Outline of Gene Expression in the Liver
Totally 5,369, 5,548, and 5,395 genes were found in the livers on the 1st, 3rd, and 5th day after MnCl2 treatment (Supplementary file 1–3). Figure 1A-C indicated the UMAP; Fig. 1D-F revealed the transcript expression; Fig. 1G-I represented moderated T statistic; Fig. 1J-L represented mean-variance trend; and Fig. 1M-O revealed the volcano plot for the DEGs in the livers on the 1st, 3rd, and 5th day after MnCl2 incubation, respectively. The number of overlapping DEGs in the liver on the 1st, 3rd, and 5th day after MnCl2 exposure was indicated in Fig. 1S with Venn diagram.
Moreover, 222 to 494 DEGs were identified in the livers on the 1st, 3rd, and 5th day after MnCl2 treatment (Table 1, Supplementary file 4–6). Compared with the livers in control, 143, 226, and 340 genes were up-regulated, whereas 79, 125, and 154 genes were down-regulated in MnCl2-treated liver on the 1st, 3rd, and 5th day after MnCl2 exposure, respectively (Table 1). In addition, the top 30 up- and down-regulated genes in MnCl2-treated liver on the 1st, 3rd, and 5th day after MnCl2 incubation were shown in Table 2–7, respectively. Notably, the overlapping DEGs identified in the livers among the 1st, 3rd, and 5th day treated with MnCl2 were revealed in Table 8. For instance, MKI67 encodes a nuclear protein that is associated with and may be necessary for cellular proliferation. CDKN1A encodes a potent cyclin-dependent kinase inhibitor that binds to and inhibits the activity of cyclin-cyclin-dependent kinase 2 or 4 complexes, and thus functions as a regulator of cell cycle progression at G1. LCN2 encodes a protein that transports small hydrophobic molecules such as lipids, steroid hormones and retinoids. The protein encoded by this gene is a neutrophil gelatinase-associated lipocalin and plays a role in innate immunity by limiting bacterial growth.
Table 2. The top 30 upregulated genes in the liver treated with MnCl2 on the 1st day
Gene symbol
|
Log2FC
|
P Value
|
Description
|
LCN2
|
5.77
|
7.98E-06
|
Lipocalin 2
|
LBP
|
3.43
|
2.77E-09
|
Lipopolysaccharide binding protein
|
S100A9
|
2.99
|
3.74E-06
|
S100 calcium binding protein A9
|
ACCN4
|
2.92
|
1.08E-07
|
Amiloride-sensitive cation channel 4, Pituitary
|
CTSH
|
2.87
|
3.63E-02
|
Cathepsin H
|
A2M
|
2.86
|
1.47E-04
|
Alpha-2-macroglobulin
|
ORM1
|
2.79
|
6.30E-08
|
Orosomucoid 1
|
TMEM111
|
2.49
|
4.34E-03
|
Transmembrane protein 111
|
HTR2B
|
2.43
|
2.88E-02
|
5-hydroxytryptamine receptor 2B
|
RRAGB
|
2.36
|
1.02E-02
|
Ras-related GTP binding B
|
SERPINA7
|
2.33
|
4.09E-03
|
Serine peptidase inhibitor, Clade A, Member 7
|
CHRNB4
|
2.28
|
1.46E-04
|
Cholinergic receptor, Nicotinic, Beta polypeptide 4
|
VDR
|
2.20
|
1.75E-03
|
Vitamin D receptor
|
CCL2
|
2.11
|
3.12E-03
|
Chemokine (C-C motif) ligand 2
|
EML2
|
2.04
|
1.90E-03
|
Echinoderm microtubule associated protein like 2
|
STX7
|
2.02
|
6.39E-03
|
Syntaxin 7
|
LOC290651
|
1.95
|
8.80E-06
|
Similar to myo-inositol 1-phosphate synthase A1
|
PLAUR
|
1.93
|
3.06E-02
|
Plasminogen activator, Urokinase receptor
|
JUNB
|
1.93
|
3.78E-03
|
Jun-B oncogene
|
CD244
|
1.88
|
3.63E-03
|
CD244 natural killer cell receptor 2B4
|
FCNB
|
1.86
|
9.92E-07
|
Ficolin B
|
CXCL10
|
1.86
|
7.05E-05
|
Chemokine (C-X-C motif) ligand 10
|
SRD5A2
|
1.84
|
5.21E-03
|
Steroid 5-alpha-reductase 2
|
FZD5
|
1.84
|
3.95E-02
|
Frizzled homolog 5 (Drosophila)
|
LMNB1
|
1.83
|
6.12E-03
|
Lamin B1
|
FGB
|
1.72
|
4.46E-04
|
Fibrinogen, B beta polypeptide
|
C5
|
1.71
|
3.48E-03
|
Complement component 5
|
GBP2
|
1.71
|
4.16E-02
|
Guanylate nucleotide binding protein 2
