DNA methylation, microRNAs, and their crosstalk as potential biomarkers in hepatocellular carcinoma

Table 1 List of genes commonly silenced by DNA hypermethylation in primary hepatocellular carcinoma tumor samples

Gene	Cellular function	Freq	Ref.
14-3-3 ε	Cell cycle, mitogenic signaling	89%	[47]
APC	Cell proliferation, migration, apoptosis	44%-71%	[48-52]
ARHI	Cell proliferation and invasion	47%	[53]
ASS	Cell cycle and cell invasion	-	[54]
BASP1	Apoptosis	50%	[55]
BLU	Apoptosis	81%	[56]
CADM1	Cell adhesion and cell differentiation	41%	[57]
CASP8	Apoptosis	34%	[58]
CAV1	Cell cycle and proliferation	56%	[59]
CCND2	Cell cycle	24%-68%	[48,50]
CDH1	Cell adhesion and metastasis	34%	[52]
CDKN2A	Cell cycle	30%-70%	[49,50,60,61]
CFTR	Intercellular transport	77%-98%	[50,62]
CHFR	Cell cycle, protein degradation	35%	[63]
COX-2	Immune response, cell migration	35%	[64]
CSRP1	Cell proliferation and differentiation	56%	[59]
DKK1	Cell proliferation	36%	[52]
DKK3	Cell differentiation, embryonic development	-	[65]
DLC-1	Cell proliferation	24%-35%	[52,66]
DLEC1	Cell proliferation	71%	[67]
E2F1	Cell proliferation, migration, and differentiation	71%	[68]
FAM43B	Cell proliferation	60%	[69]
FBLN1	Cell adhesion and migration	50%	[70]
FHIT	Cell proliferation and apoptosis	65%	[56]
GSTP1	Cell metabolism and detoxification	70%-76%	[50,60,61,71]
HAI-2/PB	Cell proliferation and invasion	80%	[72]
HDPR1	Cell proliferation, differentiation, cellular signaling	51%	[73]
HINT1	Apoptosis	55%	[74]
IGFBP-3	Cell proliferation and growth	33%	[75]
KL	Cell proliferation and growth	81%	[76]
KLK10	Cell proliferation, survival, and cellular signaling	55%	[77]
LIFR	Signal transduction	48%	[78]
MAGE-A1	Cell differentiation, embryonic development	53%	[79]
MAGE-A3	Cell differentiation, embryonic development	74%	[79]
MAT1A	Cell metabolism	85%	[80]
MT1G	Cell proliferation and apoptosis	60%	[81]
MTM1	Cell differentiation	100%	[82]
MTSS1	Cell migration and metastasis	80%	[83]
MUC2	Immune system	62%	[84]
NORE1B	Cell proliferation and growth	62%	[85]
NQO1	Cell metabolism	50%	[86]
OXGR1	Cell growth and metabolism	78%	[77]
p14ARF	Cell proliferation and apoptosis	40%-42%	[68,71]
p15INK4B	Cell cycle	22%-61%	[71,87]
P16	Cell cycle	37%-83%	[48,71,87]
P21	Cell cycle	63%	[68]
P27	Cell cycle	48%	[68]
P300	Cell proliferation and differentiation	68%	[68]
P53	Cell proliferation, apoptosis, and DNA repair	14%	[68]
P73	DNA repair and apoptosis	35%	[71]
PCDH10	Cell adhesion and migration	76%	[88]
PER3	DNA repair	60%	[89]
PRDM2	Cell metabolism	-	[90]
PTEN	Cell proliferation and migration	16%-22%	[87,91]
RASSF1A	Cell proliferation, migration, and apoptosis	64%-88%	[48,51]
RB	Cell cycle, apoptosis	24%-32%	[68,87]
RECK	Cell invasion and metastasis	55%	[92]
RELN	Cell adhesion	37%	[93]
RIZ1	Cell proliferation and apoptosis	29%-67%	[48,94,95]
RUNX-3	Apoptosis	38%-48%	[50-52]
SFRP1	Cell proliferation and differentiation	37%-45%	[51,52,96]
SFRP2	Cell proliferation and differentiation	48%-54%	[97]
SFRP5	Cell proliferation and differentiation	39%	[96]
SLIT2	Cell migration	83%	[98]
SOCS1	Cell growth and survival	39%-72%	[9,48,90,99]
SOX1	Cell proliferation	57%	[100]
SOX17	Cell differentiation and embryonic development	82%	[101]
SPARC	Cell growth and invasion	75%	[102]
SPINT2	Cell proliferation and growth	60%	[50]
SRD5A2	Cell proliferation and androgenic physiology	50%	[55]
Survivin	Apoptosis and cell proliferation	33%	[58]
TFPI2	Matrix remodelling	47%	[103]
TIP30	Apoptosis and metastasis	47%	[104]
UCHL1	Cell metabolism and protein degradation	44%	[105]
UNC5C	Cell migration	26%	[106]
Vimentin	Cell migration and signaling	56%	[107]
WIF-1	Cell proliferation	49%-61%	[52,65,108]
WT1	Cell proliferation and survival	54%	[68]
ZHX2	Cell proliferation, differentiation, and development	47%	[109]

Freq: Frequency of hypermethylation.

