Differential Expressed Genes in ECV304 Endothelial-Like Cells Infected with Herpes Simplex Virus Type 2 ()
1. Introduction
Herpes simplex virus (HSV) belongs to the α subfamily of herpesviridae [1]. Human infections caused by the herpes simplex virus (HSV) can be categorized into two distinct types: HSV-1 and HSV-2 [2] [3]. Herpes Simplex Virus type 2 (HSV-2) is implicated not only in genital and neonatal infections but also in the pathogenesis of cervical cancer and atherosclerosis (AS) [4] [5]. Furthermore, HSV-2 infection exhibits a synergistic relationship with Human Immunodeficiency Virus (HIV) infection [6]. Prior research has demonstrated the presence of HSV antigens and their corresponding DNA sequences at sites of lesions in AS, specifically within vascular smooth muscle cells (SMCs) and endothelial cells (ECs) [7]. The prevalence of positive serum HSV antibodies is markedly elevated in patients with AS. This evidence suggests that HSV infection may play a significant role in the initiation and progression of AS. The detection of HSV DNA within the vascular wall of AS further indicates a potential association between HSV infection and the development of atherosclerosis.
To date, the detailed pathological mechanisms of HSV involvement in AS, in particular the relationship of HSV with EC and SMC, the major cells of the vessel wall, in vitro is unclear. The effect of the virus on target cells in vitro could reflect the direct effect of the virus on cells in vivo. Studying the biological effects of HSV infection on vascular endothelial cells in vitro is important to elucidate the mechanisms of HSV induction.
DNA expression profiling microarrays are designed to monitor the presence or expression of various genes in samples [8] [9]. Thus, by analyzing these data, we can determine whether the tested specimen is infected with the pathogen and the extent of the infection. Microarray technology has also been used to study the effects of other viruses on ECV304 endothelial cells [10] [11]. Due to the expensive equipment and the complexity of the experiments, there are no reports on the effect of HSV on the gene expression profile of endothelial cells.
In this study, our aim is to identify the differentially expressed genes in endothelial cells induced by HSV-2, and to explore the genetic mechanisms by which HSV-2 causes vascular endothelial injury, dysfunction, and atherosclerosis. We used a high-density microarray containing 18,000 genes to compare the gene expression profiles of HSV-2-treated ECV-304. This allowed us to determine the differential expression of HSV-2-induced endothelial cell genes and to explore the genetic mechanisms by which HSV-2 causes vascular endothelial injury and dysfunction and AS.
2. Materials and Methods
2.1. Cell Culture and Virus Infection
ECV304 cells were preserved and provided by the laboratory of the Department of Nephrology, Southern Hospital. Herpes simplex virus type II (HSV-2) strain was obtained from the General Hospital of Guangzhou Military Region of the Chinese People’s Liberation Army. The HSV-2 strain was inoculated into Vero cells, and the virus was collected after 6 days and the titer was measured (50% tissue cell infection, TCID50 of 105.5/mL). ECV304 cells were maintained in a maintenance medium (MEM, Gibco, US) supplemented with 10% fetal bovine serum, glutamine, sodium bicarbonate, penicillin, and streptomycin in a 37˚C, 5% CO2 humidified incubator. Controls were added directly to the maintenance culture under the same conditions. Cultured cells and their controls were sampled at 2, 4 and 6 days after virus inoculation. Sample three times continuously within thirty minutes for subsequent experiments.
2.2. DNA Extraction and PCR
The LAT gene was amplified by PCR using the primer set: (forward: 5’-GTCAACACGGACACACTCTTTT-3’) and (reverse: 5’-CGAGGCCTGTTGGTCTTTATC-3’), which could be used to produce a 150-bp fragment. DNA was extracted from cells of the control group and infected groups at 12 h and 24 h post-infection using a DNA extraction kit for PCR detection of the HSV LAT gene. The total volume of the amplification reaction system was 50 µL, including 5 µL of DNA sample, 25 µL of 2 × Premix PCR premix buffer, 20 μmol/L of upstream and downstream primers of 1 µL each, and 18 µL of ddH2O. Cycling conditions were as follows: pre-denaturation of 5 min at 94˚C, main cycle (1 min at 94˚C, 30 s at 65˚C, 1 min at 72˚C) for 30 cycles, and extension of 5 min at 72˚C. The amplification product was identified by 1.5% agarose gels (TaKaRa company, China).
2.3. Detection of HSV Infection by Indirect Immunofluorescence
HSV-2 pp65 protein expression was detected by immunofluorescence assay. ECV304 cell crawl sheets were prepared and inoculated with virus for 24 h. Infected and uninfected cells were fixed at room temperature with a fixed solution containing 2% paraformaldehyde and 0.1% Triton X-100 for 30 min, washed twice with PBS, blocked with normal goat serum for 20 min, and washed three times with PBS. Cell crawls were incubated with mouse anti-HSV-2 pp65 (US Biological, USA) for 30 min at 37˚C, and washed five times with PBS. The secondary antibody conjugated with FITC was subsequently added and incubated for 30 min at 37˚C, washed three times with PBS and observed under a fluorescence microscope (Nikon TE-2000, US).
2.4. Microarray Hybridization and Image Analysis
Total RNA was extracted using the Trizol extraction kit (Life Technologies INC.) and the concentration and total amount of RNA was quantified by UV spectrophotometry at 260 and 280 nm. RNA samples with an A260/A280 ratio between 1.8 and 2.0 were selected. Total RNA from uninfected and infected ECV304 cells was reverse transcribed using the RNA Fluorescent Linear Amplification Kit (Agilent) and labeled with Cy5-dCTP and Cy3-dCTP, respectively. Cy5-labeled and Cy3-labeled targets were purified using Rneasy Mini Spin Columns (Qiagen) and then mixed and hybridized to Oligo Microarray Kit (Qiagen). The hybridization volume was 400 µL and consisted of 0.75 µg of each Cy3-labeled and Cy5-labeled linearly amplified cRNA, 50µL of 10 × control target and 225 µL of 2 × hybridization buffer. The mixture was vibrated and centrifuged, pipetted onto a cover glass, and covered with Agilent human 1B oligonucleotide chip. The hybridization box was hybridized at 60˚C and 4 rpm for 16 h, washed twice in 6 × SSC and 0.005% Triton X-102 solution at room temperature for 10 min each time, and then wash it in cold solution (0.1 × SSC, 0.005% Triton X-102) for 5 min. The slides were dried with nitrogen and stored in dark. The chip is scanned in the Agilent 2565BA gene chip scanner (Agilent, Palo Alto, CA, USA). The default parameters of the scanner are used for parameter setting. The scanned data are analyzed and homogenized by Feature extraction software. According to Cy3 (g processed signal) and Cy5 (r processed signal) Log ratio P-Value, P < 0.01 indicated a significant difference in gene expression between the virus-infected group and the control group. G processed signal < r processed signal was defined as up-regulation of gene expression, and g processed signal > r processed signal was defined as down-regulation of gene expression.