|
LDLR
|
1.70
|
2.33E-03
|
Low density lipoprotein receptor
|
GRIP2
|
1.69
|
1.20E-02
|
Glutamate receptor interacting protein 2
|
Table 3. The top 30 downregulated genes in the liver treated with MnCl2 on the 1st day
Gene symbol
|
Log2FC
|
P Value
|
Description
|
LOC368066
|
-2.18
|
8.01E-03
|
Similar to indolethylamine N-methyltransferase
|
EPHX2
|
-2.09
|
1.09E-02
|
Epoxide hydrolase 2, Cytoplasmic
|
SDS
|
-2.05
|
2.08E-03
|
Serine dehydratase
|
CIB2
|
-2.02
|
2.01E-02
|
Calcium and integrin binding family member 2
|
EIIH
|
-1.94
|
1.15E-05
|
Hepatic protein EIIH
|
GUCY2G
|
-1.92
|
1.90E-03
|
Guanylate cyclase 2g
|
STAC3
|
-1.89
|
6.51E-03
|
SH3 and cysteine rich domain 3
|
ZFP354A
|
-1.81
|
2.45E-02
|
Zinc finger protein 354A
|
SP4
|
-1.76
|
9.82E-03
|
Sp4 transcription factor
|
HSD11B2
|
-1.72
|
9.76E-03
|
Hydroxysteroid 11-beta dehydrogenase 2
|
CDKN3
|
-1.71
|
1.04E-02
|
Cyclin-dependent kinase inhibitor 3
|
SPBC24
|
-1.69
|
1.46E-04
|
Spindle pole body component 24 homolog
|
CML3
|
-1.66
|
1.61E-03
|
Camello-like 3
|
SLC2A5
|
-1.65
|
1.12E-03
|
Solute carrier family 2, Member 5
|
INPP4B
|
-1.62
|
3.09E-02
|
Inositol polyphosphate-4-phosphatase, Type II
|
LOC680687
|
-1.56
|
3.00E-02
|
Hypothetical protein LOC680687
|
POFUT2
|
-1.56
|
3.05E-02
|
Protein O-fucosyltransferase 2
|
DIO3
|
-1.48
|
3.31E-02
|
Deiodinase, Iodothyronine, Type III
|
CDKN1A
|
-1.47
|
3.07E-03
|
Cyclin-dependent kinase inhibitor 1A
|
CASQ2
|
-1.46
|
1.90E-03
|
Calsequestrin 2
|
RGD1307009
|
-1.45
|
1.17E-03
|
Similar to retinoid x receptor interacting protein
|
JUNDP2
|
-1.45
|
1.13E-02
|
Jun dimerization protein 2
|
ZFYVE20
|
-1.42
|
4.97E-05
|
Zinc finger, FYVE domain containing 20
|
G6PC
|
-1.33
|
4.74E-03
|
Glucose-6-phosphatase, Catalytic
|
TSC22D3
|
-1.32
|
5.79E-03
|
TSC22 domain family 3
|
LOC679907
|
-1.31
|
1.00E-02
|
Similar to mitochondria-associated granulocyte macrophage CSF signaling molecule
|
MED19
|
-1.31
|
3.61E-05
|
Mediator of RNA polymerase II transcription, Subunit 19 homolog
|
MXD3
|
-1.31
|
6.06E-03
|
Max dimerization protein 3
|
CALB1
|
-1.30
|
1.99E-02
|
Calbindin 1
|
RGS17
|
-1.29
|
3.11E-02
|
Regulator of G-protein signaling 17
|
Table 4. The top 30 upregulated genes in the liver treated with MnCl2 on the 3rd day
Gene symbol
|
Log2FC
|
P Value
|
Description
|
CTSH
|
3.60
|
2.30E-02
|
Cathepsin H
|
LCN2
|
3.04
|
1.09E-04
|
Lipocalin 2
|
TMEM111
|
3.03
|
1.34E-05
|
Transmembrane protein 111
|
EGR1
|
2.69
|
8.32E-04
|
Early growth response 1
|
CYP7A1
|
2.44
|
6.72E-04
|
Cytochrome P450, Family 7, subfamily a, polypeptide 1
|
PER1
|
2.43
|
4.06E-07
|
Period homolog 1
|
IGFBP2
|
2.39
|
6.69E-03
|
Insulin-like growth factor binding protein 2
|
ACOX2
|
2.22
|
5.94E-03
|
Acyl-Coenzyme A oxidase 2, branched chain
|
ZP3
|
2.17
|
2.37E-04
|
Zona pellucida glycoprotein 3
|
MOBP
|
2.16
|
7.91E-05
|
Myelin-associated oligodendrocytic basic protein
|
ZFP354A
|
2.15
|
4.37E-04
|
Zinc finger protein 354A
|
SYT11
|
1.98
|
3.12E-05
|
Synaptotagmin XI
|
SLC22A12
|
1.97
|
1.04E-02
|
Solute carrier family 22, member 12
|
SQLE
|
1.96
|
9.58E-04
|
Squalene epoxidase
|
GDF15
|
1.94
|
8.40E-03
|
Growth differentiation factor 15
|
ACCN4
|
1.91
|
1.55E-02
|
Amiloride-sensitive cation channel 4, pituitary
|
P2RX2
|
1.88
|
2.38E-02
|
Purinergic receptor P2X, ligand-gated ion channel, 2
|
IHPK2
|
1.87
|
7.91E-05
|
Inositol hexaphosphate kinase 2
|
CNGA4
|
1.86
|
9.10E-03
|
Cyclic nucleotide gated channel alpha 4
|
ASNS
|
1.84
|
3.54E-04
|
Asparagine synthetase
|
AIPL1
|
1.74
|
7.60E-04
|
Aryl hydrocarbon receptor-interacting protein-like 1
|
CALCR
