03639903
60CALX_HUMAN(P27824) Calnexin592011.03639903
61CAP1_HUMAN(Q01518) Adenylyl cyclase-associated protein 1475011.03639903
62CAZA1_HUMAN(P52907) F-actin-capping protein subunit α-1286011.03639903
63CCNT1_HUMAN(O60563) Cyclin-T1726011.03639903
64CGNL1_HUMAN(Q0VF96) Cingulin-like protein 11302011.03639903
65CISY_HUMAN(O75390) Citrate synthase, mitochondrial466011.03639903
66CLIP1_HUMAN(P30622) CAP-Gly domain-containing linker protein 11438011.03639903
67CN37_HUMAN(P09543) 2′,3′-cyclic-nucleotide 3′-phosphodiesterase421011.03639903
68CTSR1_HUMAN(Q8NEC5) Cation channel sperm-associated protein 1780011.03639903
69CX057_HUMAN(Q6NSI4) Uncharacterized protein CXorf57855011.03639903
70DDX21_HUMAN(Q9NR30) Nucleolar RNA helicase 2783011.03639903
71DYXC1_HUMAN(Q8WXU2) Dyslexia susceptibility 1 candidate gene 1 protein420011.03639903
72EFTU_HUMAN(P49411) Elongation factor Tu, mitochondrial452011.03639903
73EHD1_HUMAN(Q9H4M9) EH domain-containing protein 1534011.03639903
74EIF3H_HUMAN(O15372) Eukaryotic translation initiation factor 3 subunit H352011.03639903
75EHD1_HUMAN(Q9H4M9) EH domain-containing protein 1534011.03639903
76FETUA_HUMAN(P02765) α-2-HS-glycoprotein367011.03639903
77HIP1R_HUMAN(O75146) Huntingtin-interacting protein 1-related protein1068011.03639903
78HMGN1_HUMAN(P05114) Non-histone chromosomal protein HMG-14100011.03639903
79HMGN4_HUMAN(O00479) High mobility group nucleosome-binding domain- containing protein 490011.03639903
80HNRH3_HUMAN(P31942) Heterogeneous nuclear ribonucleoprotein H3346011.03639903
81IDE_HUMAN(P14735) Insulin-degrading enzyme1019011.03639903
82IMA2_HUMAN(P52292) Importin subunit α-2529011.03639903
83IMDH2_HUMAN(P12268) Inosine-5′-monophosphate dehydrogenase 2514011.03639903
84K1C26_HUMAN(Q7Z3Y9) Keratin, type I cytoskeletal 26468011.03639903
85K6PL_HUMAN(P17858) 6-phosphofructokinase, liver type780011.03639903
86MYH11_HUMAN(P35749) Myosin-111972011.03639903
87MYH14_HUMAN(Q7Z406) Myosin-141995011.03639903
88PAR10_HUMAN(Q53GL7) Poly [ADP-ribose] polymerase 101025011.03639903
89PI3R4_HUMAN(Q99570) Phosphoinositide 3-kinase regulatory subunit 41358011.03639903
90PRDX4_HUMAN(Q13162) Peroxiredoxin-4271011.03639903
91PRS6A_HUMAN(P17980) 26S protease regulatory subunit 6A439011.03639903
92PSB3_HUMAN(P49720) Proteasome subunit β type-3205011.03639903
93PTN22_HUMAN(Q9Y2R2) Tyrosine-protein phosphatase non-receptor type 22807011.03639903
94RAB10_HUMAN(P61026) Ras-related protein Rab-10200011.03639903
95RBBP7_HUMAN(Q16576) Histone-binding protein RBBP7425011.03639903
