Vol.4, No.2, 80-83 (2013)Journal of Biophysical Chemistry doi:10.4236/jbpc.2013.42011
Differential protein expression between EBV-positive and negative epithelial cells
Haibo Yu1,2*, Lian Zhao3*, Qijia Yan1*, Lielian Zuo1*, Zhengyuan Yu1, Wei Xiong1, Xiaoling Li1, Shourong Sheng3, Zhaojian Gong1, Jianhong Lu1#, Guiyuan Li1#
1Cancer Research Institute, Central South University, Changsha, China;
#Corresponding Author:***************,*************
2Institute of Metabolism and Endocrinology, The Second Xiangya Hospital and the Diabetes Center, Metabolic Syndrome Research Center, Key laboratory of Diabetes Immunology, Ministry of Education, Central South University, Changsha, China
3Department of Gastroenterology, Third Xiangya Hospital, Central South University, Changsha, China
Received 27 February 2013; revised 29 March 2013; accepted 7 April 2013
Copyright © 2013 Haibo Yu et al. This is an open access article distributed under the Creative Commo
ns Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
Epstein Barr virus infection is believed to play a role in the development of nasopharyngeal car- cinoma. In order to investigate the function of EBV in epithelial cell, proteomic methods were used to find and identify the differential proteins and expected to elucidate the mechanism of EBV. Altered protein expressions were found be- tween 293 cell (HEK293) and EBV infected cell (293-EBV). In this study, we separated differen- tial expressed proteins using 2D-DIGE method while matrix-assisted laser desorption/ionization tandem time of flight mass spectrometry (MAL- DI-TOF-MS) method was used to identify pro- teins. The results showed that 14 proteins were up regulated and 3 proteins were down regu- lated in 293-EBV cells. Bioinformatic analysis showed that these proteins are involved in cell proliferation, metastasis, apoptosis, metabolism, and signal transduction. Western blotting anal- ysis was further carried out to verify the MS re- sults. Thus, EBV may exert its functions by me- diating differential expression of these pro- teins.
Keywords:EBV; Differential In-Gelelectrophoresis (DIGE); Mass Spectrometry
1. INTRODUCTION
Epstein Barr virus infection has been believed to play a key role in the development of many tumors such as nasopharyngeal carcinoma (NPC) which prevalently acc- rus in southern China and Southeast Asia. In our pre- vious study, the proliferation rate of epithelial cell was faster after transfection with EBV genome. The growth of xenografts was also enhanced after the transfected cells were injected into nude mice in vivo. The mechanism of EBV is still unclear, thus in this work powerful proteo- mic technologies were used to elucidate the potential roles of EBV. As it is known, protein is the ultimate life performer. How does EBV regulate the protein profile of epithelial cell? Our study uses the differential in-gel elec- trophoresis DIGE and MALDI-TOF-MS（Matrix-assisted laser desorption/ionization time of flight mass spectro- metry) to select and identify differential expressed pro- tein compared 293 cells with 293-EBV cells. The results are analyzed to illuminate the mechanism of EBV in pro- moting cell proliferation and differentiation in protein level, which supply a new target and clue for cure and prognostic of EBV associated cancer.
2. Materials and methods
2.1. Cell Types and Protein Preparation
293 and The EBV-infected cell, 293-EBV, were pre- served and propagated in our laboratory. They were cul- tured in DMEM medium (GIBCO) supplemented with 10% fetal bovine serum, 5% CO2 atmosphere at 37˚C. Cells were collected during the exponential growth phase. DMEM (Dulbecco’s Modified Eagle’s medium) pur- chased from Hgclone company; 2D-Clean up protein pu- rification kit and 2D-Quant Kit purchased from the Amer- sham Biosciences company. The cells were extracted with DIGE lysis buffer. After lysed for 40 min on ice, the supernatant was transferred into a new microcentrifuge tube. The concentration of protein was determined with
*These authors contributed equally to this work. #Corresponding author.
H. B. Yu et al. / Journal of Biophysical Chemistry 4 (2013) 80-83 81
Bio-Rad protein assay reagent.
