Epigenetic basis of hepatocellular carcinoma: A network-based integrative meta-analysis

Table 1 Datasets used for the meta-analysis

No	GEO Accession	PubMed ID	HCC samples	Adjacent tissue samples
1		21500188	13	12
2		243066621	45	45
3		253762921	22	22
4	GSE592602	25945129	8	8
5	GSE297202	21747116	12	12
6	GSE180812	20165882	20	20
7	GSE37988	22234943	62	62
8	GSE44970	24012984	20	8
9	GSE54503	23208076	66	66
10	GSE57956	25093504	59	59
11	GSE607532	25294808	27	27

¹Only genes are reported, without information on CpG sites;

²Differential methylation data analysis performed in-house.

Table 2 Availability of clinicopathological information on the 11 datasets used for the integrative analysis of genome-wide DNA methylation

Clinical-pathological information	n = 11	%
Cirrhosis status in HCC samples	6	55
Child-Pugh/MELD Score	3	27
HCC Etiology	10	91
Alphafetoprotein level	5	45
Tumor grade	5	45
Tumor stage	5	45
Survival data	3	27

Table 3 Clinicopathological information of the individual datasets and methodology used for methylation analysis

Dataset	Year	PMID	GEO dataset	HCC (n)	Controls (n)	Liver cirrhosis in HCC samples	Etiology of liver disease (n)	Method
1	2011	21500188		13	12	Y (12)	HBV (3), HCV (4), alcoholic (6)	Human methylation 27 DNA analysis bead-chip
2	2014	24306662		45	45	Y (120), N (34)	HBV (149), HCV (1), nonviral (4)	Illumina GoldenGate Methylation Beadarray Cancer Panel I
3	2014	25376292		22	22	N/A	HBV (1), HCV (9), alcohol (4), other (8)	Infinium Humanmethylation 27 Beadchip
4	2015	25945129	GSE59260	8	8	N/A	HBV-HCV-(8)	Nimblegen Human DNA Methylation 3 x 720K CpG Island PI
5	2011	21747116	GSE29720	12	12	N/A	N/A	Agilent-017075 human hg 18 promoter 800-200
6	2010	20165882	GSE18081	20	20	Y (20)	HCV (20)	Illumina Golden Gate Methylation Beadarray Cancer Panel I
7	2012	22234943	GSE37988	62	62	N/A	HBV-HCV-(7), HBV + HCV-(36), HBV-HCV+(6), HBV + HCV+(13)	Illumina Human Methylation27 Beadchip
8	2013	24012984	GSE44970	20	8	N/A	HCV (8)	Human Methylation27 Beadchip
9	2013	23208076	GSE54503	66	66	Y (48), N (17), missing (1)	HBV-HCV-(19), HCV (19), HBV (13), HBV + HCV (4), missing (11)	Infinium Human Methylation 450K Beadchip
10	2014	25093504	GSE57956	59	59	Y (37), N (21)	HBV+(36), HBV-(23)	Intinium Ilumanmethylation27 Beadchip
11	2014	25294808	GSE60753	27	27	Y (26), N (1)	HBV (1), HCV (7), alcohol (9), other (10)	Iinfinium -450K Human Methy lation Beadchip

Table 4 Top canonical pathways identified by IPA for the genes corresponding to CpG sites in promoter

Ingenuity canonical pathways	-log (P-value)	Molecules
G-protein coupled receptor signaling	3.84E+00	DRD5, GNA11, VIPR2, ADCY5, ADRB1, CNR1, PIK3R5, NPY1R, FPR1, FFAR3, NFKBID, MC2R, PDPK1, GRM4, MC3R, CXCR2, PRKAR1B, DRD4, PDE6B, HCAR2, DUSP4, PTGDR
Transcriptional regulatory network in embryonic stem cells	3.42E+00	MYF5, SIX3, PAX6, GBX2, CDYL, FOXD3, ONECUT1, FOXC1
cAMP-mediated signaling	3.22E+00	DRD5, VIPR2, ADCY5, ADRB1, CNR1, NPY1R, FPR1, FFAR3, MC2R, GRM4, MC3R, CXCR2, PRKAR1B, DRD4, PDE6B, HCAR2, DUSP4, PTGDR

Table 5 Canonical pathways identified by IPA for the genes with methylation differences in the body of the gene in hepatocellular carcinoma

Ingenuity canonical pathways	-log (P-value)	Molecules
Aryl hydrocarbon receptor signaling	2.48E+00	GSTM1, CCND2, TFF1, ALDH1L2, GSTM2, TP73, GSTP1, ALDH3A1
G-protein coupled receptor signaling	2.03E+00	CAMK2B, RGS7, GABBR1, PDE4D, ADCY2, PDE1C, ADRA1D, NPR3, PDE10A, GRM6, PRKCG
cAMP-mediated signaling	1.73E+00	CAMK2B, RGS7, GABBR1, PDE4D, ADCY2, PDE1C, NPR3, PDE10A, GRM6