96RL18A_HUMAN(Q02543) 60S ribosomal protein L18a176011.03639903
97RL27A_HUMAN(P46776) 60S ribosomal protein L27a148011.03639903
98RL34_HUMAN(P49207) 60S ribosomal protein L34117011.03639903
99RL3L_HUMAN(Q92901) 60S ribosomal protein L3-like407011.03639903
100RL40_HUMAN(P62987) Ubiquitin-60S ribosomal protein L40128011.03639903
101ROAA_HUMAN(Q99729) Heterogeneous nuclear ribonucleoprotein A/B332011.03639903
102SET_HUMAN(Q01105) Protein SET290011.03639903
103SODC_HUMAN(P00441) Superoxide dismutase [Cu-Zn]154011.03639903
104SPSY_HUMAN(P52788) Spermine synthase366011.03639903
105SYRC_HUMAN(P54136) Arginyl-tRNA synthetase, cytoplasmic660011.03639903
106TADBP_HUMAN(Q13148) TAR DNA-binding protein 43414011.03639903
107TALDO_HUMAN(P37837) Transaldolase337011.03639903
108TECT2_HUMAN(Q96GX1) Tectonic-2697011.03639903
109TEBP_HUMAN(Q15185) Prostaglandin E synthase 3160011.03639903
110TPD54_HUMAN(O43399) Tumor protein D54206011.03639903
111UGPA_HUMAN(Q16851) UTP - glucose-1-phosphate uridylyltransferase508011.03639903
112WDR53_HUMAN(Q7Z5U6) WD repeat-containing protein 53358011.03639903
113ZC12A_HUMAN(Q5D1E8) Ribonuclease ZC3H12A599011.03639903
114RL22_HUMAN(P35268) 60S ribosomal protein L22128011.03639903
115RL13_HUMAN(P26373) 60S ribosomal protein L13211011.03639903
116RL23_HUMAN(P62829) 60S ribosomal protein L23140011.03639903
117RS11_HUMAN(P62280) 40S ribosomal protein S11158011.03639903
118SUMO2_HUMAN(P61956) Small ubiquitin-related modifier 295011.03639903
119TFR1_HUMAN(P02786) Transferrin receptor protein 1760011.03639903
120RL11_HUMAN(P62913) 60S ribosomal protein L11178011.03639903
121EF1A2_HUMAN(Q05639) Elongation factor 1-α 246310191.04422777
122CBX1_HUMAN(P83916) Chromobox protein homolog 1185131.10688593
123RL12_HUMAN(P30050) 60S ribosomal protein L12165131.10688593
124RAN_HUMAN(P62826) GTP-binding nuclear protein Ran216131.10688593
125PDIA6_HUMAN(Q15084) Protein disulfide-isomerase A6440131.10688593
126RL10L_HUMAN(Q96L21) 60S ribosomal protein L10-like214131.10688593
127SRC8_HUMAN(Q14247) Src substrate cortactin550131.10688593
128RL8_HUMAN(P62917) 60S ribosomal protein L8257131.10688593
129RS23_HUMAN(P62266) 40S ribosomal protein S23143131.10688593
130RS13_HUMAN(P62277) 40S ribosomal protein S13151131.10688593
131RL24_HUMAN(P83731) 60S ribosomal protein L24157131.10688593
132RL37A_HUMAN(P61513) 60S ribosomal protein L37a92251.13371212
133PAL4A_HUMAN(Q9Y536) Peptidylprolyl cis-trans isomerase A-like 4A/B/C164251.13371212