2.5. Validation of Microarray Results by Real-Time qPCR
Total RNA was extracted from ECV304 cells infected with HSV-2 and the control group for 6 h. Prepare the RT reaction solution (on ice) as per the reverse transcription kit. The reverse transcription was carried out under the following conditions: 10 min at 42˚C, and 2 min at 95˚C. The chip was verified by real-time qPCR, and a Rotor Gene 3000 Fluorescence quantitative detection system was detected by the SYBR RT-PCR kit. The genes to be tested and the sequence of GAPDH primers are shown in Table 1. The final volume of the 20 μL reaction mixture consisted of PCR mixture, diluted cDNA, and specific primers for two up-regulated genes and two down-regulated genes selected from the differentially expressed genes. The PCR reaction was carried out under the following conditions: an initial denaturation of 2 min at 95˚C; 30 cycles, each cycle consisting of 2 min at 95˚C, 94˚C, 1 min at 54˚C, and 1 min at 72˚C. At the end of the procedure, the specificity of the primer group was confirmed by the analysis of the neutrophilic curve. RT-PCR results were compared at different dilutions (100, 10−1, 10−2, 10−3, 10−4, 10−5 copies/reaction) to estimate the copy number of the target gene and GAPDH mRNA. Taking the housekeeping gene GAPDH as the internal standard (IS), the quantitative results of GAPDH in the HSV-infection Group and the Control Group were calculated through the standard curve of the housekeeping gene, and the error of RNA quantity (relative quantity) was calculated. Each target gene was quantified by its standard curve, and the errors of the two groups of housekeeping genes were corrected. The corrected quantitative results of the target gene were compared to obtain the relative ratio of the two.
Table 1. Specific primers used in qRT-PCR analysis.
Gene name |
Forward primer (5’ - 3’) |
Downstream primers (5’ - 3’) |
GAPDH |
GCACCGTCA AGGCTGAGA AC |
ATGGTGGTGAAGACGCCAGT |
SP100 |
AAAGTTGAGTGCCAAGCCCAAG |
TCTAAGGGCTCATCAACGTCAGTG |
RPS24 |
GACAACTGGCTTTGGCATGATTTA |
CCA ACA TTGGCCTTTGCAGTC |
HNRPA1 |
AGGCTGGCAGATACG TTCGTC |
CCTCAGGCTCTCATCAGTTGTTTC |
RTP801 |
GCAGGACGCACTTGTCTTAGCA |
CCA AAGGCTAGGCATGGTGAG |
2.6. Statistical Processing
Statistical analysis was performed using SPSS10.0. One-Way ANOVA test was used to compare group means and differences between multiple groups. A P-value < 0.05 was considered significant.
3. Results
3.1. LAT Gene Testing
The HSV LAT gene is the first gene expressed after the virus invades the host cell. Since the presence of viral mRNA in the host cell is an important indicator of viral replication, the detection of viral LAT mRNA expression indicates active HSV infection. In the present study, PCR amplification of HSV-2 LAT gene after infection of cells with virus and viral supernatant was consistent with the expected results, indicating the possibility of direct virus infection to cells (Figure 1).
M: DNA Marker; 1, 4: Control; 2, 3: 12 h Post-infection; 5, 6: 24 h Post-infection.
Figure 1. Electrophoreosis of PCR products of HSV-2 LAT gene.
3.2. Detection of LAT Protein
Two days after viral infection, most cells showed a large fluorescent signal around the nucleus (Figure 2(A)). The control group had a homogeneous cell background with no strong fluorescent signal (Figure 2(B)). This finding suggests that HSV-2 can infect ECV304 cells and proliferate in their cytoplasm.
Figure 2. Immunofluorescence was used to detect the expression of the LAT gene in ECV304 cells 2 days after ECV304 infection (200×). (A) Control group; (B) ECV304 cells (green fluorescence showed positive LAT protein in infected cells).
3.3. Identifying the Quality of RNA
The RNA quantification results are as follows: The RNA of the control group is 1149 ng/µL, with an A260/A280 ratio of 1.8. The RNA of the HSV-2 infection group is also 1149 ng/µL, with an A260/A280 ratio of 1.81. The samples were subjected to agarose gel electrophoresis to identify the quality of RNA, and electrophoresis showed three clear RNA bands with visible 28S, 18S and 5S, and the samples were of high purity and integrity, in accordance with the DNA microarray requirements (Figure 3). Qualified samples were stored at −80˚C.
M: Marker; 1: Control; 2: HSV-2 infection.
Figure 3. Electrophoresis result of total RNA sample.
3.4. Quality Control of Microarray Hybridization
In the Agilent 2565BA gene microarray scanner, the microarrays were scanned with the default parameters of the scanner, and the scanned data were analyzed and homogenized using the feature extraction software. The scanned results of the hybridized gene expression profiling microarrays were in accordance with the standard, with high signal intensity and uniform background (Figure 4).
Figure 4. Microarray hybridization scanning picture.
3.5. Scatter Plots of Hybridizing Signal
Figure 5. Scatter plot of Cy3/Cy5 hybridization intensity in the microarray. The fluorescence intensity of the chip was analyzed by scattergraph. Taking process signal (Cy3) as ordinate and process signal (Cy5) as abscissa, a scatter plot of fluorescence intensity of all points was drawn.
Gene Chip scatter plot analysis elucidates the intensity of fluorescent hybridization signals. As depicted in Figure 5, the horizontal axis represents Cy3 fluorescence intensity values, while the vertical axis represents Cy5 fluorescence intensity values. Each data point on this plot signifies the hybridization signal emanating from a specific gene locus on the microarray. The color of these data points conveys information about differential expression: yellow indicates no differential expression, whereas blue or red hues signify differential expression. The ECV-304 cells, subject to experimental (Cy3) and control (Cy5) conditions, exhibit a two-color fluorescent marker overlay. When these two fluorescent signals overlap at a single point, the resultant color provides insights into gene expression trends. Specifically, a stronger Cy3 signal renders the point green, suggesting an up-regulation trend. Conversely, a stronger Cy5 signal results in a red point, indicating a down-regulation trend. When the intensities of both signals are comparable, the point appears yellow.
3.6. Differential Expressed Genes in HSV-2 Infected ECV304 Cells
When the fluorescence signal reaches a certain intensity, data points with ratios greater than 1.37 or less than 0.7 are selected. Data points with signal intensities greater than 5 × 108 were considered valid data points, and data points with ratios greater than 1.37 or less than 0.7 were considered to have significant expression changes. Based on the selection criteria, 17,575 valid data points were extracted from the microarray results, and a total of 462 differentially expressed genes were extracted. 318 genes were up-regulated (ratio more than 1.37); 144 genes were down-regulated (ratio less than 0.7) (Table 2). Positive values indicate up-regulation, while negative values describe down-regulation.
Table 2. Differential display of genes expressed in ECV 304 infected with HSV-2.
