Differential expression of genes during aflatoxin B1-induced hepatocarcinogenesis in tree shrews

Table 1 Sources of total RNA samples

Number and type of samples	Animal treatment
HCC tissue
1 Tree shrew in AFB1 group died of HCC at the 88th week of treatment with an amount of AFB1 up to 10.4 mg.
2 Tree shrew in AFB1 group died of HCC at the 89th week of treatment with an amount of AFB1 up to 11.3 mg.
HCC-surrounding non-cancerous liver tissue
3 Autopsy tissue from the same animal and at the same time as #1
4 Autopsy tissue from the same animal and at the same time as #2.
Liver tissue taken before HCC developed
5 Biopsy tissue from the same animal as #1, at the 30th week of AFB1-treatment with a total amount of 3.1 mg AFB1.
6 Biopsy tissue from the same animal as #2, at the 30th week of AFB1-treatment with a total amount of 3.2 mg AFB1.
Liver tissue taken before AFB1-treatment
7 Biopsy tissue mixture from 3 animals before they were grouped and treated (0th week of the experiment).
Homochronous control
8 Biopsy tissue mixture from 2 animals in control group at the 90th week of the experiment.

Table 2 Differential expressions of important genes

Coordinate	Genbank	Gene Name	1	2	3	4	5	6
			BC/BA	NCL/BC	HCC/NCL	HCC/BC	HCC/BA	HCC/C
Oncogenes & tumor suppressors (1/57, 1.8%)
A1i	X12795	v-erbA related protein 3 (EAR3)			↑(3.56)	↑(7.45)	↑(4.80)
Cell Cycle Regulators (1/41, 2.4%)
A6i	L26584	cell division cycle 25 homolog (CDC25)				↑(3.43)		↑(5.92)
Ion channels & transport proteins (0/3)
Modulators, effectors & intracellular transducers (5/88, 5.7%)
B1f	M62424	thrombin receptor (TR)	↓(0.15)	↑(5.64)		↑(10.75)		↑(6.52)
B1l*	X74764	neurotrophic tyrosine kinase		↑(5.50)	↑(2.63)	↑(14.05)	↑(25.36)
		receptor-related 3
B2j	D21878	bone marrow stromal antigen 1 (BST-1)	↓(0.15)		↑(11.14)			↑(3.40)
B4l	U07707	epidermal growth factor receptor	↓(0.40)			↑(2.87)		↑(2.18)
		substrate 15 (EPS15)
B5m*	M65066	cAMP-dependent protein kinase type I	↓(0.02)	↑(4.03)	↑(11.35)	↑(51.59)
		beta regulatory subunit (PRKAR1B)
Stress response proteins (1/7, 14.3%)
B7m	M11717	heat shock 70-kDa protein 1 (HSP70.1)	↓(3.54)	↑(0.26)		↑(0.26)		↑(0.17)
Apoptosis-associated proteins (15/64, 23.4%)
C1g	M14694	p53 cellular tumor antigen			↓(0.35)			↓(0.10)