134RL18_HUMAN(Q07020) 60S ribosomal protein L18188251.13371212
135K1C9_HUMAN(P35527) Keratin, type I cytoskeletal 96238171.17638585
136TBB1_HUMAN(Q9H4B7) Tubulin β-1 chain451381.31408061
137PAIRB_HUMAN(Q8NC51) Plasminogen activator inhibitor 1 RNA-binding protein408261.34868122
138CATD_HUMAN(P07339) Cathepsin D412021.56775608
139FLNB_HUMAN(O75369) Filamin-B2602021.56775608
140MYL6B_HUMAN(P14649) Myosin light chain 6B208021.56775608
141PSB2_HUMAN(P49721) Proteasome subunit β type-2201021.56775608
142RS7_HUMAN(P62081) 40S ribosomal protein S7194021.56775608
143IQGA1_HUMAN(P46940) Ras GTPase-activating-like protein IQGAP11657021.56775608
144DX39B_HUMAN(Q13838) Spliceosome RNA helicase DDX39B428021.56775608
145GANAB_HUMAN(Q9BS14)Neutral α-glucosidase AB944021.56775608
146H13_HUMAN(P16402) Histone H1.3221021.56775608
147H14_HUMAN(P10412) Histone H1.4219021.56775608
148HNRH2_HUMAN(P55795) Heterogeneous nuclear ribonucleoprotein H2449021.56775608
149ATPA_HUMAN(P25705) ATP synthase subunit α, mitochondrial553021.56775608
150H31T_HUMAN(Q16695) Histone H3.1t136021.56775608
151RL10A_HUMAN(P62906) 60S ribosomal protein L10a217021.56775608
152H12_HUMAN(P16403) Histone H1.2213021.56775608
153TBB8_HUMAN(Q3ZCM7) Tubulin β-8 chain4445151.57505162
154EF1D_HUMAN(P29692) Elongation factor 1-δ281151.6649664
155RLA0L_HUMAN(Q8NHW5) 60S acidic ribosomal protein P0-like317161.8799355
156RS8_HUMAN(P62241) 40S ribosomal protein S8208031.95562202
157CCD50_HUMAN(Q8IVM0) Coiled-coil domain-containing protein 50306031.95562202
158K2C6A_HUMAN(P02538) Keratin, type II cytoskeletal 6A564031.95562202
159MT1X_HUMAN(P80297) Metallothionein-1X61031.95562202
160RL17_HUMAN(P18621) 60S ribosomal protein L17184031.95562202
161RS26_HUMAN(P62854) 40S ribosomal protein S26115031.95562202
162H15_HUMAN(P16401) Histone H1.5226031.95562202
163ATPB_HUMAN(P06576) ATP synthase subunit β, mitochondrial529172.06719342
164ADT1_HUMAN(P12235) ADP/ATP translocase 1298042.26131991
165H2A1_HUMAN(P0C0S8) Histone H2A type 1130042.26131991
166RS6_HUMAN(P62753) 40S ribosomal protein S6249042.26131991
167H2B1B_HUMAN(P33778) Histone H2B type 1-B126042.26131991
168FLNA_HUMAN(P21333) Filamin-A2647192.38204237
169H2B1M_HUMAN(Q99879) Histone H2B type 1-M126062.72867159
170H2AX_HUMAN(P16104) Histone H2A.x143072.9159295
171H2A1B_HUMAN(P04908) Histone H2A type 1-B/E130093.23077846
172H2A1A_HUMAN(Q96QV6) Histone H2A type 1-A131093.23077846
173H2B1C_HUMAN(P62807) Histone H2B type 1-C/E/F/G/I1260113.48962935
174ACTA_HUMAN(P62736) Actin, aortic smooth muscle3770153.90067941