Gene Symbol |
Ratio (g/r) |
Gene Symbol |
Ratio (g/r) |
Gene Symbol |
Ratio (g/r) |
IL9 |
111.7176 |
PSMA7 |
4.3616 |
RPS5 |
3.1848 |
NAG-7 |
36.2037 |
RPS23 |
4.3444 |
HOXA3 |
3.1625 |
ATP6V0A2 |
30.6709 |
UQCRH |
4.2248 |
PTMA |
3.1382 |
I_1100038 |
8.7079 |
HSPC016 |
4.1982 |
VIM |
3.0837 |
RPLP1 |
8.2655 |
RPL34 |
4.1579 |
RPL7A |
3.0814 |
RPS10 |
7.8284 |
I_1000200 |
4.1499 |
LAMR1 |
3.0735 |
MT2A |
7.4679 |
MT1E |
4.0840 |
RPL7 |
3.0290 |
I_1109418 |
7.0383 |
RPL10A |
4.0734 |
RPL13A |
3.0256 |
FTH1 |
6.9516 |
ZFPM1 |
4.0569 |
APP |
2.9978 |
RPS20 |
6.7500 |
RPL35 |
3.9349 |
RPS24 |
2.9796 |
RPL36A |
6.6287 |
RPS11 |
3.9325 |
LU |
2.9455 |
RPL19 |
6.4475 |
RPS14 |
3.9083 |
PABPC1 |
2.9446 |
RPL38 |
6.2619 |
RPS25 |
3.8745 |
IGFBP7 |
2.9329 |
RPL23A |
6.1302 |
RPL8 |
3.8569 |
ANAPC11 |
2.8797 |
RPL26 |
6.0291 |
MT1B |
3.8338 |
BTF3 |
2.8327 |
RPS27A |
5.9390 |
UBC |
3.7614 |
NSEP1 |
2.8307 |
RPS29 |
5.8372 |
RPS17 |
3.7474 |
RPL11 |
2.8280 |
S100A6 |
5.6553 |
EEF2 |
3.7165 |
RPS8 |
2.7911 |
RPS18 |
5.6445 |
MIF |
3.6554 |
RPLP0 |
2.7563 |
RPL27 |
5.5780 |
NQO1 |
3.6068 |
I_962007 |
2.7405 |
EEF1B2 |
5.5078 |
OAZ1 |
3.5915 |
I_1847600.FL1 |
2.7162 |
RPL31 |
5.4858 |
MT1H |
3.4805 |
I_958592 |
2.7154 |
RPL37A |
5.4226 |
RPS15A |
3.4709 |
GPX1 |
2.7026 |
SUI1 |
5.4220 |
RPL39 |
3.4672 |
MT1A |
2.6876 |
RPL26 |
5.3642 |
LRRN1 |
3.4660 |
HIST1H4C |
2.6689 |
I_1002391 |
5.3147 |
CALM2 |
3.4604 |
RPL30 |
2.6677 |
CAM-KIIN |
5.3061 |
RPL24 |
3.4533 |
PTMS |
2.6490 |
I_963838 |
5.2654 |
RPS28 |
3.4451 |
I_966336 |
2.6435 |
RPL12 |
5.2032 |
MAP3K10 |
3.4417 |
I_1109768 |
2.6310 |
RPL9 |
5.0539 |
RPS6 |
3.3936 |
KIAA0616 |
2.6284 |
LOC51142 |
5.0194 |
NME2 |
3.3849 |
NM_000983.2 |
2.6241 |
RPS21 |
5.0185 |
RPL41 |
3.3441 |
FLJ22184 |
2.6229 |
ATP5E |
4.9273 |
NM_139020.1 |
3.3386 |
HES7 |
2.6205 |
RPS12 |
4.9041 |
TUBA6 |
3.3351 |
EIF3S3 |
2.6144 |
RPL37 |
4.8491 |
RPS16 |
3.3208 |
NM_178438.1 |
2.6101 |
C21orf6 |
4.7727 |
MT1K |
3.3205 |
GAPD |
2.6071 |
RPS27 |
4.7202 |
RPL27A |
3.2840 |
GLTSCR2 |
2.6041 |
RPL35A |
4.6320 |
I_963575 |
3.2436 |
MTCO2 |
2.6002 |
NM_006082.1 |
4.4970 |
RPL17 |
3.2110 |
VGF |
2.5959 |
RPS13 |
4.4756 |
RPL23 |
3.1938 |
NM_178430.1 |
2.5736 |
RPS2 |
2.5501 |
HSPA5 |
2.1935 |
DTYMK |
1.9531 |
PP2447 |
2.5492 |
EBNA1BP2 |
2.1859 |
TMSB10 |
1.9490 |
H2AFZ |
2.5349 |
NM_178511.1 |
2.1853 |
COX6A1 |
1.9465 |
RPS15 |
2.5000 |
NM_000398.3 |
2.1832 |
I_1201840 |
1.9410 |
MT1J |
2.4955 |
EEF1D |
2.1763 |
S100A10 |
1.9405 |
RPS3A |
2.4749 |
I_1000395 |
2.1550 |
TUBA1 |
1.9351 |
ID1 |
2.4594 |
MTND1 |
2.1545 |
PROL2 |
1.9144 |
GRIN2D |
2.4444 |
NDUFC2 |
2.1482 |
MRPS24 |
1.8799 |
HCN2 |
2.4259 |
I_961758 |
2.1462 |
ATP5G3 |
1.8573 |
FAU |
2.4240 |
MGC14353 |
2.1321 |
NM_003017.2 |
1.8535 |
HES1 |
2.4212 |
FBL |
2.1311 |
H3F3B |
1.8280 |
NDUFB2 |
2.4136 |
DUX4 |
2.1310 |
DIA1 |
1.8203 |
NM_001743.3 |
2.4102 |
NM_005251.1 |
2.1301 |
PRO0478 |
1.8193 |
PARD6G |
2.3801 |
DKFZp434N0650 |
2.1238 |
LOC51219 |
1.8170 |
NDUFS5 |
2.3533 |
LSAMP |
2.1204 |
GNG11 |
1.8153 |
RPL21 |
2.3337 |
RPL32 |
2.1155 |
EGR1 |
1.8146 |
CKS2 |
2.3328 |
COX7C |
2.1142 |
HSPA1A |
1.8122 |
SFRS9 |
2.3299 |
I_1152056 |
2.1133 |
NM_145293.1 |
1.8055 |
I_959447 |
2.3260 |
TPT1 |
2.1102 |
SSR2 |
1.8048 |
MT1X |
2.3242 |
SNK |
2.1065 |
H2AV |
1.7792 |
DKFZp434N074 |
2.3097 |
MTND3 |
2.1002 |
LDHB |
1.7750 |
FLJ14464 |
2.3068 |
SP100 |
2.0971 |
I_1000283 |
1.7707 |
STMN1 |
2.3054 |
ATP5O |
2.0886 |
RPL14 |
1.7664 |
DBI |
2.3020 |
COX7A2 |
2.0827 |
DAP |
1.7645 |
HSPA8 |
2.2911 |
I_931617 |
2.0797 |
I_964413 |
1.7492 |
DRD4 |
2.2910 |
COX8 |
2.0775 |
FTL |
1.7473 |
NM_152350.1 |
2.2761 |
SLC25A5 |
2.0500 |
CCT5 |
1.7348 |
HSPE1 |
2.2726 |
MT2A |
2.0420 |
THOC4 |
1.7294 |
HINT1 |
2.2725 |
KRT18 |
2.0291 |
RoXaN |
1.7275 |
EEF1A1 |
2.2678 |
NCL |
2.0277 |
HMGB1 |
1.7216 |
CASKIN1 |
2.2615 |
ARF1 |
2.0275 |
DKFZp762E1312 |
1.7183 |
H2AFX |
2.2557 |
CKLFSF3 |
2.0187 |
NOLA2 |
1.7032 |
POLR2L |
2.2444 |
NNMT |
2.0158 |
TXN |
1.7009 |
GSTP1 |
2.2436 |
RAB34 |
2.0139 |
ATP5L |
1.6978 |
LGALS1 |
2.2373 |
TK1 |
1.9980 |
I_1000105 |
1.6892 |
LBX1 |
2.2227 |
RPL4 |
1.9735 |
ZFP36L2 |
1.6889 |
VHL |
2.2149 |
I_960618 |
1.9724 |
I_931932 |
1.6873 |
RPS4X |
2.2147 |
HIST1H4L |
