Transient and etiology-related transcription regulation in cirrhosis prior to hepatocellular carcinoma occurrence

Table 1 Biological and clinical data from patients with cirrhosis alone, HCC-associated cirrhosis and controls

Patient1	Number	Male/Female	Age2	Pathology3	Etiology4	Metavir5
						A0	A1	A2	A3
A1 to A7	7	3/4	48.4 ± 3.2	CIR	ALC	0	4	1	2
A15 to A24	10	7/3	50.4 ± 7.5	CIR	ALC	0	8	2	0
PA1 to PA10	10	8/2	67.1 ± 8.0	CIR + HCC	ALC	2	5	3	0
PA21 to PA29	9	9/0	60.9 ± 8.2	CIR + HCC	ALC	1	7	1	0
V8 to V14	7	6/1	57.4 ± 8.9	CIR	HCV	0	2	4	1
V25 to V34	10	5/5	55.7 ± 16.0	CIR	HCV	0	5	3	2
PV11 to PV20	10	6/4	71.5 ± 5.1	CIR + HCC	HCV	0	1	6	3
PV30 to PV35	6	2/4	66.2 ± 9.5	CIR + HCC	HCV	0	1	3	2
CL1 to CL8	8	3/5	59.9 ± 15.9	No CIR, no HCC6	--	7	1	0	0
CL9 to CL18	10	2/8	49.5 ± 14.0	No CIR, no HCC6	--	9	1	0	0

¹HCC-free alcoholic cirrhosis; PA: Peritumoral alcoholic cirrhosis; V: HCC-free HCV cirrhosis; PV: Peritumoral HCV cirrhosis; CL: Control without any detectable fibrosis. Underlined samples were studied by microarray and qRT-PCR; No underlining, independent cohort of samples studied by qRT-PCR only.

²mean ± SD.

³CIR: Cirrhosis; CIR + HCC: Peritumoral cirrhosis.

⁴HCV: Hepatitis C virus infection; ALC: Alcoholism; --: None.

⁵From left to right, number of patients with a given score of inflammation A0-A3. Difference in METAVIR score in HCC-free cirrhosis between all V vs A patients, P = 0.17 (Mann and Whitney’s test); In peritumoral cirrhosis between all PV vs PA patients, P = 0.005.

⁶Histologically normal liver sampled at a distance from a benign liver tumor or from a metastasis of non-hepatic cancer.

Table 2 Oligonucleotides for qRT-PCR

Oligonucleotides	Forward	Reverse	Amplicon size (bp)
ACSM2	AAATCCCGACAAGACAGCAG	CTGATCACAGCCGTCTCAAC	201
GSTA1/2	TCTGCAGAAGATTTGGACAAG	TCAATCAGGGCTCTCTCCTT	170
ADH4	GTCTGCTTGGATGTGGGTTT	TGATTCTGGAAGCTCCTGCT	150
HSCARG	GAAACTGGTGGTGGTTTTCG	CATCTTGGTCTCCCTGCACT	170
AHNAK	CAAAGGGAAACACACCGACT	GCTCTCAGCAGTCAATGCAA	207
HSD17B6	TCTGGGGACTGGTGAACAAT	GTGCTCTCCTCACCAAAGGA	150
APOH	CCGAGGAGGGATGAGAAAGT	AGAATCAGCGCCATTCAGAT	193
IFI27	CCAAGCTTAAGACGGTGAGG	AAAACTACGGCAGAGCCAGA	196
ARID1A	CTACGCTGCCACGTGTGTAT	GTACAGCATCGCACCAAGAG	187
MT1G	TCCTGCAAGTGCAAAGAGTG	ACTTCTCCGATGCCCCTTT	118
ARL2BP	AGGATGAAGTGGCTGGTGAC	GGAAGCTGGCAGAGAAGATG	170
ORM2	TTATATCGCATCGGCCTTTC	CCGCTGGACATTCAGGTAAC	172
ATP5G2	TTGTCTCCACTCCCTCCTTG	TGTGTCGATGTCCCTTGAAA	191
PLG	GTAGGTGGTCCCTGGTGCTA	CCTACAACCCTTCCAGGACA	137
CYP3A4	CTTTGGAAGTGGACCCAGAA	CGGGTTTTTCTGGTTGAAGA	164
STAT3	CCCCATACCTGAAGACCAAG	CTCCGAGGTCAACTCCATGT	185
CYP2E1	TCAAGCCATTTTCCACAGGA	CGATATCCTTTGGGTCAACGA	129
TIMP1	AATTCCGACCTCGTCATCAG	GTTGTGGGACCTGTGGAAGT	195
DPF2	CTCCTGGCTCACTCTTACGG	AAGGGGATTTTGGAGGTAGG	211
18S	GTGGAGCGATTTGTCTGGTT	CGCTGAGCCAGTCAGTGTAG	200
DPM1	GCAGTCCACGACAGAACAAA	CATCTGGGCTTCCATCATCT	150