[i] The expression levels of the 174 proteins were altered more than 2-fold as a result of lumican upregulation.

Table II

Differentially expressed proteins in lumican downregulated PANC-1 cells.

Table II

Differentially expressed proteins in lumican downregulated PANC-1 cells.

Spectral counting

No.IDAccession no. and descriptionNo. of amino acidsNCsiLumFold-change (Rsc)
1ACTG_HUMAN(P63261) Actin, cytoplasmic 2375190−4.1933172
2H2B1D_HUMAN(P58876) Histone H2B type 1-D12670−2.8944219
3H2A2A_HUMAN(Q6FI13) Histone H2A type 2-A13040−2.2414896
4HNRPU_HUMAN(Q00839) Heterogeneous nuclear ribonucleoprotein U82530−1.9363502
5H2B1M_HUMAN(Q99879) Histone H2B type 1-M12630−1.9363502
6KRT81_HUMAN(Q14533) Keratin, type II cuticular Hb150530−1.9363502
7RL10_HUMAN(P27635) 60S ribosomal protein L1021430−1.9363502
8K2C6B_HUMAN(P04259) Keratin, type II cytoskeletal 6B56461−1.8595343
9CAPZB_HUMAN(P47756) F-actin-capping protein subunit β27720−1.5490424
10FUBP2_HUMAN(Q92945) Far upstream element-binding protein 271120−1.5490424
11IPO7_HUMAN(O95373) Importin-7103820−1.5490424
12K2C6C_HUMAN(P48668) Keratin, type II cytoskeletal 6C56420−1.5490424
13ML12A_HUMAN(P19105) Myosin regulatory light chain 12A17120−1.5490424
14NUCB1_HUMAN(Q02818) Nucleobindin-146120−1.5490424
15EF1B_HUMAN(P24534) Elongation factor 1-β22520−1.5490424
16NDKA_HUMAN(P15531) Nucleoside diphosphate kinase A15220−1.5490424
17VINC_HUMAN(P18206) Vinculin113472−1.5155258
18PDIA3_HUMAN(P30101) Protein disulfide-isomerase A350541−1.3933005
19K2C5_HUMAN(P13647) Keratin, type II cytoskeletal 559083−1.2936552
20K1C19_HUMAN(P08727) Keratin, type I cytoskeletal 19400115−1.1429912
21ECH1_HUMAN(Q13011) δ(3,5)-δ(2,4)-dienoyl-CoA isomerase, mitochondrial32852−1.1144173
22PAIRB_HUMAN(Q8NC51) Plasminogen activator inhibitor 1 RNA-binding protein40852−1.1144173
23RS3_HUMAN(P23396) 40S ribosomal protein S324331−1.0881611
24CRIP1_HUMAN(P50238) Cysteine-rich protein 17731−1.0881611
25RS28_HUMAN(P62857) 40S ribosomal protein S286931−1.0881611
26RS13_HUMAN(P62277) 40S ribosomal protein S1315131−1.0881611
27RS27A_HUMAN(P62979) Ubiquitin-40S ribosomal protein S27a15631−1.0881611
28CAPG_HUMAN(P40121) Macrophage-capping protein34810−1.0182433
29CDC42_HUMAN(P60953) Cell division control protein 42 homolog19110−1.0182433
30CX6B1_HUMAN(P14854) Cytochrome c oxidase subunit 6B18610−1.0182433
31GDIA_HUMAN(P31150) Rab GDP dissociation inhibitor α44710−1.0182433
32IQGA1_HUMAN(P46940) Ras GTPase-activating-like protein IQGAP1165710−1.0182433
33RL12_HUMAN(P30050) 60S ribosomal protein L1216510−1.0182433
34RL35A_HUMAN(P18077) 60S ribosomal protein L35a11010−1.0182433
35SQSTM_HUMAN(Q13501) Sequestosome-144010−1.0182433
36RL10L_HUMAN(Q96L21) 60S ribosomal protein L10-like21410−1.0182433
37RS14_HUMAN(P62263) 40S ribosomal protein S1415110−1.0182433
38AGR3_HUMAN(Q8TD06) Anterior gradient protein 3 homolog16610−1.0182433
39ALMS1_HUMAN(Q8TCU4) Alstrom syndrome protein 1416710−1.0182433
40AP3D1_HUMAN(O14617) AP-3 complex subunit δ-1115310−1.0182433
41BI2L2_HUMAN(Q6UXY1) Brain-specific angiogenesis inhibitor 1-associated protein 2-like protein 252910−1.0182433
42CH041_HUMAN(Q6NXR4) Uncharacterized protein C8orf4150810−1.0182433
43CORO7_HUMAN(P57737) Coronin-792510−1.0182433
44CSPG4_HUMAN(Q6UVK1) Chondroitin sulfate proteoglycan 4232210−1.0182433