1.9623 |
NM_003358.1 |
1.6863 |
CCT4 |
2.1969 |
SNRPG |
1.9589 |
NXT1 |
1.6851 |
SEPW1 |
1.6782 |
NM_173609.1 |
1.5329 |
RBM8A |
0.7166 |
TPSG1 |
1.6712 |
I_964798 |
1.5291 |
SNRPD1 |
0.7151 |
SLC25A3 |
1.6579 |
PCNA |
1.5230 |
WBSCR1 |
0.7001 |
FLJ20308 |
1.6578 |
PCBP1 |
1.5212 |
APPBP1 |
0.6981 |
POLR2A |
1.6538 |
CMT2 |
1.5142 |
PSMB6 |
0.6954 |
HNRPM |
1.6532 |
YWHAB |
1.5060 |
PYCR1 |
0.6948 |
RPL10 |
1.6495 |
TNFRSF1A |
1.5048 |
HSPCB |
0.6936 |
PLP2 |
1.6392 |
NDUFB4 |
1.5034 |
ANXA2 |
0.6895 |
TEBP |
1.6368 |
EIF3S9 |
1.4982 |
DNAJC9 |
0.6855 |
NM_003769.1 |
1.6275 |
C20orf24 |
1.4963 |
CCT3 |
0.6848 |
MT1H |
1.6255 |
MRPS12 |
1.4917 |
GARS |
0.6845 |
UNRIP |
1.6048 |
NM_022833.1 |
1.4869 |
PDHA1 |
0.6839 |
RPL36AL |
1.6005 |
SERF2 |
1.4865 |
NDUFS6 |
0.6838 |
OCSP |
1.5976 |
SLC21A12 |
1.4849 |
SF3B1 |
0.6758 |
QP-C |
1.5974 |
I_957363 |
1.4813 |
I_1110347 |
0.6753 |
I_932488 |
1.5964 |
LOC51685 |
1.4707 |
I_1109809 |
0.6738 |
UBL1 |
1.5957 |
NACA |
1.4686 |
MCM3 |
0.6704 |
U2AF1 |
1.5939 |
DNCL1 |
1.4640 |
ITGAM |
0.6673 |
TGFA |
1.5907 |
MORF4L2 |
1.4540 |
MTHFD2 |
0.6672 |
SEC61B |
1.5839 |
I_951081 |
1.4475 |
FLJ11323 |
0.6591 |
RPL15 |
1.5826 |
LAMP1 |
1.4449 |
LMNA |
0.6591 |
HIST2H2AA |
1.5816 |
BLCAP |
1.4415 |
HSPA9B |
0.6588 |
SF1 |
1.5815 |
UQCR |
1.4393 |
MGC4308 |
0.6568 |
NME1 |
1.5814 |
NM_178352.1 |
1.4379 |
PPIA |
0.6507 |
CDC42 |
1.5759 |
AKAP2 |
1.4374 |
NEUGRIN |
0.6498 |
MGC10974 |
1.5756 |
RPL5 |
1.4328 |
PSA |
0.6488 |
SFPQ |
1.5729 |
MGAT1 |
1.4283 |
NASP |
0.6393 |
FHL2 |
1.5616 |
PSME2 |
1.4211 |
CD59 |
0.6383 |
NDUFS3 |
1.5587 |
HIST3H3 |
1.4201 |
FLJ23209 |
0.6341 |
RPS3 |
1.5585 |
I_1152035 |
1.4183 |
RRM1 |
0.6339 |
HIS1 |
1.5579 |
NM_138425.1 |
1.4156 |
PSMC1 |
0.6310 |
EEF1G |
1.5558 |
I_1000097 |
1.4115 |
RARS |
0.6260 |
PC4 |
1.5514 |
PTTG1 |
1.4052 |
GSTTLp28 |
0.6246 |
NEDD8 |
1.5479 |
PABPC3 |
1.4025 |
SFRS7 |
0.6226 |
PCBP2 |
1.5460 |
I_932347 |
1.3941 |
DKFZp566H0824 |
0.6220 |
PSMB1 |
1.5439 |
CCT2 |
1.3919 |
RHO |
0.6208 |
SOD1 |
1.5437 |
LENG5 |
1.3876 |
SNRPA1 |
0.6155 |
I_930805 |
1.5384 |
EIF2S2 |
1.3812 |
STMN4 |
0.6154 |
ZFP36L1 |
1.5363 |
CBS |
1.3766 |
|
|
LOC147700 |
0.6118 |
PFDN2 |
0.5141 |
YEA |
0.0009 |
I_960911 |
0.6109 |
EIF3S2 |
0.5094 |
BAIAP1 |
0.0008 |
PCK2 |
0.6105 |
I_960077 |
0.4929 |
HSD3B7 |
0.0008 |
PAICS |
0.6084 |
DDB1 |
0.4832 |
ALDOC |
0.0008 |
HNRPDL |
0.6054 |
I_1000514 |
0.4793 |
I_964921.FL2 |
0.0008 |
FLJ90165 |
0.6008 |
I_1110080 |
0.4735 |
I_1000255 |
0.0008 |
UBCE7IP5 |
0.5999 |
EIF4A1 |
0.4678 |
GML |
0.0008 |
I_931957 |
0.5981 |
I_1000329 |
0.4639 |
WBP4 |
0.0008 |
MRPL22 |
0.5980 |
HUMAUANTIG |
0.4554 |
DNASE2 |
0.0008 |
I_1000009 |
0.5951 |
ANXA1 |
0.4480 |
PPIL4 |
0.0008 |
GP2 |
0.5926 |
ACTB |
0.4374 |
NEU4 |
0.0008 |
RBM3 |
0.5903 |
TPM1 |
0.4280 |
TRNT1 |
0.0008 |
I_1151867 |
0.5899 |
C20orf97 |
0.4213 |
MTERF |
0.0008 |
PSMB7 |
0.5887 |
CLIC1 |
0.3868 |
MFNG |
0.0007 |
CEBPB |
0.5884 |
NM_173624.1 |
0.3830 |
MGC21738 |
0.0007 |
I_928538 |
0.5884 |
HSPCA |
0.3799 |
GGCX |
0.0007 |
I_965066 |
0.5844 |
HNRPA1 |
0.3795 |
ZNF333 |
0.0007 |
I_1109622 |
0.5842 |
RTP801 |
0.3263 |
FLJ20333 |
0.0007 |
FLJ20700 |
0.5794 |
CELSR1 |
0.0017 |
ZNF215 |
0.0007 |
ARHGEF15 |
0.5783 |
FLJ20695 |
0.0014 |
CACH-1 |
0.0007 |
FLJ10097 |
0.5759 |
SORCS3 |
0.0013 |
G22P1 |
0.0007 |
FLJ21174 |
0.5743 |
SPP1 |
0.0012 |
ACAS2L |
0.0007 |
CENPF |
0.5694 |
PIGO |
0.0011 |
FUT5 |
0.0007 |
I_962171 |
0.5617 |
I_1110369 |
0.0011 |
I_1000094 |
0.0006 |
SSRP1 |
0.5593 |
PROSC |
0.0011 |
LOC51185 |
0.0006 |
I_962014 |
0.5588 |
I_961649 |
0.0010 |
NM_144963.1 |
0.0006 |
RPL6 |
0.5583 |
eQC |
0.0010 |
BRUNOL6 |
0.0006 |
IL30 |
0.5571 |
MCCC2 |
0.0010 |
NM_138350.1 |
0.0006 |
I_965611 |
0.5539 |
I_966078 |
0.0009 |
C22orf2 |
0.0006 |
HEC |
0.5538 |
I_963210 |
0.0009 |
ZNF267 |
0.0006 |
THOC3 |
0.5445 |
I_934625 |
0.0009 |
PIG3 |
0.0005 |
NM_006088.2 |
0.5425 |
DBR1 |
0.0009 |
SPP1 |
0.0005 |
RA410 |
0.5376 |
SCGB1A1 |
0.0009 |
NM_145300.1 |
0.0005 |
HMGN2 |
0.5271 |
MLANA |
0.0009 |
I_931899 |
0.0003 |
DNAJA1 |
0.5266 |
ANKRA2 |
0.0009 |
|
|
CYCS |
0.5248 |
NM_173501.1 |
0.0009 |
|
|
3.7. Verification of Gene Chip Results
Table 3. Relative quantitation results of real time RT-PCR.