|
1.72
|
2.13E-02
|
Calcitonin receptor
|
LOC312030
|
1.70
|
1.92E-03
|
Similar to splicing factor, Arginine/serine-rich 2, interacting protein
|
FGF2
|
1.69
|
8.79E-03
|
Fibroblast growth factor 2
|
GRP
|
1.67
|
1.27E-02
|
Gastrin releasing peptide
|
PPARA
|
1.66
|
8.97E-03
|
Peroxisome proliferator activated receptor alpha
|
SSTR4
|
1.65
|
8.71E-03
|
Somatostatin receptor 4
|
NPY1R
|
1.65
|
6.74E-03
|
Neuropeptide Y receptor Y1
|
AP2B1
|
1.64
|
2.10E-02
|
Adaptor-related protein complex 2, beta 1 subunit
|
PKD2
|
1.63
|
5.23E-03
|
Polycystic kidney disease 2
|
Table 5. The top 30 downregulated genes in the liver treated with MnCl
2on the 3rd day
Gene symbol
|
Log2FC
|
P Value
|
Description
|
SLC25A30
|
-3.18
|
5.71E-07
|
Solute carrier family 25, member 30
|
EPHA3
|
-3.17
|
1.05E-02
|
Eph receptor A3
|
ZFX
|
-3.11
|
2.38E-03
|
Zinc finger protein X-linked
|
RGD1564008
|
-2.90
|
9.53E-07
|
Similar to dapper 1
|
FOS
|
-2.84
|
1.14E-02
|
FBJ murine osteosarcoma viral oncogene homolog
|
SCD2
|
-2.17
|
4.36E-03
|
Stearoyl-Coenzyme A desaturase 2
|
MYO1E
|
-2.10
|
7.93E-04
|
Myosin IE
|
SP4
|
-2.07
|
4.43E-03
|
Sp4 transcription factor
|
RGD1564876
|
-2.04
|
3.59E-02
|
Similar to Solute carrier family 35, Member E3
|
SYNJ1
|
-1.95
|
4.85E-02
|
Synaptojanin 1
|
ADORA2B
|
-1.94
|
1.09E-02
|
Adenosine A2B receptor
|
G0S2
|
-1.87
|
7.00E-04
|
G0/G1 switch gene 2
|
TTC7B
|
-1.82
|
1.68E-02
|
Tetratricopeptide repeat domain 7B
|
LOC361473
|
-1.79
|
1.40E-02
|
Similar to mitochondrial translational release factor 1-like
|
STAC3
|
-1.77
|
1.05E-03
|
SH3 and cysteine rich domain 3
|
SSX2IP
|
-1.76
|
2.45E-03
|
Synovial sarcoma, X breakpoint 2 interacting protein
|
BTBD3
|
-1.76
|
3.35E-02
|
BTB (POZ) domain containing 3
|
RGD1560913
|
-1.73
|
2.92E-02
|
Similar to expressed sequence AW413625
|
TGFBR1
|
-1.72
|
1.64E-02
|
Transforming growth factor, beta receptor 1
|
SLC39A10
|
-1.68
|
4.37E-03
|
Solute carrier family 39, member 10
|
SULT2B1
|
-1.65
|
2.26E-02
|
Sulfotransferase family, cytosolic, 2B, Member 1
|
MSLN
|
-1.61
|
2.86E-02
|
Mesothelin
|
PPARG
|
-1.61
|
1.92E-02
|
Peroxisome proliferator activated receptor gamma
|
COL27A1
|
-1.58
|
3.40E-03
|
Procollagen, Type XXVII, alpha 1
|
HSD11B2
|
-1.57
|
7.31E-03
|
Hydroxysteroid 11-beta dehydrogenase 2
|
LOC498564
|
-1.55
|
4.80E-02
|
Similar to integrin, beta-like 1
|
PLK1
|
-1.55
|
5.92E-03
|
Polo-like kinase 1
|
NCALD
|
-1.53
|
9.69E-04
|
Neurocalcin delta
|
PSMA3L
|
-1.51
|
3.07E-02
|
Proteasome subunit alpha type 3-like
|
SLC1A6
|
-1.49
|
1.23E-02
|
Solute carrier family 1, member 6
|
Table 6. The top 30 upregulated genes in the liver treated with MnCl2 on the 5th day
Gene symbol
|
Log2FC
|
P Value
|
Description
|
P2RX2
|
2.76
|
1.73E-04
|
Purinergic receptor P2X, Ligand-gated ion channel, 2
|
CNGA4
|
2.70
|
5.17E-05
|
Cyclic nucleotide gated channel alpha 4
|
PPP2R2D
|
2.53
|
9.48E-03
|
Protein phosphatase 2, Regulatory subunit B, Delta isoform
|
SLC22A12
|
2.49
|
1.91E-04
|
Solute carrier family 22 (organic anion/cation transporter), Member 12
|
LOC286989
|
2.43
|
1.02E-02
|
UDP-glucuronosyltransferase
|
ZP3
|
2.37
|
1.62E-05
|
Zona pellucida glycoprotein 3
|
MOBP
|
2.37
|
1.45E-04
|
Myelin-associated oligodendrocytic basic protein
|
LOC312030
|
2.31
|
1.70E-04
|
Similar to splicing factor, Arginine/serine-rich 2, Interacting protein
|
UCN
|
2.29
|
4.16E-02
|
Urocortin
|
ZFP354A
|
2.27
|
3.01E-04
|
Zinc finger protein 354A
|
IBSP
|
2.25
|
1.99E-04
|
Integrin binding sialoprotein
|
KCNT1
|
2.21
|
2.00E-02
|
Potassium channel, Subfamily T, Member 1
|
GRP
|
2.17
|
3.72E-02
|