2.2. Two-Dimensional Fluorescence
Difference Gel
Electrophoresis (2D-DIGE)
For two-dimensional fluorescence difference gel ele- ctrophoresis (2D-DIGE), the IPG-strip was re-hydrated with sample at 30 V for 12 hours, and then IEF was con- ducted at 500 V for 1 hour, 1000 V for 1 hour, 10,000 v for 8 h, 500 v for 10 h. After IEF, the strips were equili- brated in equilibration buff
er Afterward, the IPG strips were electrophoresed on 12.5% acrylamide gels in the second dimension with electrophoresis parameter 3 w per gel for 30 min then 16 w per gel. The gels were scanned with a Typhoon 9400 fluorescence scanner (GE Health- care). DeCyder 2D soft was used to match and select the different expressed proteins (folds > 1.8). Then, prepara- tion of gel to cut different expressed proteins and the dif- ferent expressed proteins identified by MALDI-TOF- MS/MS.
2.3. Western Blotting
Western blotting was carried out as described previ- ously. Antibody to HSP-70,  -actin was purchased from Sigma-Aldrich (St Louis, MO).
2.4. Statistical Analysis
SPSS11.5 software was adopted to do student’s t-test analysis. All data showed by X ± S. p < 0.05 means that the difference was statistically significant.
3. Results
3.1. Protein Expression Profile in 293 and
293-EBV Cells
We obtained the images of proteins from 293 cells, 293-EBV cells and from the internal standard using dif- ferent emission filters on the Typhoon 9400 fluorescence scanner. The obtained images were analyzed by DeCyder 5.0 software. We compared the protein expression in each group, and the results of the matched spots from the dif-ferent gels were analyzed by Student’s t test. We found 17 protein spots which showed consistent expression differences (fold > 1.8) between the two groups (Figure 1, Table 1). The differentially expressed proteins were selected and analyzed by the MALD I-TOF-MS/MS. The PMF are obtained and identified by FlexAnalysis 3.0 software from NCBInr data. Take the number of 1940 protein spot （Raichu404X ）as an example showed in  Fig- ures 2-4. Raichu404X was up-regulated significantly in 293-EBV cell. To validate the results of MALDI-TOF/ MS/MS, two different expressed proteins are detected by
western blot between 293 cell line and 293-EBV cell
(a)
(b)
Figure 1. Overlapping image of three different emission fil- ters. Red circles represent the differentially expressed pro- teins detected (a) separated proteins dye overlay with Cy2, Cy3, Cy5 (b) elected and labeled significant difference pro- teins.
(A)                    (B)
Figure 2. (A) Raichu404X (the spot number 1940) protein ex- pression level in the 293 cell line (control). (B) Raichu404X protein expression level in the 293-EBV cell line. As a graphic showed, the spot 1940 field within the magnified boxes (top- left) was up-regulated in 293-EBV cell line; the same result was showed in b (three-dimension graphics).
line. The different expressed Raichu404X and Hsp70 protein were validated by Western blot. The results are consistent with results of MALD-I-TOF/MS/MSAs which it is known. Raichu404X and Hsp70 proteins are closely associated with carcinogenesis. They are involved in se- veral biological f
unctions including signal transduction, Cell migration, DNA synthesis, cell proliferation, cell apoptosis and invasion of many cancers.
4. Discussion
According to DIGE analysis, 14 up-regulated proteins and 3 down-regulated proteins in the 293-EBV , the dif- ferently expressed protein spots were chosen and identi-
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Table 1. The identified differentially expressed proteins between 293 and 293-EBV cells.
Spot No.
Accession No.