Table 3 Performance of various, unsupervised or supervised classification tools for HCC-free cirrhosis samples

Samples2	Tool1
	UHC (%)	SVM (%)	KNNC (%)
A1-A7 + A15-A24	653	100	100
V8-V14 + V25-V34	71	80	70
CL1-CL18	100	70	80
All test samples	79	83	83

¹Unsupervised hierarchical clustering (UHC) was done with 52 cirrhotic or CL samples. Supervised training/testing procedures (KNNC; SVM) were each done by first separating these 52 samples into 22 training (A1-A7, V8-V14 and CL1-CL8) and 30 test samples (A15-A24, V25-V34 and CL9-CL18).

²20 transcript levels were measured in total RNA from every sample by qRT-PCR. These transcripts had the most significant difference in levels (Bonferroni-corrected ANOVA, P < 0.05) between alcoholism vs HCV vs CLs (ACSM2, ADH4, AHNAK, APOH, ARID1A, ARL2BP, ATP5G2, CYP2E1, CYP3A4, DPF2, DPM1, GSTA 1-2, HSCARG, HSD17B6, IFI27, MT1G, ORM2, PLG, STAT3, TIMP1).

³% of properly classified test samples.

Table 4 Over-representation of functional subsets in our list of 70 transcripts

Cell proliferation1(O = 7; E = 2.66; P = 0.012)	Regulation of cell migration (O = 2; E = 0.15; P = 0.009)	Blood vesseldevelopment (O = 3; E = 0.41; P = 0.007)	Lipid metabolism (O = 9; E = 3.55; P = 0.007)	Antigen processing and presentation (O = 3; E = 0.25; P = 0.001)	Acute inflammatory response (O = 4; E = 0.58; P = 0.002)	NADH dehydrogenase activity (O = 3; E = 0.36; P = 0.005)	Oxidoreductaseactivity (O = 3; E = 0.25; P = 0.001)
JAG1 (Hs.5908813)	JAG1 (Hs.590881)	JAG1 (Hs.590881)	CLU (Hs.436657)	HLA-A (Hs.181224)	CLU (Hs.436657)	NDUFA6 (Hs.274416)	CYP2E1 (Hs.12907)
IGFBP7 (Hs.479808)	PLG (Hs.143436)	PLG (Hs.143436)	CYP2J2 (Hs.152096)	HLA-B (Hs.77961)	ORM1 (Hs. 567311)	NDUFB10 (Hs.513266)	CYP2J2 (Hs.152096)
IL12RB2 (Hs.479347)		CUL7 (Hs.520136)	CYP3A4 (Hs.567254)	CD74 (Hs.591258)	ORM2 (Hs.522356)	NDUFS4 (Hs.528222)	CYP3A4 (Hs.567254)
PLG (Hs.143436)			HMGCS2 (Hs.59889)		STAT3 (Hs.463059)
TIMP1 (Hs.522632)			APOB (Hs.120759)
ARHGEF1 (Hs.438429)			HSD17B6 (Hs.524513)
CD74 (Hs.591258)			DPM1 (Hs.301898)
			LARGE (Hs.474667)
			CD74 (Hs.591258)

¹This transcript subset coding for proteins with a related function was identified by gene ontology with the GOTM tool;

²Significance of enrichment for the GO category between transcript number observed (O) and expected (E) in this cate gory;

³Hs. number: Unique transcript identifier.