|
IS (GAPDH) |
Target Gene |
Relative |
Quantitative Results (copies) |
Relative (X) |
Quantitative Results (Y) (copies) |
Quantitative results
after correction (Y/X) |
SP100 gene |
Control |
12,698,991 |
1 |
8,212,159 |
8212159.0 |
1 |
Infect |
6,223,228 |
0.490 |
8,800,188 |
17957484.0 |
2.187 |
RPS24 gene |
Control |
12,698,991 |
1 |
12,835,309 |
12835309.0 |
1 |
Infect |
6,223,228 |
0.490 |
10,901,163 |
22244690.7 |
1.733 |
RTP801 gene |
Control |
12,698,991 |
1 |
479358511 |
479358511.0 |
1 |
Infect |
6,223,228 |
0.490 |
49557264 |
101125541.9 |
0.211 |
HNRPA1 gene |
Control |
12,698,991 |
1 |
30478756 |
30478756.0 |
1 |
Infect |
6,223,228 |
0.490 |
24397428 |
49784894.2 |
1.633 |
Table 4. Compared the Results of Real Time RT-PCR and Microarray.
|
Real Time RT-PCR Quantitative Results |
Gene chip test results |
SP100 gene |
2.187 |
2.097 |
RPS24 gene |
1.733 |
2.980 |
RTP801 gene |
0.211 |
0.326 |
HNRPA1 gene |
1.633 |
0.380 |
Four genes were selected and validated by real-time quantitative PCR using double-stranded DNA combined with SYBR Green I. The quantitative results were generally consistent with the gene microarray detection results, which illustrated the reliability of the human whole-genome oligonucleotide expression profiling microarray in screening differentially expressed genes (Table 3 & Table 4).
3.8. Analysis of Differential Genes Involved in Biological Processes
The differential genes were uploaded to the Internet bioinformatics analysis professional website to analyze the biological processes involved in the differential genes (https://panther.appliedbiosystems.com/), and it was found that 462 differential genes were involved in a total of 237 biological processes, 79 of which were significantly different (P < 0.05), as shown in Table 5. The results showed that 73 genes related to protein synthesis, such as RPS, RPL, EEF and SUI1, were generally up-regulated 6 h after HSV-2 infection of ECV304 cells. HSPA8, HSPE1, CCT2, CCT4 and CCT5 genes related to protein elongation and folding were up-regulated, and 9 genes such as HSPCB, HSPCA, CCT3 and PPIA were down-regulated; 12 genes related to cell signaling such as CASK, APP and HINT1 were up-regulated, and 5 genes such as RHO and CELSR1 were down-regulated.
Table 5. Analysis of gene expression profiles in ECV 304 infected with HSV-2.
№ |
Biological process |
Unigene ID |
Entrez Gene |
Symbol |
chip Ratio |
1 |
protein biosynthesis |
356502 |
6176 |
RPLP1 |
8.2655 |
406620 |
6204 |
RPS10 |
7.8284 |
8102 |
6224 |
RPS20 |
6.7500 |
432485 |
6173 |
RPL36A |
6.6287 |
381061 |
6143 |
RPL19 |
6.4475 |
380953 |
6169 |
RPL38 |
6.2619 |
419463 |
6147 |
RPL23A |
6.1302 |
482144 |
6154 |
RPL26 |
6.0291 |
311640 |
6233 |
RPS27A |
5.9390 |
156367 |
6235 |
RPS29 |
5.8372 |
546290 |
6222 |
RPS18 |
5.6445 |
514196 |
6155 |
RPL27 |
5.5780 |
421608 |
1933 |
EEF1B2 |
5.5078 |
523670 |
6160 |
RPL31 |
5.4858 |
433701 |
6168 |
RPL37A |
5.4226 |
150580 |
10209 |
SUI1 |
5.4220 |
408054 |
6136 |
RPL12 |
5.2032 |
412370 |
6133 |
RPL9 |
5.0539 |
190968 |
6227 |
RPS21 |
5.0185 |
546289 |
6206 |
RPS12 |
4.9041 |
80545 |
6167 |
RPL37 |
4.8491 |
546291 |
6232 |
RPS27 |
4.7202 |
182825 |
11224 |
RPL35 |
4.6320 |
529631 |
6165 |
RPL35A |
4.6320 |
446588 |
6207 |
RPS13 |
4.4756 |
386384 |
6228 |
RPS23 |
4.3444 |
438227 |
6164 |
RPL34 |
4.1579 |
148340 |
4736 |
RPL10A |
4.0734 |
433529 |
6205 |
RPS11 |
3.9325 |
381126 |
6208 |
RPS14 |
3.9083 |
178551 |
6132 |
RPL8 |
3.8569 |
433427 |
6218 |
RPS17 |
3.7474 |
515070 |
1938 |
EEF2 |
3.7165 |
370504 |
6210 |
RPS15A |
3.4709 |
546284 |
6170 |
RPL39 |
3.4672 |
547172 |
6152 |
RPL24 |
3.4533 |
153177 |
6234 |
RPS28 |
3.4451 |
408073 |
6194 |
RPS6 |
3.3936 |
381172 |
6171 |
RPL41 |
3.3441 |
397609 |
6217 |
RPS16 |
3.3208 |
523463 |
6157 |
RPL27A |
3.2840 |
374588 |
6139 |
RPL17 |
3.2110 |
406300 |
9349 |
RPL23 |
3.1938 |
378103 |
6193 |
RPS5 |
3.1848 |
499839 |
6130 |
RPL7A |
3.0814 |
421257 |
6129 |
RPL7 |
3.0290 |
546356 |
23521 |
RPL13A |
3.0256 |
356794 |
6229 |
RPS24 |
2.9796 |
388664 |
6135 |
RPL11 |
2.8280 |
546285 |
6175 |
RPLP0 |
2.7563 |
400295 |
6156 |
RPL30 |
2.6677 |
492599 |
8667 |
EIF3S3 |
2.6144 |
356366 |
6187 |
RPS2 |
2.5501 |
406683 |
6209 |
RPS15 |
2.5000 |
356572 |
6189 |
RPS3A |
2.4749 |
387208 |
2197 |
FAU |
2.4240 |
381123 |
6144 |
RPL21 |
2.3337 |
520703 |
1915 |
EEF1A1 |
2.2678 |
446628 |
6191 |
RPS4X |
2.2147 |
333388 |
1936 |
EEF1D |
2.1763 |
265174 |
6161 |
RPL32 |
2.1155 |
186350 |
6124 |
RPL4 |
1.9735 |
446522 |
9045 |
RPL14 |
1.7664 |
401929 |
6134 |
RPL10 |
1.6495 |
444749 |
6166 |
RPL36AL |
1.6005 |
381219 |
6138 |
RPL15 |
1.5826 |
546286 |
6188 |
RPS3 |
1.5585 |
144835 |
1937 |
EEF1G |
1.5558 |
371001 |
8662 |
EIF3S9 |
1.4982 |
411125 |
6183 |
MRPS12 |
1.4917 |
505735 |
4666 |
NACA |
1.4686 |
532359 |
6125 |
RPL5 |
1.4328 |
429180 |
8894 |
EIF2S2 |
1.3812 |
520943 |
7458 |
WBSCR1 |
0.7001 |
404321 |
2617 |
GARS |
0.6845 |
506215 |
5917 |
RARS |
0.6260 |
483924 |
29093 |
MRPL22 |
0.5980 |
546283 |
6128 |
RPL6 |
0.5583 |
530096 |
8668 |
EIF3S2 |
0.5094 |
129673 |
1973 |
EIF4A1 |
0.4678 |
2 |
signal transduction |
546291 |
6232 |
RPS27 |
4.7202 |
466743 |
4294 |
MAP3K10 |
3.4417 |
434980 |
351 |
APP |
2.9978 |
155048 |
4059 |