Gastrin releasing peptide
|
VDR
|
2.17
|
4.24E-04
|
Vitamin D receptor
|
PLXNA3
|
2.08
|
2.89E-05
|
Plexin A3
|
S100A9
|
2.04
|
1.73E-04
|
S100 calcium binding protein A9 (calgranulin B)
|
TMEM111
|
2.03
|
2.78E-02
|
Transmembrane protein 111
|
RRAD
|
2.01
|
4.31E-03
|
Ras-related associated with diabetes
|
RGD1562236
|
1.99
|
4.83E-04
|
Similar to breast cancer membrane protein 101 (predicted)
|
ALPK3
|
1.96
|
2.80E-04
|
Alpha-kinase 3 (predicted)
|
FGD4
|
1.94
|
2.24E-03
|
FYVE, RhoGEF and PH domain containing 4
|
GDF15
|
1.94
|
1.78E-03
|
Growth differentiation factor 15
|
RRAGB
|
1.92
|
2.68E-02
|
Ras-related GTP binding B
|
SYT11
|
1.91
|
3.95E-05
|
Synaptotagmin XI
|
FZD5
|
1.90
|
6.18E-03
|
Frizzled homolog 5 (Drosophila)
|
HSPB3
|
1.89
|
5.40E-04
|
Heat shock 27kDa protein 3
|
AP2B1
|
1.88
|
6.07E-05
|
Adaptor-related protein complex 2, Beta 1 subunit
|
PKD2
|
1.87
|
1.55E-03
|
Polycystic kidney disease 2 (predicted)
|
SAG
|
1.86
|
8.25E-04
|
Retinal S-antigen
|
NPY1R
|
1.84
|
9.07E-05
|
Neuropeptide Y receptor Y1
|
Table 7. The top 30 downregulated genes in the liver treated with MnCl2 on the 5th day
Gene symbol
|
Log2FC
|
P Value
|
Description
|
MYLPF
|
-2.94
|
9.58E-04
|
Myosin light chain, Phosphorylatable, Fast skeletal muscle
|
MTHFR
|
-2.70
|
1.89E-03
|
5,10-methylenetetrahydrofolate reductase
|
HK2
|
-2.41
|
7.82E-03
|
Hexokinase 2
|
SMCP
|
-2.40
|
3.84E-02
|
Sperm mitochondria-associated cysteine-rich protein
|
TOR3A
|
-2.19
|
2.09E-04
|
Torsin family 3, member A
|
ITGB7
|
-2.15
|
1.70E-02
|
Integrin, beta 7
|
CHKA
|
-2.12
|
4.21E-05
|
Choline kinase alpha
|
TXN2
|
-2.08
|
6.81E-03
|
Thioredoxin 2
|
RGD1307414
|
-2.05
|
5.34E-04
|
Similar to RIKEN cDNA 1110067D22
|
RGD1307009
|
-1.97
|
5.74E-04
|
Similar to retinoid x receptor interacting protein
|
RGD1359349
|
-1.94
|
1.11E-04
|
Similar to hypothetical protein MNC34760
|
RGD1561792
|
-1.89
|
3.18E-03
|
Similar to CG14903-PA
|
ARNTL
|
-1.82
|
5.36E-04
|
Aryl hydrocarbon receptor nuclear translocator-like
|
LOC499693
|
-1.80
|
1.30E-02
|
Similar to amylase 2, pancreatic
|
HMBS
|
-1.80
|
3.58E-02
|
Hydroxymethylbilane synthase
|
HIBCH
|
-1.80
|
2.57E-02
|
3-hydroxyisobutyryl-Coenzyme A hydrolase
|
DIO3
|
-1.76
|
4.16E-04
|
Deiodinase, Iodothyronine, type III
|
GALNTL1
|
-1.75
|
1.16E-02
|
UDP-N-acetyl-alpha-D-galactosamine:polypeptide
N-acetylgalactosaminyltransferase-like 1
|
RGD1307157
|
-1.75
|
5.18E-03
|
Similar to DDM36
|
ENSA
|
-1.73
|
9.05E-03
|
Endosulfine alpha
|
KDELR3
|
-1.69
|
5.40E-03
|
KDEL endoplasmic reticulum protein retention receptor 3
|
SLC34A2
|
-1.69
|
1.94E-04
|
Solute carrier family 34, member 2
|
SOCS2
|
-1.68
|
1.88E-03
|
Suppressor of cytokine signaling 2
|
RGD1549725
|
-1.68
|
1.10E-02
|
Similar to RIKEN cDNA 9430083G14
|
MYC
|
-1.65
|
3.25E-02
|
Myelocytomatosis viral oncogene homolog
|
MBP
|
-1.64
|
9.58E-05
|
Myelin basic protein
|
GPATC1
|
-1.63
|
2.17E-02
|
G patch domain containing 1
|
MKI67
|
-1.61
|
1.16E-02
|
Antigen identified by monoclonal antibody Ki-67
|
CPT1B
|
-1.60
|
3.19E-04
|
Carnitine palmitoyltransferase 1b
|
RGD1561025
|
-1.59
|
3.80E-03
|
Kelch/ankyrin repeat containing cyclin A1 interacting protein isoform A
|
Table 8. The overlapping DEGs identified in the liver among the 1st, 3rd, and 5th day treated with MnCl2 and their functions
Gene symbol
|
Full name
|
Functions
|
MKI67
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Antigen identified by monoclonal antibody Ki-67
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MKI67 encodes a nuclear protein that is associated with and may be necessary for cellular proliferation.