C1n	L07414	TNF-related activation protein (TRAP)			↓(0.42)			↓(0.24)
C3c	M25627	glutathione S-transferase A1 (GSTA1)				↓(0.29)		↓(0.23)
C3j	M13228	N-myc proto-oncogene	↑(181.47)		↓(0.30)		↑(148.98)	↓(0.18)
C3k	U25994	receptor (TNFRSF)-interacting	↑(4.61)		↓(0.25)			↓(0.06)
		serine-threonine kinase 1 (RIPK1)
C3n	L11015	tumor necrosis factor C (TNFC)			↓(0.24)			↓(0.08)
C4c*	U15172	NIP1 (NIP1)			↓(0.08)		↓(0.16)	↓(0.20)
C4d*	U15174	BCL2/adenovirus E1B 19-kDa-			↓(0.31)		↓(0.34)	↓(0.21)
		interacting protein 3 (BNIP3)
C4f	U23765	BCL2 homologous			↓(0.28)			↓(0.12)
		antagonist/killer 1 (BAK1)
C4h	U29680	BCL2-related protein A1 (BCL2A1)		↑(8.19)	↓(0.16)			↓(0.09)
C4k	U45880	inhibitor of apoptosis protein 3 (IAP3)	↑(4.53)		↓(0.42)			↓(0.23)
C4m*	U57059	TNF-related apoptosis inducing	↓(0.26)				↓(0.20)	↓(0.40)
		ligand (TRAIL)
C5c	Y09392	WSL protein+TRAMP+Apo-3+			↓(0.39)			↓(0.20)
		death domain receptor 3 (DDR3)
C5f	U39613	cysteine protease ICE-LAP3			↓(0.13)			↓(0.03)
C5h	U71364	cytoplasmic antiproteinase 3 (CAP3)		↑(44.85)	↓(0.28)			↓(0.13)
DNA synthesis, repair & recombinant proteins (15/34, 44.1%)
C5k*	L07540	replication factor C 36-kDa	↓(0.24)	↑(6.73)	↓(0.26)		↓(0.26)	↓(0.17)
		subunit (RFC36)
C6a	M30938	nuclear factor IV			↓(0.25)			↓(0.07)
C6b	M31899	xeroderma pigmentosum group B			↓(0.06)			↓(0.04)
		complementing protein (XPB-ERCC3)
C6c	M32865	70-kDa thyroid autoantigen (TLAA)			↓(0.44)			↓(0.20)
C6e	M63488	replication protein A 70-kDa			↓(0.25)			↓(0.07)
		subunit (RPA70)
C6l	J04088	DNA topoisomerase II alpha (TOP2A)		↑(4.00)	↓(0.33)			↓(0.30)
C7a	M13194	excision repair cross-complementing			↓(0.26)			↓(0.09)
		rodent repair deficiency
		complementation group 1 (ERCC1)
C7b	M13267	cytosolic superoxide dismutase 1 (SOD1)				↓(0.39)		↓(0.24)
C7c	U07418	colon cancer nonpolyposis type			↓(0.30)			↓(0.10)
		2 protein (COCA2)
C7d*	D21090	UV excision repair protein RAD23			↓(0.26)		↓(0.44)	↓(0.08)
		homolog B (hHR23B; XPC repair-
		complementing complex 58-kDa protein)
C7g	S40706	growth arrest & DNA damage-inducible			↓(0.29)			↓(0.15)
		protein 153 (GADD153)
C7i	U12134	DNA damage repair & recombination		↑(6.02)	↓(0.27)			↓(0.12)
		protein 52 homolog (RAD52)
C7k	U63139	DNA damage repair & recombination			↓(0.18)			↓(0.08)
		protein 50 homolog (RAD50)