LU |
2.9455 |
445015 |
2906 |
GRIN2D |
2.4444 |
99922 |
1815 |
DRD4 |
2.2910 |
483305 |
3094 |
HINT1 |
2.2725 |
530863 |
57524 |
CASKIN1 |
2.2615 |
83381 |
2791 |
GNG11 |
1.8153 |
435136 |
7295 |
TXN |
1.7009 |
50425 |
10728 |
TEBP |
1.6368 |
279594 |
7132 |
TNFRSF1A |
1.5048 |
460978 |
8883 |
APPBP1 |
0.6981 |
247565 |
6010 |
RHO |
0.6208 |
523560 |
3320 |
HSPCA |
0.3799 |
252387 |
9620 |
CELSR1 |
0.0017 |
523732 |
7356 |
SCGB1A1 |
0.0009 |
3 |
protein folding |
180414 |
3312 |
HSPA8 |
2.2911 |
1197 |
3336 |
HSPE1 |
2.2726 |
421509 |
10575 |
CCT4 |
2.1969 |
520028 |
3303 |
HSPA1A |
1.8122 |
1600 |
22948 |
CCT5 |
1.7348 |
534385 |
10189 |
THOC4 |
1.7294 |
50425 |
10728 |
TEBP |
1.6368 |
189772 |
10576 |
CCT2 |
1.3919 |
509736 |
3326 |
HSPCB |
0.6936 |
523037 |
23234 |
DNAJC9 |
0.6855 |
491494 |
7203 |
CCT3 |
0.6848 |
184233 |
3313 |
HSPA9B |
0.6588 |
356331 |
5478 |
PPIA |
0.6507 |
445203 |
3301 |
DNAJA1 |
0.5266 |
492516 |
5202 |
PFDN2 |
0.5141 |
523560 |
3320 |
HSPCA |
0.3799 |
551568 |
85313 |
PPIL4 |
0.0008 |
4 |
response to unfolded protein |
180414 |
3312 |
HSPA8 |
2.2911 |
1197 |
3336 |
HSPE1 |
2.2726 |
520028 |
3303 |
HSPA1A |
1.8122 |
509736 |
3326 |
HSPCB |
0.6936 |
445203 |
3301 |
DNAJA1 |
0.5266 |
523560 |
3320 |
HSPCA |
0.3799 |
5 |
positive regulation of nitric oxide biosynthesis |
509736 |
3326 |
HSPCB |
0.6936 |
523560 |
3320 |
HSPCA |
0.3799 |
6 |
protein refolding |
523560 |
3320 |
HSPCA |
0.3799 |
7 |
RNA splicing |
516076 |
6637 |
SNRPG |
1.9589 |
365116 |
7307 |
U2AF1 |
1.5939 |
355934 |
6421 |
SFPQ |
1.5729 |
309090 |
6432 |
SFRS7 |
0.6226 |
528763 |
6627 |
SNRPA1 |
0.6155 |
8 |
nuclear mRNA splicing |
369624 |
8683 |
SFRS9 |
2.3299 |
79110 |
4691 |
NCL |
2.0277 |
534385 |
10189 |
THOC4 |
1.7294 |
365116 |
7307 |
U2AF1 |
1.5939 |
355934 |
6421 |
SFPQ |
1.5729 |
552581 |
9939 |
RBM8A |
0.7166 |
464734 |
6632 |
SNRPD1 |
0.7151 |
471011 |
23451 |
SF3B1 |
0.6758 |
309090 |
6432 |
SFRS7 |
0.6226 |
527105 |
9987 |
HNRPDL |
0.6054 |
484227 |
84321 |
THOC3 |
0.5445 |
546261 |
3178 |
HNRPA1 |
0.3795 |
411300 |
11193 |
WBP4 |
0.0008 |
348342 |
60677 |
BRUNOL6 |
0.0006 |
9 |
mRNA-nucleus export |
534385 |
10189 |
THOC4 |
1.7294 |
552581 |
9939 |
RBM8A |
0.7166 |
484227 |
84321 |
THOC3 |
0.5445 |
546261 |
3178 |
HNRPA1 |
0.3795 |
10 |
mRNA metabolism |
546271 |
5094 |
PCBP2 |
1.5460 |
2853 |
5093 |
PCBP1 |
1.5212 |
458280 |
5042 |
PABPC3 |
1.4025 |
11 |
mRNA stabilization |
387804 |
26986 |
PABPC1 |
2.9446 |
12 |
RNA transcription termination |
532216 |
7978 |
MTERF |
0.0008 |
13 |
tRNA 3’-processing |
506382 |
51095 |
TRNT1 |
0.0008 |
14 |
glycyl-tRNA aminoacylation |
404321 |
2617 |
GARS |
0.6845 |
15 |
arginyl-tRNA aminoacylation |
506215 |
5917 |
RARS |
0.6260 |
16 |
translational initiation |
150580 |
10209 |
SUI1 |
5.4220 |
371001 |
8662 |
EIF3S9 |
1.4982 |
429180 |
8894 |
EIF2S2 |
1.3812 |
17 |
regulation of translational initiation |
150580 |
10209 |
SUI1 |
5.4220 |
492599 |
8667 |
EIF3S3 |
2.6144 |
520943 |
7458 |
WBSCR1 |
0.7001 |
530096 |
8668 |
EIF3S2 |
0.5094 |
18 |
regulation of translation |
150580 |
10209 |
SUI1 |
5.4220 |
520703 |
1915 |
EEF1A1 |
2.2678 |
19 |
translational elongation |
356502 |
6176 |
RPLP1 |
8.2655 |
421608 |
1933 |
EEF1B2 |
5.5078 |
546285 |
6175 |
RPLP0 |
2.7563 |
520703 |
1915 |
EEF1A1 |
2.2678 |
333388 |
1936 |
EEF1D |
2.1763 |
144835 |
1937 |
EEF1G |
1.5558 |
20 |
embryo implantation |
523732 |
7356 |
SCGB1A1 |
0.0009 |
21 |
cytoplasmic sequestering of NF-kappaB |
31210 |
602 |
BCL3 |
2.1500 |
22 |
iron ion transport |
446345 |
2495 |
FTH1 |
6.9516 |
517666 |
1727 |
DIA1 |
1.8203 |
433670 |
2512 |
FTL |
1.7473 |
23 |
ATP synthesis coupled proton transport |
546238 |
514 |
ATP5E |
4.9273 |
409140 |
539 |
ATP5O |
2.0886 |
429 |
518 |
ATP5G3 |
1.8573 |
486360 |
10632 |
ATP5L |
1.6978 |
24 |
proton transport |
201939 |
23545 |
ATP6V0A2 |
30.6709 |
546238 |
514 |
ATP5E |
4.9273 |
409140 |
539 |
ATP5O |
2.0886 |
429 |
518 |
ATP5G3 |
1.8573 |
486360 |
10632 |
ATP5L |
1.6978 |
25 |
mitochondrial electron transport |
324250 |
4708 |
NDUFB2 |
2.4136 |
472185 |
4725 |
NDUFS5 |
2.3533 |
407860 |
4718 |
NDUFC2 |
2.1482 |
502528 |
4722 |
NDUFS3 |
1.5587 |
304613 |
4710 |
NDUFB4 |
1.5034 |
408257 |
4726 |
NDUFS6 |
0.6838 |
26 |
mitochondrial electron transport |
481571 |
7388 |
UQCRH |
4.2248 |
27 |
ribosomal protein-nucleus import |
406300 |
9349 |
RPL23 |
3.1938 |
28 |
cysteine biosynthesis from serine |
533013 |
875 |
CBS |
1.3766 |
29 |
bile acid biosynthesis |
460618 |
80270 |
HSD3B7 |
0.0008 |
30 |
DNA metabolism |
471873 |
1841 |
DTYMK |
1.9531 |
350966 |
9232 |
PTTG1 |
1.4052 |
118243 |
1777 |
DNASE2 |
0.0008 |
31 |
DNA replication and chromosome cycle |
350966 |
9232 |
PTTG1 |
1.4052 |
497741 |
1063 |
CENPF |
0.5694 |
32 |
base-excision repair, DNA ligation |
434102 |
3146 |
HMGB1 |
1.7216 |
33 |
regulation of DNA replication |
147433 |
5111 |
PCNA |
1.5230 |
34 |
DNA damage response |
86161 |
2765 |
GML |
0.0008 |
35 |
dTTP biosynthesis |
471873 |
1841 |
DTYMK |
1.9531 |
36 |
nucleotide metabolism |
463456 |
4831 |
NME2 |
3.3849 |
118638 |
4830 |
NME1 |
1.5814 |
522099 |
84720 |
PIGO |
0.0011 |
37 |
CTP biosynthesis |
463456 |
4831 |
NME2 |
3.3849 |
118638 |
4830 |
NME1 |
1.5814 |
38 |