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HSD11B2
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Hydroxysteroid 11-beta dehydrogenase 2
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There are at least two isozymes of the corticosteroid 11-beta-dehydrogenase, a microsomal enzyme complex responsible for the interconversion of cortisol and cortisone. The type I isozyme has both 11-beta-dehydrogenase and 11-oxoreductase activities.
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CDKN1A
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Cyclin-dependent kinase inhibitor 1A
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CDKN1A encodes a potent cyclin-dependent kinase inhibitor that binds to and inhibits the activity of cyclin-cyclin-dependent kinase2 or -cyclin-dependent kinase4 complexes, and thus functions as a regulator of cell cycle progression at G1.
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SULT2B1
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Sulfotransferase family, cytosolic, 2B, member 1
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SULT2B1 protein catalyze the sulfate conjugation of many hormones, neurotransmitters, drugs, and xenobiotic compounds. These cytosolic enzymes are different in their tissue distributions and substrate specificities.
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PER2
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Period homolog 2
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PER2 encodes components of the circadian rhythms of locomotor activity, metabolism, and behavior.
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CELSR2
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Cadherin EGF LAG seven-pass G-type receptor 2
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CELSR2 protein is receptor involved in contact-mediated communication, with cadherin domains acting as homophilic binding regions and the EGF-like domains involved in cell adhesion and receptor-ligand interactions.
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CCR2
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Chemokine (C-C motif) receptor 2
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CCR2 is essential component of the nuclear pore complex that seems to be required for NPC assembly and maintenance and is a receptor for monocyte chemoattractant protein-1 and is involved in monocyte infiltration in inflammatory diseases.
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ORM1
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Orosomucoid 1
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ORM1 functions as transport protein in the blood stream and appears to modulate the activity of the immune system during the acute-phase reaction. ORM1 may be involved in aspects of immunosuppression.
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CHRNB4
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Cholinergic receptor, nicotinic, Beta polypeptide 4
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CHRNB4 encodes one of the beta subunits of the nicotinic acetylcholine receptor superfamily which form ligand-gated ion channels with a central pore that forms a cation channel.
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ELMOD1
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ELMO domain containing 1
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ELMOD1 plays an essential role in the initiation of DNA synthesis. During the S phase of the cell cycle, the DNA polymerase alpha complex is recruited to DNA at the replicative forks and is predicted to be involved in positive regulation of GTPase activity.
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POLA2
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Polymerase, alpha 2
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POLA2 enable DNA binding activity and is involved in DNA replication, synthesis of RNA primer. It participates in ion transport events as addition of plasmolipin to lipid bilayers induces the formation of ion channels.
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PLLP
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Plasma membrane proteolipid
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PLLP appears to be involved in myelination and participates in ion transport events as addition of plasmolipin to lipid bilayers induces the formation of ion channels.
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ACCN4
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Amiloride-sensitive cation channel 4, pituitary
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ACCN4 is acid sensing ion channel subunit family member 4 and cation channel with high affinity for sodium.
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SLC9A2
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Solute carrier family 9, member 2
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SLC9A2 is involved in sodium-ion transport by exchanging intracellular hydrogen ions to external sodium ions and help in the regulation of cell pH and volume. SLC9A2 is associated with pH regulation to eliminate acids generated by active metabolism or to counter adverse environmental conditions.
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LCN2
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Lipocalin 2
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LCN2 encodes a protein that transport small hydrophobic molecules such as lipids, steroid hormones and retinoids. The protein encoded by this gene is a neutrophil gelatinase-associated lipocalin and plays a role in innate immunity by limiting bacterial growth.
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HTR2B
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5-hydroxytryptamine receptor 2B
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This gene functions as a receptor for various ergot alkaloid derivatives and psychoactive substances.
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GRPR
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Gastrin releasing peptide receptor
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GRPR regulates numerous functions of the gastrointestinal and central nervous systems, including release of gastrointestinal hormones, smooth muscle cell contraction, and epithelial cell proliferation and is a potent mitogen for neoplastic tissues.
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NPY1R
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Neuropeptide Y receptor Y1
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NPY1R belongs to the G-protein-coupled receptor superfamily. The encoded transmembrane protein mediates the function of neuropeptide Y (NPY), a neurotransmitter, and peptide YY, a gastrointestinal hormone.