45CXL14_HUMAN(O95715) C-X-C motif chemokine 1411110−1.0182433
46DDX17_HUMAN(Q92841) Probable ATP-dependent RNA helicase DDX1765010−1.0182433
47DNJB1_HUMAN(P25685) DnaJ homolog subfamily B member 134010−1.0182433
48EIF3C_HUMAN(Q99613) Eukaryotic translation initiation factor 3 subunit C91310−1.0182433
49FER_HUMAN(P16591) Tyrosine-protein kinase Fer82210−1.0182433
50GELS_HUMAN(P06396) Gelsolin78210−1.0182433
51INSRR_HUMAN(P14616) Insulin receptor-related protein129710−1.0182433
52KRT85_HUMAN(P78386) Keratin, type II cuticular Hb550710−1.0182433
53MT1X_HUMAN(P80297) MT-1X6110−1.0182433
54NALP6_HUMAN(P59044) NACHT, LRR and PYD domains-containing protein 689210−1.0182433
55PLEC_HUMAN(Q15149) Plectin468410−1.0182433
56PRS8_HUMAN(P62195) 26S protease regulatory subunit 840610−1.0182433
57PUR2_HUMAN(P22102) Trifunctional purine biosynthetic protein adenosine-3101010−1.0182433
58RFX6_HUMAN(Q8HWS3) DNA-binding protein RFX692810−1.0182433
59RS17_HUMAN(P08708) 40S ribosomal protein S1713510−1.0182433
60RUNX1_HUMAN(Q01196) Runt-related transcription factor 145310−1.0182433
61SEPT9_HUMAN(Q9UHD8) Septin-958610−1.0182433
62SRSF4_HUMAN(Q08170) Serine/arginine-rich splicing factor 449410−1.0182433
63SUMO3_HUMAN(P55854) Small ubiquitin-related modifier 310310−1.0182433
64TIM13_HUMAN(Q9Y5L4) Mitochondrial import inner membrane translocase subunit Tim139510−1.0182433
65UGDH_HUMAN(O60701) UDP-glucose 6-dehydrogenase49410−1.0182433
66WDHD1_HUMAN(O75717) WD repeat and HMG-box DNA-binding protein 1112910−1.0182433
67ZZEF1_HUMAN(O43149) Zinc finger ZZ-type and EF-hand domain-containing protein 1296110−1.0182433
68RL22_HUMAN(P35268) 60S ribosomal protein L2212810−1.0182433
69BASP1_HUMAN(P80723) Brain acid soluble protein 122710−1.0182433
70SPSY_HUMAN(P52788) Spermine synthase36610−1.0182433
71RS25_HUMAN(P62851) 40S ribosomal protein S2512510−1.0182433
72RS9_HUMAN(P46781) 40S ribosomal protein S919410−1.0182433
73HIP1R_HUMAN(O75146) Huntingtin-interacting protein 1-related protein106810−1.0182433
74RL15_HUMAN(P61313) 60S ribosomal protein L1520410−1.0182433
75G6PI_HUMAN(P06744) Glucose-6-phosphate isomerase55810−1.0182433
76TERA_HUMAN(P55072) Transitional endoplasmic reticulum ATPase806141.05291534
77RS5_HUMAN(P46782) 40S ribosomal protein S5204141.05291534
78PCBP3_HUMAN(P57721) Poly(rC)-binding protein 3371141.05291534
79K2C74_HUMAN(Q7RTS7) Keratin, type II cytoskeletal 745294111.0534109
80CH60_HUMAN(P10809) 60 kDa heat shock protein, mitochondrial5737201.19690092
81RAN_HUMAN(P62826) GTP-binding nuclear protein Ran216021.20893401
82ADT2_HUMAN(P05141) ADP/ATP translocase 2298021.20893401
83CAZA1_HUMAN(P52907) F-actin-capping protein subunit α-1286021.20893401
84G6PD_HUMAN(P11413) Glucose-6-phosphate 1-dehydrogenase515021.20893401
85H2AV_HUMAN(Q71UI9) Histone H2A.V128021.20893401
86H2B1H_HUMAN(Q93079) Histone H2B type 1-H126021.20893401
87H2B1J_HUMAN(P06899) Histone H2B type 1-J126021.20893401
88H2B1K_HUMAN(O60814) Histone H2B type 1-K126021.20893401
89HCD2_HUMAN(Q99714) 3-hydroxyacyl-CoA dehydrogenase type-2261021.20893401
90MT1A_HUMAN(P04731) Metallothionein-1A61021.20893401
91RL18A_HUMAN(Q02543) 60S ribosomal protein L18a176021.20893401
92RL21_HUMAN(P46778) 60S ribosomal protein L21160021.20893401
93RS7_HUMAN(P62081) 40S ribosomal protein S7194021.20893401