C7m	X84740	DNA ligase III (LIG3)		↑(4.23)	↓(0.21)			↓(0.08)
C7n	X90392	muscle-specific DNase I-like (DNL1L)		↓(5.36)	↓(0.22)			↓(0.12)
Transcription factors & DNA-bingding proteins (8/98, 8.2%)
D3a	D26155	SWI/SNF-related matrix-associated	↓(0.19)	↑(3.85)		↑(4.58)		↑(2.55)
		actin-dependent regulator of chromatin
		subfamily A member 2 (SMARCA2)
D4b*	M36542	octamer-binding transcription		↑(2.64)		↑(8.32)	↑(7.33)	↑(3.30)
		factor 2 (OCT2)
D4e	M62810	mitochondrial transcription				↑(8.04)		↑(25.07)
		factor 1 (MTTF1)
D4j	M76541	transcriptional repressor protein	↓(0.32)	↑(2.73)		↑(3.74)		↑(3.10)
		yin & yang 1 (YY1)
D5a*	M83234	nuclease-sensitive element			↑(2.59)	↑(3.24)	↑(2.75)	↑(5.21)
		DNA-binding protein (NSEP)
D5f	M96824	nucleobindin 1 (NUCB1)	↓(0.24)	↑(2.64)		↑(3.00)		↑(2.37)
D6h*	U10324	nuclear factor NF90				↑(3.69)	(2.34)	↑(2.90)
D6l	U14575	activator of RNA decay (ARD-1)	↑(72.46)	↑(2.53)			↑(93.75)
Growth factor & chemokine receptors (1/25, 4.0%)
E1h	L06623	endothelin receptor type B (ETB)	↓(0.37)	↑(4.46)		↑(2.07)		↑(2.78)
Interleukin & interferon receptors (0/18)
Hormone receptors (0/4)
Neurotransmitter receptors (0/11)
Cell surface antigens & adhesion proteins (3/40, 7.5%)
E5l	M23197	myeloid cell surface CD33 antigen		↑(6.68)		↑(5.75)		↑(3.26)
E6f*	M74777	dipeptidyl peptidase IV (DPP IV);			↓(0.32)		↓(0.36)	↓(0.28)
		T-cell activation CD26
E7b	M35198	integrin beta 6 (ITGB6)		↑(5.44)	↓(0.44)			↓(0.36)
Growth factors, cytokines & chemokines (20/64, 31.3%)
F1f	X03438	granulocyte colony-stimulating	↑(8.95)	↑(3.32)	↓(0.05)	↓(0.18)		↓(0.06)
		factor (GCSF)
F1j*	M16552	thrombomodulin (THBD)			↓(0.10)		↓(0.24)	↓(0.23)
F2a	D16431	hepatoma-derived growth factor (HDGF)			↓(0.39)			↓(0.28)
F2c	J04130	T-cell activation protein 2 (AT2)			↓(0.17)			↓(0.20)
F2e	K03515	neuroleukin (NLK)	↑(9.09)		↓(0.40)			↓(0.26)
F2f	L12260	neuregulin 1 (NRG1)	↑(21.23)		↓(0.10)		↑(3.76)	↓(0.13)
F2g	L12261	heregulin-beta3		↑(6.10)	↓(0.10)			↓(0.14)
F2h	L36052	thrombopoietin (THPO)		↑(7.26)	↓(0.04)	↓(0.24)		↓(0.06)
F2m	M22491	bone morphogenetic protein 3 (BMP3)			↓(0.05)			↓(0.08)
F3a	M24545	small inducible cytokine subfamily			↓(0.36)			↓(0.34)
		A member 2 (SCYA2)
F3d	M30704	amphiregulin (AR)	↑(3.69)		↓(0.17)	↓(0.41)		↓(0.12)