Protein name Level
Function Fold p-value
574 gi|62896815
Heat shock 70 kDa protein 8
isoform 2 variant Up Cellcycle, proliferation 3.78 0.0018 598 gi|42543698 Chain A, the crystal structure of
the human Hsp70 Atpase
domain Up Cellcycle, proliferation 3.546 0.0024 701 gi|83754516
Chain B, crystal structure of the
Chip-Ubc13-Uev1a complex Up Ubiquitylation 2.83 0.0028 760 gi|4506753
RuvB-like 1
Up Proliferation    1.86 0.036 853 gi|119597640
Protein disulfide isomerase family
A, member 3, isoform CRA_a Up Metabolism 1.85 0.021 920 gi|5453603
Chaperonin containing TCP1,
subunit 2
Down
Cellcyclecytoskeletal proteins
1.89 0.00083 1248 gi|34234 Laminin-binding protein Up Invasion and metastasis    1.96
0.05
1862 gi|5668560
Serine/threonine phosphatase 1 gamma Up Cellcycle, proliferation 2.16 0.041 1878 gi|62324
Epstein-Barr nuclear antigen
[Human herpesvirus ] Up Generegulation,
extrachromosomal replication    6.51 0.00021 1926 gi|70995211
Peroxisomal enoyl-coenzyme A
hydratase-like protein Up Metabolism the fatty acid beta-oxidation pathway    2.95 0.022 1940 gi|14595132 Raichu404X [Homo sapiens] Up RAS signal pathway 22.22 8.40E-05 1979
gi|6755214
Proteasome activator subunit 3
Up
Cell cycle, proliferation
2.14
0.0013
2071 gi|6912586 6 phosphogluconolactonase Up Metabolism
2.46 0.017
2081 gi|5803013 Endoplasmic reticulum protein 29 isoform 1 precursor [Homo
sapiens
Up Stress 8.13 0.00085 2479 gi|4758504 Hydroxysteroid (17-beta)
dehydrogenase 10 isoform 1
[Homo sapiens]
Up Metabolism steroid hormones
and fats
1.86 0.045
3392 gi|3891774 Crystal structure of a human low
molecular weight
phosphotyrosyl phosphatase Down
Metabolism 2.16 0.0056 3600 gi|4584423
AKAP450 protein Down
Immunity/induce Tcell to  combine
2.17 0.014
fied as heat shock 70, Chip-Ubc13-Uev1a, RuvB-like 1, protein disulfide isomerase, TCP1, laminin-binding pro- tein, serine/threonine phosphatase, Epstein-Barr nuclear antigen, peroxisomal enoyl-coenzyme A hydratase-like protein, Raichu404X, proteasome activator subunit 3, 6- phosphogluconolactonase, endoplasmic reticulum protein 29 isoform 1 precursor, hydroxysteroid (17-beta) dehy- drogenase 10 isoform 1, Human Low Molecular Weight Phosphotyrosyl Phosphatase and AKAP450 protein. The identified proteins involved in numerous crucial intra- cellular functions such as cell cycle and apoptosis, regu- lation of signal transduction pathways, cell proliferation and so on. Since it has been suggested that Raichu404X
contains Ras interaction domain and that Cdc42Hs is a member in Ras/MAPK pathways Raichu404X is differ- ently expressed in stem cells [1]. The function of Rai- chu404X needs further investigated. Hsp70 as central components of the cellular chaperone network, are fre- quently recruited by viruses and is important factors in developmental and pathological processes such as o
nco- genesis, neurodegenerative and autoimmune diseases, vi- ral infections and aging [2-5]. Hsp70 interacts with key regulators of many signal transduction pathways control- ling cell homeostasis, proliferation, differentiation and cell death and Hsp70 are ubiquitous molecular chaperones that function in a myriad of biological processes, modulating
H. B. Yu et al. / Journal of Biophysical Chemistry 4 (2013) 80-83
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Figure 3. (a) Database searches of the masses of the tryp- sinized peptides identified the protein based on the 11 spikes from A to K. (b) Mass spectrometric characterization of R- aichu404X.
Figure 4. Westernblot results show the Rai-
chu404X and Hsp70 proteins are up-regulated
in 293-EBV cell line. (A) Control (293) (B) 29
3-EBV.
polypeptide folding, degradation and translocation across membranes, and protein–protein interactions [6-10]. Hsp70 and other members of the Hsp family have been shown to inhibit apoptosis at several different stages [7,9]. This work will provide us significant clue to clarify the hap- pen of EBV associated diseases and new molecular will be used for prognosis and drug targets of clinic.
5. ACKNOWLEDGEMENTS
This work was supported by Projects of Hunan Provincial Natural Science Foundation of China (11JJ2043), the National Natural Science Foundation of China (81171931), the Research and Development Pro- ject of Hunan Development and Reform Commission (2009-13910), Graduate Degree Thesis Innovation Foundation of Central South Uni- versity (2010ybfz102), Doctor Innovation
Research Project of Hunan Province (CX2010B040).
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