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TMEM111
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Transmembrane protein 111
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TMEM111 is ER membrane protein complex subunit 3 and belongs to the EMC3 family.
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S100A9
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S100 calcium binding protein A9
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S100A9 is a calcium- and zinc-binding protein which plays a prominent role in the regulation of inflammatory processes and immune response. It can induce neutrophil chemotaxis, adhesion, and can increase the bactericidal activity of neutrophils by promoting phagocytosis via activation of SYK, PI3K/AKT, and ERK1/2.
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PLXNA3
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Plexin A3
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PLXNA3 is necessary for signaling by class 3 semaphorins and subsequent remodeling of the cytoskeleton. It may play a role in regulating semaphorin-mediated programmed cell death in the developing nervous system.
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VDR
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Vitamin D receptor
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VDR plays a central role in calcium homeostasis. The VDR-RXR heterodimers bind to specific response elements on DNA and activate the transcription of vitamin D3-responsive target genes.
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ZFP354A
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Zinc finger protein 354A
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ZFP354A may play a role in renal development and may also be involved in the repair of the kidney after ischemia-reperfusion. It was involved in regulation of transcription by RNA polymerase II.
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CNGA4
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Cyclic nucleotide gated channel alpha 4
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CNGA4 is a modulatory subunit of vertebrate cyclic nucleotide-gated membrane channels that transduce odorant signals.
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GO Enrichment for the DEGs
As indicated in Fig. 2A, the DEGs identified in the livers on the 1st day after MnCl2 incubation might be related to cellular calcium ion homeostasis, inflammatory response, innate immune response, cell differentiation, apoptotic process, cell adhesion, the positive regulation of cell migration, angiogenesis, vasoconstriction, cell proliferation, and ERK1 and ERK2 cascade; the responses to xenobiotic stimulus, drug, hypoxia, organic cyclic compound, lipopolysaccharide (LPS) nutrient levels, bacterium, estradiol, peptide hormone, and glucocorticoid; the regulation of cell cycle, cell shape, cell proliferation, and apoptotic process; the negative regulation of apoptotic process, and cell proliferation; cellular response to LPS, organic cyclic compound, and tumor necrosis factor.
Also, the DEGs identified in the livers on the 3rd day after MnCl2 exposure might be associated with calcium-mediated signaling, cell proliferation, cellular calcium ion homeostasis, and signal transduction; cellular response to calcium ion and hypoxia; chemical synaptic transmission, ERK1 and ERK2 cascade, fatty acid metabolic process, inflammatory response, and ion transmembrane transport; the negative regulations of apoptotic process and cell proliferation; positive regulation of apoptotic process, cell growth, cell migration, cell proliferation, and gene expression; regulations of cell proliferation, gene expression, activity, drug, hypoxia, nutrient, nutrient levels, and xenobiotic stimulus (Fig. 2B).
Moreover, the DEGs identified in the livers on the 5th day after MnCl2 incubation might be related to apoptotic process, cell differentiation, chemical synaptic transmission, cell proliferation, inflammatory response, protein phosphorylation, signal transduction, and wound healing; the negative regulation of apoptotic process, cell proliferation, gene expression, and transcription; the positive regulation of cell migration, cell proliferation, gene expression, MAPK cascade, protein phosphorylation, and transcription; the regulations of gene expression, transcription, drug, estradiol, ethanol, glucocorticoid, hypoxia, LPS, nutrient levels, organic cyclic compound, and xenobiotic stimulus (Fig. 2C). Notably, the overlapping DEGs identified in the livers on the 1st, 3rd, and 5th day after MnCl2 incubation might participate in acute-phase response, animal organ regeneration, cellular calcium ion homeostasis, ion transmembrane transport, response to drug, hyperoxia, hypoxia, and xenobiotic stimulus; the positive regulation of reactive oxygen species (ROS) and metabolic process (Fig. 2D).
In addition, the expression profiles of the DEGs in the livers on the 1st day after exposure in apoptotic process, cell adhesion, response to xenobiotic stimulus, inflammatory response, negative regulation of cell proliferation, and negative regulation of apoptotic process were shown in Fig. 2E-J, respectively. Also, the expression profiles of the DEGs on the 3st day in response to xenobiotic stimulus; negative regulation of cell proliferation; positive regulation of apoptotic process; fatty acid metabolic process; inflammatory response; and ion transmembrane transport were revealed in Fig. 2K-P, respectively.
KEGG Enrichment for the DEGs
As indicated in Fig. 3A, the DEGs identified in the livers on the 1st day after MnCl2 incubation participated in many signaling pathways, including complement and coagulation cascades, neuroactive ligand-receptor interaction, chemokine, cAMP, hepatitis C, IL-17, NF-kappa B, PI3K-Akt, TNF, MAPK, necroptosis, AMPK, RNA transport, toxoplasmosis, bile secretion, cell adhesion molecules, hepatitis B, natural killer cell mediated cytotoxicity, protein processing in endoplasmic reticulum, serotonergic synapse, systemic lupus erythematosus, toll-like receptor, and cytokine-cytokine receptor interaction signaling pathways.