F3i	M57502	small inducible cytokine subfamily			↓(0.22)			↓(0.27)
		A member 1 (SCYA1)
F3l	M60718	hepatocyte growth factor (HGF)			↓(0.20)			↓(0.30)
F4c	M74178	hepatocyte growth factor-like protein	↓(0.31)	↑(5.11)		↑(4.62)		↓(2.28)
F4e	M92934	connective tissue growth factor (CTGF)	↑(3.86)	↑(6.00)	↓(0.30)		↑(5.44)	↓(0.24)
F4h	U43142	vascular endothelial growth	↑(8.75)	↑(3.82)	↓(0.14)		↑(4.81)	↓(0.15)
		factor C (VEGFC)
F4i	X02530	small inducible cytokine subfamily	↑(5.60)	↑(4.14)	↓(0.14)		↑(3.17)	↓(0.15)
		B member 10 (SCYB10)
F4k	X06234	calgranulin A (CALA)			↓(0.21)			↓(0.21)
F4n	X51943	AFGF + HBGF-1 + ECGF-beta			↓(0.10)			↓(0.19)
F5e	X79929	OX40 ligand (OX40L)	↑(5.42)		↓(0.11)	↓(0.31)		↓(0.09)
Interleukins & interferons (12/20, 60.0%)
F5j	J04156	interleukin 7 (IL7)		↑(3.62)	↓(0.25)			↓(0.27)
F5k	A14844	interleukin 2 (IL2)	↑(11.82)		↓(0.14)	↓(0.16)		↓(0.13)
F5l	X02851	interleukin 1 alpha (IL1-alpha)			↓(0.19)			↓(0.24)
F5n	M14743	interleukin 3 (IL3)		↑(11.18)	↓(0.08)			↓(0.06)
F6a	M13982	interleukin 4 (IL4)	↑(2.87)		↓(0.26)			↓(0.22)
F6b*	X04602	interleukin 6 (IL6)		↑(3.37)	↑(2.73)	↑(9.47)	↑(12.41)
F6d	M28622	interferon beta 1 (IFN-beta 1)			↓(0.17)			↓(0.16)
F6g	L06801	interleukin 13 (IL13)	↓(0.20)	↑(5.04)		↑(5.90)		↓(2.36)
F6i	M57765	interleukin 11 (IL11)		↑(4.15)	↓(0.11)	↓(0.40)		↓(0.10)
F6j	M65290	interleukin 12 beta subunit (IL12-beta)	↑(2.71)		↓(0.19)			↓(0.18)
F6k	M65291	interleukin 12 alpha subunit	↑(3.78)		↓(0.17)			↓(0.14)
		(IL12-alpha)
F6l	U14407	interleukin 15 (IL15)		↑(3.36)	↓(0.24)			↓(0.30)
Hormones (7/14, 50.0%)
F7d	A06925	prorelaxin H2 (RLN2)			↓(0.23)			↓(0.16)
F7f	J04040	glucagon (GCG)		↑(5.75)	↓(0.17)			↓(0.12)
F7i	M31159	insulin-like growth factor-binding			↓(0.35)			↓(0.35)
		protein 3 ( IGFBP3)
F7j	M68867	cellular retinoic acid-binding		↑(2.84)	↓(0.27)			↓(0.23)
		protein II (CRABP2)
F7l	X58022	corticotropin-releasing	↑(5.00)	↑(2.97)	↓(0.12)	↓(0.36)		↓(0.16)
		factor-binding protein
F7m	U08098	estrogen sulfotransferase (STE; EST)	↑(5.62)	↑(3.44)			↑(12.98)
F7n	X54469	preprotachykinin beta (beta-PPT)	↑(15.57)		↓(0.07)	↓(0.21)		↓(0.09)
Total amount		89/588, 15.1%	32	36	70	29	20	83
Up-regulation			20(62.5)	35(97.2)	5(7.1)	18(62.1)	13(65.0)	15(18.1)
Down-regulation			12(37.5)	1(2.8)	65(92.9)	11(37.9)	7(35.0)	68(81.9)