94SPT6H_HUMAN(Q7KZ85) Transcription elongation factor SPT61726021.20893401
95SRP14_HUMAN(P37108) Signal recognition particle 14 kDa protein136021.20893401
96TBA4B_HUMAN(Q9H853) Putative tubulin-like protein α-4B241021.20893401
97TCTP_HUMAN(P13693) Translationally-controlled tumor protein172021.20893401
98UBB_HUMAN(P0CG47) Polyubiquitin-B229021.20893401
99RL19_HUMAN(P84098) 60S ribosomal protein L19196021.20893401
100FKB1A_HUMAN(P62942) Peptidyl-prolyl cis-trans isomerase FKBP1A108021.20893401
101MDHM_HUMAN(P40926) Malate dehydrogenase, mitochondrial338021.20893401
102NDE1_HUMAN(Q9NXR1) Nuclear distribution protein nudE homolog 1346021.20893401
103PSA5_HUMAN(P28066) Proteasome subunit α type-5241021.20893401
104H2B1B_HUMAN(P33778) Histone H2B type 1-B126021.20893401
105HNRH2_HUMAN(P55795) Heterogeneous nuclear ribonucleoprotein H2449151.30464663
106K2C1_HUMAN(P04264) Keratin, type II cytoskeletal 1644281.34002273
107K1C9_HUMAN(P35527) Keratin, type I cytoskeletal 9623031.59614949
108RLA0L_HUMAN(Q8NHW5) 60S acidic ribosomal protein P0-like317031.59614949
109H2A1B_HUMAN(P04908) Histone H2A type 1-B/E130031.59614949
110SRC8_HUMAN(Q14247) Src substrate cortactin550031.59614949
111UBA1_HUMAN(P22314) Ubiquitin-like modifier-activating enzyme 11058031.59614949
112H2A1H_HUMAN(Q96KK5) Histone H2A type 1-H128031.59614949
113H2A2B_HUMAN(Q8IUE6) Histone H2A type 2-B130031.59614949
114HN1_HUMAN(Q9UK76) Hematological and neurological expressed 1 protein154031.59614949
115HNRPK_HUMAN(P61978) Heterogeneous nuclear ribonucleoprotein K463031.59614949
116K1C14_HUMAN(P02533) Keratin, type I cytoskeletal 14472031.59614949
117K22O_HUMAN(Q01546) Keratin, type II cytoskeletal 2 oral638031.59614949
118K6PP_HUMAN(Q01813) 6-phosphofructokinase type C784031.59614949
119POTEJ_HUMAN(P0CG39) POTE ankyrin domain family member J1038031.59614949
120RADI_HUMAN(P35241) Radixin583031.59614949
121HS904_HUMAN(Q58FG1) Putative heat shock protein HSP 90-α A44182101.62280828
122PROF1_HUMAN(P07737) Profilin-11403141.67515744
123H90B3_HUMAN(Q58FF7) Putative heat shock protein HSP 90-β-3597171.70556987
124TBB2A_HUMAN(Q13885) Tubulin β-2A chain4457331.88806959
125TIM50_HUMAN(Q3ZCQ8) Mitochondrial import inner membrane translocase subunit TIM50353041.90119651
126RL4_HUMAN(P36578) 60S ribosomal protein L4427041.90119651
127H2A1C_HUMAN(Q93077) Histone H2A type 1-C130041.90119651
128K2C6A_HUMAN(P02538) Keratin, type II cytoskeletal 6A564041.90119651
129NDK8_HUMAN(O60361) Putative nucleoside diphosphate kinase137041.90119651
130TKT_HUMAN(P29401) Transketolase623041.90119651
131H2B1O_HUMAN(P23527) Histone H2B type 1-O126052.15292781
132H2B2F_HUMAN(Q5QNW6) Histone H2B type 2-F126052.15292781
133ANXA5_HUMAN(P08758) Annexin A5320062.36724523
134H2A2C_HUMAN(Q16777) Histone H2A type 2-C129062.36724523
135H2B1L_HUMAN(Q99880) Histone H2B type 1-L126072.55385105
136HSP76_HUMAN(P17066) Heat shock 70 kDa protein 6643082.71910308
137TBA3E_HUMAN(Q6PEY2) Tubulin α-3E chain450082.71910308
138TBA3C_HUMAN(Q13748) Tubulin α-3C/D chain450092.86739459
139TBA4A_HUMAN(P68366) Tubulin α-4A chain4480143.44154556
140TBA1A_HUMAN(Q71U36) Tubulin α-1A chain4510143.44154556
141TBB8_HUMAN(Q3ZCM7) Tubulin β-8 chain4440163.61971953
142ACTC_HUMAN(P68032) Actin, α cardiac muscle 13770213.98789716
143ACTS_HUMAN(P68133) Actin, α skeletal muscle3770424.95088405