UTP biosynthesis |
463456 |
4831 |
NME2 |
3.3849 |
118638 |
4830 |
NME1 |
1.5814 |
39 |
GTP biosynthesis |
463456 |
4831 |
NME2 |
3.3849 |
118638 |
4830 |
NME1 |
1.5814 |
40 |
nucleoside triphosphate biosynthesis |
463456 |
4831 |
NME2 |
3.3849 |
118638 |
4830 |
NME1 |
1.5814 |
41 |
dTDP biosynthesis |
471873 |
1841 |
DTYMK |
1.9531 |
42 |
purine base biosynthesis |
518774 |
10606 |
PAICS |
0.6084 |
43 |
polyamine biosynthesis |
446427 |
4946 |
OAZ1 |
3.5915 |
44 |
aminoglycan biosynthesis |
519818 |
4245 |
MGAT1 |
1.4283 |
45 |
prostaglandin biosynthesis |
407995 |
4282 |
MIF |
3.6554 |
50425 |
10728 |
TEBP |
1.6368 |
46 |
“de novo” IMP biosynthesis |
518774 |
10606 |
PAICS |
0.6084 |
47 |
cysteine biosynthesis via cystathione |
533013 |
875 |
CBS |
1.3766 |
48 |
anaerobic glycolysis |
446149 |
3945 |
LDHB |
1.7750 |
49 |
intracellular sequestering of iron ion |
446345 |
2495 |
FTH1 |
6.9516 |
50 |
immune cell chemotaxis |
313 |
6696 |
SPP1 |
0.0012 |
51 |
T-helper 1 type immune response |
313 |
6696 |
SPP1 |
0.0012 |
52 |
regulation of myeloid cell differentiation |
313 |
6696 |
SPP1 |
0.0012 |
53 |
negative regulation of bone mineralization |
313 |
6696 |
SPP1 |
0.0012 |
54 |
induction of positive chemotaxis |
313 |
6696 |
SPP1 |
0.0012 |
55 |
intracellular signaling cascade |
209983 |
3925 |
STMN1 |
2.3054 |
201058 |
81551 |
STMN4 |
0.6154 |
56 |
G-protein coupled receptor protein signaling pathway |
99922 |
1815 |
DRD4 |
2.2910 |
83381 |
2791 |
GNG11 |
1.8153 |
247565 |
6010 |
RHO |
0.6208 |
57 |
sensory perception |
298198 |
123920 |
CKLFSF3 |
2.0187 |
247565 |
6010 |
RHO |
0.6208 |
58 |
rhodopsin mediated signaling |
247565 |
6010 |
RHO |
0.6208 |
59 |
phototransduction, visible light |
247565 |
6010 |
RHO |
0.6208 |
60 |
phosphoinositide-mediated signaling |
83758 |
1164 |
CKS2 |
2.3328 |
147433 |
5111 |
PCNA |
1.5230 |
61 |
cellular morphogenesis |
421597 |
7428 |
VHL |
2.2149 |
62 |
positive regulation of cell differentiation |
421597 |
7428 |
VHL |
2.2149 |
63 |
proteolysis and peptidolysis |
421597 |
7428 |
VHL |
2.2149 |
567440 |
25823 |
TPSG1 |
1.6712 |
531064 |
4738 |
NEDD8 |
1.5479 |
64 |
cellular respiration |
437060 |
54205 |
CYCS |
0.5248 |
65 |
caspase activation via cytochrome c |
437060 |
54205 |
CYCS |
0.5248 |
66 |
acetyl-CoA metabolism |
530331 |
5160 |
PDHA1 |
0.6839 |
67 |
peptidyl-glutamic acid carboxylation |
77719 |
2677 |
GGCX |
0.0007 |
68 |
spliceosome assembly |
516076 |
6637 |
SNRPG |
1.9589 |
502829 |
7536 |
SF1 |
1.5815 |
69 |
ribosome biogenesis and assembly |
499839 |
6130 |
RPL7A |
3.0814 |
546285 |
6175 |
RPLP0 |
2.7563 |
27222 |
55651 |
NOLA2 |
1.7032 |
70 |
regulation of macrophage activation |
407995 |
4282 |
MIF |
3.6554 |
71 |
regulation of viral genome replication |
356331 |
5478 |
PPIA |
0.6507 |
72 |
positive regulation of fibroblast proliferation |
275243 |
6277 |
S100A6 |
5.6553 |
73 |
positive regulation of translation |
387804 |
26986 |
PABPC1 |
2.9446 |
74 |
negative regulation of transcriptional
preinitiation complex formation |
434102 |
3146 |
HMGB1 |
1.7216 |
75 |
negative regulation of cyclin dependent protein kinase activity |
15299 |
10614 |
HIS1 |
1.5579 |
76 |
leucine catabolism |
167531 |
64087 |
MCCC2 |
0.0010 |
77 |
nascent polypeptide association |
505735 |
4666 |
NACA |
1.4686 |
78 |
oxidative phosphorylation |
481571 |
7388 |
UQCRH |
4.2248 |
79 |
copper ion homeostasis |
418241 |
4502 |
MT2A |
7.4679 |
434980 |
351 |
APP |
2.9978 |
4. Discussion
Our investigation revealed that HSV-2 is capable of infecting ECV 304 cells, leading to a cascade of modifications in gene expression. These modifications encompass variations in the expression of genes associated with cell signaling, cytoskeletal dynamics and motility, cell cycle regulation, transcription and transcription factors, protein synthesis, ion channel activity, cellular receptors, immune responses, and metabolic processes.
Gene microarrays can monitor the expression levels of thousands of genes simultaneously on a large scale to study the relationship between abundant gene expression and disease. Gene microarray technology can be used to analyze the differential expression of genes during viral infection of host cells, which is important for the treatment of diseases [12].
Previous research showed that ECV304 cells were infected with HSV-2 and morphological changes of the cells were observed by contrast microscopy and tissue staining. It was found that cell necrosis was the predominant form of cell death, and no significant apoptosis was observed. Later, Zhang et al. [13] confirmed that HSV-2 infection of ECV304 cells significantly induced apoptosis. However, the relatively simple information obtained in the single-gene model makes it more difficult to perform a comprehensive in-depth analysis. Mo et al. [14] analyzed the effect of rubella virus (RUBV), human cytomegalovirus (HCMV), and HSV-2 co-infection on mRNA accumulation in ECV304 cells by using microarray technology, and found that 80 genes were up-regulated and 19 genes were down-regulated, including VEGF, WISP2, WISP2 and COL11A2, etc. However, the effect of HSV-2 infection on the mRNA expression of ECV304 cells has not been studied by gene chip technology.