1 BA: liver tissue from animals before AFB1 treatment (0th week of the experiment). BC: liver tissue from the same HCC animals after AFB1 treatment for 30 weeks. HCC: hepatocellular carcinoma tissue from the same animal after AFB1 treatment. NCL: HCC-surrounding non-cancerous liver tissues from the same HCC animal. C: liver tissue from untreated animals taken at the time when HCC developed in AFB1 treated animals. 2 The numbers followed the name of functional classes are the amount of the important genes identified in this class and their percentage. 3 ↑: up-regulation; ↓: down-regulation. The numbers followed are the averages of the two checked samples. 4 *: Genes might be of more importance.

Table 3 Four types of differentially expressed genes

Type I, genes that up-regulated both in HCC vs NCL and NCL vs BC. Modulators, effectors & intracellular transducers

neurotrophic tyrosine kinase receptor-related 3

cAMP-dependent protein kinase type I beta regulatory

subunit (PRKAR1B)

Type II, genes that had similar expressing level in HCC and NCL but higher than that in BC.

II-1. Modulators, effectors & intracellular transducers

thrombin receptor (TR)

epidermal growth factor receptor substrate 15 (EPS15)

II-2. Transcription factors & DNA-bingding proteins

SWI/SNF-related matrix-associated actin-dependent

regulator of chromatin subfamily A member 2 (SMARCA2)

octamer-binding transcription factor 2 (OCT2)

mitochondrial transcription factor 1 (MTTF1)

transcriptional repressor protein yin & yang 1 (YY1)

nucleobindin 1 (NUCB1)

II-3. Growth factor & chemokine receptors

endothelin receptor type B ( ETB)

II-4. Cell surface antigens & adhesion proteins

myeloid cell surface CD33 antigen

II-5. Growth factors, cytokines & chemokines

hepatocyte growth factor-like protein

Type III, genes down-regulated in HCC vs NCL.

III-1. Apoptosis-associated proteins

p53 cellular tumor antigen

TNF-related activation protein (TRAP)

N-myc proto-oncogene

receptor (TNFRSF)-interacting serine-threonine kinase 1 (RIPK1)

tumor necrosis factor C (TNFC)

NIP1 (NIP1)

BCL2/adenovirus E1B 19-kDa-interacting protein 3 (BNIP3)

BCL2 homologous antagonist/killer 1 (BAK1)

inhibitor of apoptosis protein 3 (IAP3)

WSL protein + TRAMP + Apo-3 + death domain receptor 3 (DDR3)

cysteine protease ICE-LAP3

III-2. DNA synthesis, repair & recombination proteins

nuclear factor IV

xeroderma pigmentosum group B complementing

protein (XPB; ERCC3)

70-kDa thyroid autoantigen (TLAA)

replication protein A 70-kDa subunit (RPA70)

excision repair cross-complementing rodent repair deficiency

complementation group 1 (ERCC1)

colon cancer nonpolyposis type 2 protein (COCA2)

UV excision repair protein RAD23 homolog B (hHR23B; XPC repair-complementing complex 58-kDa protein)

DNA damage-inducible protein 153 (GADD153)

DNA damage repair & recombination protein 50 homolog (RAD50)

III-3. Growth factors, cytokines & chemokines

thrombomodulin (THBD)

hepatoma-derived growth factor (HDGF)

T-cell activation protein 2 (AT2)

neuroleukin (NLK)

neuregulin 1 (NRG1)

bone morphogenetic protein 3 (BMP3)

small inducible cytokine subfamily A member 2 (SCYA2)

hepatocyte growth factor (HGF)

calgranulin A (CALA)

AFGF + HBGF-1 + ECGF-beta

III-4. Interleukins & interferons

interleukin 2 (IL2)

interleukin 1 alpha (IL1-alpha)

interleukin 4 (IL4)

interferon beta 1 (IFN-beta 1)

interleukin 12 beta subunit (IL12-beta)

interleukin 12 alpha subunit (IL12-alpha)

III-5. Hormones

insulin-like growth factor-binding protein 3 ( IGFBP3)

Type IV, genes up-regulated before HCC appearance but down-regu-lated after HCC developed.

IV-1. DNA synthesis, repair & recombination proteins

replication factor C 36-kDa subunit (RFC36)

DNA topoisomerase II alpha (TOP2A)

DNA damage repair & recombination protein 52 homolog (RAD52)

DNA ligase III (LIG3)

muscle-specific DNase I-like ( DNL1L)

IV-2. Growth factors, cytokines & chemokines

granulocyte colony-stimulating factor (GCSF)

heregulin-beta3

thrombopoietin (THPO)

connective tissue growth factor (CTGF)

IV-3. Interleukins & interferons

interleukin 7 (IL7)

interleukin 3 (IL3)

interleukin 6 (IL6)

interleukin 11 (IL11)

IV-4. Hormones

cellular retinoic acid-binding protein II (CRABP2)

corticotropin-releasing factor-binding protein