[i] The expression levels of the 143 proteins were altered more than 2-fold as a result of lumican downregulation.

Table III

Correlation between lumican expression level and identified protein expression level.

Table III

Correlation between lumican expression level and identified protein expression level.

Fold-change (Rsc)

IDAccession no. and descriptionLumican upregulationLumican downregulation
ACTC_HUMAN(P68032) Actin, α cardiac muscle 1−3.7706616943.987897158
TBB2A_HUMAN(Q13885) Tubulin β-2A chain−2.9897736121.888069587
H2B1H_HUMAN(Q93079) Histone H2B type 1-H−2.8547299161.208934009
HS904_HUMAN(Q58FG1) Putative heat shock protein HSP 90-α A4−2.7058890541.622808285
TBA4A_HUMAN(P68366) Tubulin α-4A chain−2.7058890543.441545557
H2B1O_HUMAN(P23527) Histone H2B type 1-O−1.8857880342.152927808
ANXA5_HUMAN(P08758) Annexin A5−1.5801931342.367245226
H2A1H_HUMAN(Q96KK5) Histone H2A type 1-H−1.5801931341.596149489
HNRPK_HUMAN(P61978) Heterogeneous nuclear ribonucleoprotein K−1.5801931341.596149489
K22O_HUMAN(Q01546) Keratin, type II cytoskeletal 2 oral−1.5801931341.596149489
K1C14_HUMAN(P02533) Keratin, type I cytoskeletal 14−1.5040946411.596149489
H2B1J_HUMAN(P06899) Histone H2B type 1-J−1.1924300721.208934009
MDHM_HUMAN(P40926) Malate dehydrogenase, mitochondrial−1.1924300721.208934009
NDK8_HUMAN(O60361) Putative nucleoside diphosphate kinase−1.1924300721.901196513
HIP1R_HUMAN(O75146) Huntingtin-interacting protein 1-related protein1.036399032−1.018243251
RL22_HUMAN(P35268) 60S ribosomal protein L221.036399032−1.018243251
SPSY_HUMAN(P52788) Spermine synthase1.036399032−1.018243251
RL10L_HUMAN(Q96L21) 60S ribosomal protein L10-like1.106885931−1.018243251
RL12_HUMAN(P30050) 60S ribosomal protein L121.106885931−1.018243251
RS13_HUMAN(P62277) 40S ribosomal protein S131.106885931−1.088161097
PAIRB_HUMAN(Q8NC51) Plasminogen activator inhibitor 1 RNA-binding protein1.348681223−1.114417304
IQGA1_HUMAN(P46940) Ras GTPase-activating-like protein IQGAP11.567756075−1.018243251
MT1X_HUMAN(P80297) Metallothionein-1X1.955622017−1.018243251
H2B1M_HUMAN(Q99879) Histone H2B type 1-M2.728671586−1.936350191

[i] Twenty-four proteins showed correlation between lumican expression level and their expression level.

Functional annotation of proteins whose expression level is regulated by lumican

Gene ontology (GO) analyses were performed using the identified candidate proteins for each molecular function (Fig. 3), biological process (Fig. 4) and cellular component (Fig. 5) using DAVID. We also analyzed pathway terms, but no significant category was found. Functional annotations were counted by normalizing to the total number of proteins identified. Since a multifunctional protein yields more than one annotation and some proteins are not defined by GO terms yet, the total number of classified proteins resulted in more or less than 100% (Fig. 3). Major GO molecular function categories of the identified proteins were 34.8% structural molecule activity, 17.4% structural constituents of ribosomes, and 21.7% RNA binding proteins (Fig. 3).

Discussion

In the present study, we used a gel-free LC-MS-based proteomics approach to examine the effect of lumican on cell growth and invasion. Using semi-quantitative methods based on spectral counting, we successfully identified several proteins whose expression levels were altered more than 2-fold in both lumican upregulated PANC-1 cells and lumican downregulated PANC-1 cells. A limitation of spectral counting is in its accurate quantitative capacity (35). Therefore, we selected candidate proteins whose expression level was regulated by lumican using the analysis results of two types of cells; lumican upregulated cells and lumican downregulated cells. Thus, we identified 24 proteins whose Rsc values were inversely correlated among differentially expressed proteins in lumican upregulated and downregulated cells as candidate proteins (Fig. 3).

To examine the role of these candidate proteins in the effect of lumican on cell growth and invasion, we performed a functional classification of the candidate proteins by GO analysis. Although the GO terms for molecular function, biological processes, and cellular components were examined, we focused on molecular function. The molecular functions of candidate proteins were mainly classified in the ‘structural molecule activity’ category. Since structural molecule activity proteins contribute to the formation of complexes within or outside of the cell, such candidate proteins may be related to cell invasion regulated by lumican.