Also, as shown in Fig. 3B, the DEGs identified in the livers on the 3rd day after MnCl2 exposure might be related to a large number of signaling pathways, including metabolic pathways, cAMP, PPAR, FoxO, IL-17, apelin, prolactin, thermogenesis, TGF-beta, calcium, PI3K-Akt, Cell cycle, relaxin, oxytocin, hepatitis B, chemokine, neuroactive ligand-receptor interaction, Cytokine-cytokine receptor interaction, bile secretion, synaptic vesicle cycle, Th17 cell differentiation, cellular senescence, regulation of actin cytoskeleton, thyroid hormone synthesis, EGFR tyrosine kinase inhibitor resistance, endocrine resistance, cholinergic synapse, serotonergic synapse, and fatty acid degradation signaling pathways.
The DEGs identified in the livers on the 5th day after MnCl2 exposure might be related to a large number of signaling pathways mainly participated in MAPK, PI3K-Akt, Hepatitis B, cAMP, IL-17, Glucagon, mTOR, ErbB, Ras, AMPK, FoxO, Jak-STAT, apelin, sphingolipid, adipocytokine, TNF, cell cycle, PPAR, endocytosis, Rap1, bile secretion, TGF-beta, Th17 cell differentiation, hepatitis C, cellular senescence, hepatocellular carcinoma, gap junction, endocrine resistance, focal adhesion, insulin resistance, non-alcoholic fatty liver disease, neuroactive ligand-receptor interaction, regulation of actin cytoskeleton, cytokine-cytokine receptor interaction, protein digestion and absorption, and natural killer cell mediated cytotoxicity signaling pathways (Fig. 3C).
Notably, the overlapping DEGs might participate in IL-17, cAMP, p53, mineral absorption, parathyroid hormone synthesis, secretion and action, transcriptional misregulation in cancer, circadian rhythm, endocrine and other factor-regulated calcium reabsorption, basal cell carcinoma, acute myeloid leukemia, non-small cell lung cancer, renal cell carcinoma, melanoma, pancreatic cancer, and platinum drug resistance (Fig. 3D).
Moreover, the expression profiles of the DEGs in the liver on the 1st day after exposure in TNF, IL-17, necroptosis, PI3K-Akt, cAMP, NF-kappa B, and AMPK signaling pathways were revealed in Fig. 3E-K, respectively. Also, the expression profiles of the DEGs in the liver on the 3rd after exposure in metabolic pathways, PPAR, PI3K-Akt, hepatitis B, and bile secretion signaling pathways were shown in Fig. 3L-P, respectively.
Reactome Enrichment for the DEGs
The DEGs identified in the livers on the 1st day after MnCl2 incubation participated in multiple signaling pathways, including metabolism, immune system, signal transduction, innate immune system, protein metabolism, small molecules transport, neutrophil degranulation, post-translational protein modification, cytokine signaling in immune system, extracellular matrix organization, generic transcription pathway, lipid metabolism, peptide ligand-binding receptors, RNA polymerase II transcription, cellular responses to external stimuli, ion channel transport, carbohydrate metabolism, steroids metabolism, neuronal system, stimuli-sensing channels, biological oxidations, cell cycle checkpoints, cell surface interactions at the vascular wall, chemokine receptors bind chemokines, complement cascade, ion homeostasis, MAPK family signaling cascades, potassium channels, and Toll-like receptor cascades (Fig. 4A).
Also, the DEGs identified in the livers on the 3rd day after MnCl2 treatment participated in multiple signaling pathways, including metabolism pathway, signal transduction, small molecule transport, immune system, lipid metabolism, innate immune system, generic transcription pathway, adaptive immune system, RNA polymerase II transcription, gene expression, peptide ligand-binding receptors, bile salt transport, steroid metabolism, transmission across chemical synapses, extracellular matrix organization, cytokine signaling in immune system, neurotransmitter release cycle, inorganic cations/anions transport, extracellular matrix degradation, fatty acid metabolism, cilium assembly, biological oxidations, organelle biogenesis and maintenance, and MAPK family signaling cascades (Fig. 4B).
Likewise, the DEGs identified in the livers on the 5th day after MnCl2 incubation participated in multiple signaling pathways, including metabolism, signal transduction, immune system, small molecule transport, hemostasis, lipid metabolism, gene expression, innate immune system, GPCR downstream signaling, RNA polymerase II transcription, cytokine signaling in immune system, signaling by interleukins, GPCR ligand binding, MAPK family signaling cascades, transmission across chemical synapses, platelet activation, signaling and aggregation, bile salt transport, MAPK1/MAPK3 signaling, cellular responses to external stimuli, developmental biology, RAF/MAP kinase cascade, FLT3 signaling, steroids metabolism, platelet degranulation, extracellular matrix organization, and cellular responses to stress (Fig. 4C).
Notably, overlapping DEGs exposure participated in small molecules transport, neutrophil degranulation, RNA polymerase II transcription, gene expression, innate immune system, immune system, vitamin D metabolism, sodium/proton exchangers, AKT phosphorylates targets in the cytosol, hedgehog ligand biogenesis, G1/S DNA damage checkpoints, cytosolic sulfonation of small molecules, nuclear receptor transcription pathway, iron uptake and transport, G1/S transition, DNA damage, signaling by hedgehog, mitotic G1-G1/S phases, Phase II-conjugation of compounds, S Phase, and stimuli-sensing channels (Fig. 4D).