In this study, we used the ECV304 endothelial cell line as a model and human genome-wide oligonucleotide microarray to investigate the inhibitory effect of HSV-2 on ECV304 cells and to elucidate the expression of related genes. HSV-2 may play a role in signal transduction and regulation of gene expression in this process. The results showed that a total of 462 genes were significantly differentially expressed in the experimental group compared with the control group, of which 318 genes were up-regulated and 144 genes were down-regulated. The results showed that differential genes were involved in 237 biological processes, 79 of which were significantly different (P < 0.05).
These genes include cell signaling-related genes, cytoskeleton and motility-related genes, cell cycle-related genes, protein synthesis-related genes, transcription and transcription factor-related genes, and cell receptor-related genes. The results of the four differentially expressed genes validated by RT-PCR were generally consistent with those of the differentially expressed genes analyzed by gene microarray, indicating the reliability of the gene microarray data. Several genes that may be involved in endothelial damage and atherosclerosis caused by HSV-2 infection, which were identified for the first time in this study, are discussed below.
Heat shock proteins (HSP) are a family of proteins with important physiological functions that are highly conserved in evolution [15]. HSP can be classified into several families such as HSP110, HSP90, HSP70, HSP60, small molecule HSP and ubiquitin according to their molecular weight and degree of homology [16] [17]. Under adverse conditions such as stress, HSP can induce cell production, improve cell resistance, and play a role in stress protection, so it is also known as stress protein (SP). The main physiological functions of HSP are to promote and maintain the correct folding of new polypeptide chains [18], to participate in cell damage and repair [19], and to regulate cell growth, division, differentiation and death [20] [21]. Our results show that HSV-2 also induces HSP production in ECV304 cells after infection, suggesting that upregulation of HSP may be involved in some HSV-2-mediated cell biological damage.
The ribosome is an important organelle in the cell responsible for protein synthesis and consists of four rRNAs and 80 ribosomal proteins (RP) [22] [23]. RP is an important component of the ribosome and plays an important role in the translation process of the ribosome, such as folding of rRNA to form a functional three-dimensional structure; adjusting the spatial conformation of the ribosome during protein synthesis; and catalyzing protein synthesis in concert with rRNA at the binding site of the ribosome. In our study, the expression of 36 RPL genes and 23 RPS genes related to protein synthesis was elevated, and only the expression of RPL6 gene was decreased, indicating that the ribosomal protein gene is extensively involved in virus-cell interaction during the early stages of HSV-2 infection of ECV304 cells.
The S100 family of proteins is a group of EF-chiral calcium-binding proteins that play a variety of biological roles in vivo through regulation of calcium ions and interaction with target proteins, participating in cell cycle activities, cell differentiation, tumor growth, and extracellular matrix secretory activities [24]. Its distribution is cell- and tissue-specific [25], and several S100 members are abnormally expressed in tumors and are closely associated with tumor infiltration and metastasis. S100A6 is called calcyclin and used to be also known as 2A9, 5B10, and PRA. Chromosome 1q21 has been found to be altered in certain cancers or precancerous lesions, such as breast cancer, lymphoma and leukemia. From the specific expression of the biological function of the S100 protein family in tumors and its chromosomal localization, it can be found that it is closely related to tumors, of which S100A6 has increased expression in most tumor tissues. The results of this study showed that HSV-2 infection of ECV304 cells induced a 5.6-fold increase in cellular S100A6 gene expression. Combined with the results of many studies at home and abroad, which also showed that the development of cervical cancer may be related to HSV-2 infection, it suggests that the S100A6 gene may play some role in the development of cervical cancer. The aberrant expression of S100A6 is intricately associated with cellular proliferation and differentiation processes. Upon stimulation of quiescent cells by serum, epidermal growth factor, platelet-derived growth factor, nerve growth factor, retinoic acid, ischemic injury, and other physiological or pathological stimuli, there is a marked increase in the intracellular levels of S100A6 [26]. Existing literature indicates that S100A6 is significantly overexpressed in a variety of proliferative cell types, including ras-transformed NIH3T3 cells, SV40-transformed mouse fibroblasts, and various human malignancies such as acute myeloid leukemia, endometrial cancer, breast cancer, lung cancer, colorectal tumors, thyroid tumors, malignant fibrous histiocytoma, melanoma, neuroblastoma, squamous cell carcinoma of the oral mucosa, as well as in diverse epithelial-derived tumor cell lines. These cells exhibit elevated S100A6 expression levels in comparison to their differentiated or growth-inhibited counterparts [27].
Osteopontin (OPN) is a secreted acidic glycoprotein with multiple functions, classified as extracellular matrix, which promotes cell adhesion and migration [28]. OPN is present in human blood, urine, breast milk and other body fluids, as well as in the gastrointestinal tract, bladder, pancreas, lungs, bronchi and other tissues [29]. In our study, the expression of the Spp1 gene was significantly reduced, which may be associated with HSV-2 infection, suggesting that the Spp1 gene may play some role in the process of HSV-2 infection.
Calcium/calmodulin-dependent serine protein kinase (CASK) is a family of membrane-associated guanylate kinases. It was first cloned in nematodes [30], and its homologs were subsequently found in Drosophila and mammals [31]. CASK has several distinct protein binding domains and binds to other proteins to form protein complexes involved in the construction of the cell membrane protein backbone, cell signaling, gene regulation and many other important cellular physiological processes [32]. Current research on CASK is focused on the brain nervous system [33] and embryonic development [34]. With the accumulation of research data and advances in proteomics technology, it has become possible to understand the molecular network of various proteins that CASK interacts with in different cells and their related functions. Meanwhile, the establishment of knockout models of this protein in mammals will help to understand the role of CASK in overall biological development as well as in related diseases.
5. Conclusion
Among all biological processes induced by HSV-2 infection of ECV304 cells, 462 differential genes were found to be involved in a total of 237 biological processes, 79 of which were significantly different (P < 0.05). These mainly included biological processes such as protein synthesis, signaling, protein folding, RNA splicing, ion channels, ribosome synthesis and assembly, cellular respiration, DNA and mRNA metabolism. Further analysis revealed that a variety of genes among these differentially expressed genes may be involved in HSV-causing endothelial damage, cervical cancer and atherosclerosis, while the role of most other differentially expressed genes identified for the first time in the pathogenesis of HSV is unclear. Nevertheless, screening for aberrantly expressed genes by gene microarray provides valuable clues for an in-depth study of the mechanisms by which HSV causes endothelial injury and atherosclerosis.
Acknowledgements
We are grateful to Changwen Ke, director of the Institute of Microbiology Laboratory, Guangdong Center for Disease Control, for providing a high-quality laboratory and excellent instruments and equipment for the study of this topic, as well as giving valuable suggestions and enthusiastic guidance.
Funding
This work is supported by grants from the National Natural Science Foundation of China (No.31760716; 31560681), and the Project of Jiangxi Province (No. 20151BBF60007; 20171ACB21028).
Abbreviations
HSV |
Herpes simplex virus |
AS |
Atherosclerosis |
ECV-304 cells |
Human umbilical vein endothelial cells |
HSV-2 |
Herpes simplex virus type 2 |
RP |
Ribosomal proteins |
CASK |
Calcium/calmodulin-dependent serine protein kinase |