Annexin A5 expression may be regulated by lumican since Annexin A5 expression was negatively correlated with the lumican expression level. Annexin A5, also known as Annexin V, is widely known as a marker of early stage apoptosis (36,37). We previously demonstrated that the PDAC cell line secretes 70-kDa glycosylated lumican and that this secreted lumican stimulates cell growth (28). Thus, the induction of cell growth by lumican may be related to an inhibition of apoptosis through Annexin A5 expression. However, previous reports suggest that lumican plays an important role in apoptosis induction (22,3840). This discrepancy may be derived from the differences between glycosylated lumican secreted from the PDAC cell line and other cells.

Furthermore, MT-1X expression levels positively correlated with the lumican expression level. MT family proteins are encoded by 10 functional isoforms (MT-1A, MT-1B, MT-1E, MT-1F, MT-1G, MT-1H, MT-1X, MT-2A, MT-3 and MT-4), and seven non-functional isoforms (MT-1C, MT-1D, MT-1I, MT-1J, MT-1K, MT-L and MT-2B). MT family proteins are involved in essential metal homeostasis, cellular free radical scavenging, cell proliferation, apoptosis, and metal detoxification. MT-1X and MT-2A transcripts were significantly upregulated under hypoxia in human prostate cancer cell lines, and siRNA-MT-2A treatment inhibited cell growth and induction of apoptosis, but an effect of MT-1X on cell growth and apoptosis was not demonstrated (41). Zinc is an abundant metal in the human prostate, and zinc inhibits cell growth and induces apoptosis in human prostate cancer cell lines (42,43). These findings suggest that MT-2A may play an important role in cell growth and apoptosis in prostate cancer through intracellular zinc homeostasis. MT-1X function in cancer cells, particularly PDAC, is not well understood. MT-1X is a known zinc-binding protein, and MT-1X mRNA expression was induced, as well as MT-2A, under hypoxic conditions (41). Thus, MT-1X may inhibit apoptosis as well as MT-2A. Furthermore, Ryu et al(44) suggested that MT-1E could enhance the migration and invasion of human glioma cells by inducing MMP-9 activation. MT-1X may have functions resembling MT-1E, since MMP-9 is a zinc-requiring enzyme, and MT-1X is classified into isoforms such as MT-1E. As mentioned above, it may be postulated that MT-1X plays an important role in cell growth and invasion by lumican. Further study is required to validate the expression levels of these candidate proteins, and to clarify the effect of candidate proteins, including MT-1X, on cell growth and invasion that are affected by lumican.

In conclusion, we identified more than 400 proteins from both lumican upregulated and lumican downregulated cells using global shotgun proteomics. A label-free semi-quantitative method based on spectral counting led to 24 candidate proteins whose expression was regulated by lumican. These candidate proteins included apoptosis-related and invasion-related proteins. Therefore, lumican may be involved in cell growth and invasion by altering the expression of these proteins.

Acknowledgements

The authors thank K. Teduka and T. Fujii for their technical assistance (Department of Pathology, Integrative Oncological Pathology). The present study was supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science to T.Y. (C, no. 24591019) and Z.N. (C, no. 23590477).

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October 2013
Volume 30 Issue 4

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Online ISSN:1791-2431

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Copy and paste a formatted citation
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Spandidos Publications style
Yamamoto T, Kudo M, Peng W and Naito Z: Analysis of protein expression regulated by lumican in PANC‑1 cells using shotgun proteomics. Oncol Rep 30: 1609-1621, 2013.
APA
Yamamoto, T., Kudo, M., Peng, W., & Naito, Z. (2013). Analysis of protein expression regulated by lumican in PANC‑1 cells using shotgun proteomics. Oncology Reports, 30, 1609-1621. https://doi.org/10.3892/or.2013.2612
MLA
Yamamoto, T., Kudo, M., Peng, W., Naito, Z."Analysis of protein expression regulated by lumican in PANC‑1 cells using shotgun proteomics". Oncology Reports 30.4 (2013): 1609-1621.
Chicago
Yamamoto, T., Kudo, M., Peng, W., Naito, Z."Analysis of protein expression regulated by lumican in PANC‑1 cells using shotgun proteomics". Oncology Reports 30, no. 4 (2013): 1609-1621. https://doi.org/10.3892/or.2013.2612