PANTHER Enrichment for the DEGs
The DEGs identified in the livers on the 1st day after MnCl2 incubation participated in multiple signaling pathways, including 2-arachidonoylglycerol biosynthesis, apoptosis signaling pathway, blood coagulation, cholesterol biosynthesis, circadian clock system, inflammation mediated by chemokine and cytokine, plasminogen activating cascade, and vitamin D metabolism signaling pathways (Fig. 5A).
Likewise, the DEGs obtained in the livers on the 3rd day after MnCl2 treatment were associated with various signaling pathways, including p53, interleukin, insulin/IGF, ionotropic glutamate receptor, nicotinic acetylcholine receptor, pyruvate metabolism, circadian clock system, metabotropic glutamate receptor group III, 2-arachidonoylglycerol biosynthesis, muscarinic acetylcholine receptor 1, 2, 3 and 4 signaling pathways (Fig. 5B).
Also, the DEGs identified in the livers on the 5th day after MnCl2 incubation participated in multiple signaling pathways, including Ras, p53, TGF-beta, FGF, Vitamin D, B cell activation, interleukin, angiogenesis, ionotropic glutamate receptor, PDGF, Nicotinic acetylcholine receptor, T cell activation, apoptosis, circadian clock system, blood coagulation, opioid proenkephalin, alpha adrenergic receptor, adrenaline and noradrenaline biosynthesis, beta3 adrenergic receptor, interferon-gamma, insulin/IGF pathway-protein kinase B, metabotropic glutamate receptor group II, beta1 adrenergic receptor, and oxidative stress response signaling pathways (Fig. 5C).
Notably, overlapping DEGs exposure participated in p53, interleukin, vitamin D metabolism, circadian clock system, and p53 pathway feedback loops 2 signaling pathway (Fig. 5D).
Protein Classification for the DEGs
The DEGs identified in the livers on the 1st day after MnCl2 incubation participated in multiple signaling pathways, including amylase, HSP90 family chaperone, cyclase, chemokine, extracellular matrix glycoprotein, non-receptor tyrosine protein kinase, complement component, basic leucine zipper transcription factor, kinase activator, ligand-gated ion channel, cytokine, calmodulin-related, calcium-binding protein, voltage-gated ion channel, extracellular matrix protein, kinase modulator, ion channel, cysteine protease, lyase, transporter, intercellular signal molecule, protein-binding activity modulator, and gene-specific transcriptional regulator (Fig. 6A).
Moreover, the DEGs identified in the livers on the 3rd day after MnCl2 incubation participated in multiple signaling pathways, including ligand-gated ion channel, Rel homology transcription factor, C4 zinc finger nuclear receptor, ligase, growth factor, membrane trafficking regulatory protein, secondary carrier transporter, ion channel, transporter, G-protein coupled receptor, gene-specific transcriptional regulator, DNA-binding transcription factor, and translational protein (Fig. 6B).
In addition, the DEGs identified in the livers on the 5th day after MnCl2 incubation participated in multiple signaling pathways, including DNA polymerase processivity factor, ligand-gated ion channel, basic helix-loop-helix transcription factor, secondary carrier transporter, basic leucine zipper transcription factor, ion channel, phospholipase, reductase, voltage-gated ion channel, transporter, growth factor, non-receptor serine/threonine protein kinase, G-protein coupled receptor, DNA-binding transcription factor, and gene-specific transcriptional regulator (Fig. 6C). Notably, the overlapping DEGs might participate in kinase inhibitor, transporter, DNA-directed DNA polymerase, ion channel, and ligand-gated ion channel (Fig. 6D).
Integration of the PPI Network
To further discover the key genes linked to MnCl2-induced liver toxicity, PPI networks of the DEGs were constructed (Fig. 7A-C). As shown in Fig. 7A, MnCl2-induced hepatotoxicity on the 1st day after MnCl2 incubation included STAT3, ICAM1, CCL2, CXCL10, JAK2, IRF1, TNFRSF1A, HSPA4, CXCL9, JAK3, LCN2, KLKB1, and MAP3K1. As shown in Fig. 7B, MnCl2-induced liver toxicity on the 3rd day after MnCl2 incubation included KRAS, FOS, PPARG, FGF2, FOXO3, EGR1, CAV1, SMAD1, SMAD5, CDKN1A, RET, TGFBR1, and PPARA. In addition, MnCl2-induced liver toxicity on the 5th day after MnCl2 incubation included IL6, TNF, FN1, FGF2, MYC, MET, HGF, PDGFRB, ERBB2, PROM1, TGFB1, and KIT (Fig. 7C).
Hub Genes and their Functions
The hub genes associated with MnCl2 incubation-induced liver toxicity were obtained based on the MCC method. As shown in Fig. 8A, MnCl2-induced liver toxicity on the 1st day after MnCl2 incubation included STAT3, ICAM1, CCL2, CXCL10, JAK2 and IRF1, et al. Similarly, MnCl2-induced liver toxicity on the 3rd day included KRAS, FOS, PPARG, FGF2, FOXO3, EGR1 and CAV1, et al (Fig. 8B). Moreover, MnCl2-induced liver toxicity on the 5st day included IL6, TNF, FN1, FGF2, MYC, MET, HGF, PDGFRB, ERBB2, PROM1 and MBP, et al (Fig. 8C).