Table 2 MicroRNA genes targeted by DNA hypermethylation in hepatocellular carcinoma

MicroRNA	Validated gene targets in HCC	Biological functions	Ref.
miR-1	FoxP1, MET, HDAC4	Regulates cell growth, replication and clonogenic survival	[164-166]
miR-10a	HOXB3, HOXA3, HOXA1, HOXD10, USF2, HOXD4	Regulates embryonic development and cell differentiation	[167]
miR-124	CDK6, VIM, SMYD3, E2F6, IQGAP1	Regulates cell cycle progression (G1-S checkpoint), apoptosis, and metastasis	[166,168]
miR-1247	ADAM15, CIT, MMP24	Regulates cell proliferation and migration	[166]
miR-125b	PIGF, MMP2, MMP9, SIRT7, LIN28B	Regulates cell proliferation, anchorage-independent growth, cell migration, invasion, and angiogenesis	[169]
miR-129-2	SOX4, VCP, IκBα	Regulates apoptosis	[166,170]
miR-132	AKT1, CTNNB1, CCND1	Regulates cell proliferation	[171]
miR-203	ABCE1, CDK6	Regulates cell growth and cell cycle progression	[168]
miR-320	NRP1, CTNNB1	Regulates cell migration, proliferation, and metasatasis	[167]
miR-335	ROCK1, MAPK1, LRG1, MYCN	Regulates migration and cell proliferation	[172]
miR-596	LGALS3BP, FOXP1, IGF2BP2	Regulates cell growth and induces apoptosis	[166]
miR-663	JUNB, JUND,	Regulates cell proliferation	[165]
miR-9	MTHFD2, HOXD1, MMP14	Regulates cell proliferation, invasion, metastasis, and angiogenesis	[166]

HCC: Hepatocellular carcinoma.

Table 3 Pathways and molecular functions regulated by methylation-silenced microRNA gene targets

No.	Panther	Genes	Gene	Contrib
GO molecular function1
1	Binding (GO:0005488)	2799	40.60%	33.80%
2	Catalytic activity (GO:0003824)	2126	30.80%	25.70%
3	Transcription regulator activity (GO:0030528)	920	13.30%	11.10%
4	Receptor activity (GO:0004872)	696	10.10%	8.40%
5	Structural molecule activity (GO:0005198)	558	8.10%	6.70%
6	Enzyme regulator activity (GO:0030234)	494	7.20%	6.00%
7	Transporter activity (GO:0005215)	418	6.10%	5.00%
8	Ion channel activity (GO:0005216)	152	2.20%	1.80%
9	Translation regulator activity (GO:0045182)	57	0.80%	0.70%
10	Motor activity (GO:0003774)	53	0.80%	0.60%
GO Biological process2
1	Metabolic process (GO:0008152)	3277	47.50%	20.80%
2	Cellular process (GO:0009987)	2564	37.20%	16.30%
3	Cell communication (GO:0007154)	1793	26.00%	11.40%
4	Developmental process (GO:0032502)	1315	19.10%	8.40%
5	Transport (GO:0006810)	1190	17.30%	7.60%
6	Immune system process (GO:0002376)	956	13.90%	6.10%
7	System process (GO:0003008)	927	13.40%	5.90%
8	Cell cycle (GO:0007049)	797	11.60%	5.10%
9	Cellular component organization (GO:0016043)	630	9.10%	4.00%
10	Response to stimulus (GO:0050896)	613	8.90%	3.90%
GO cellular component3
1	Intracellular (GO:0005622)	486	7.10%	50.40%
2	Extracellular region (GO:0005576)	233	3.40%	24.10%
3	Plasma membrane (GO:0005886)	86	1.20%	8.90%
4	Ribonucleoprotein complex (GO:0030529)	84	1.20%	8.70%
5	Protein complex (GO:0043234)	76	1.10%	7.90%
GO protein class4
1	Nucleic acid binding (PC00171)	1144	16.60%	12.60%
2	Transcription factor (PC00218)	920	13.30%	10.20%
3	Hydrolase (PC00121)	691	10.00%	7.60%
4	Receptor (PC00197)	690	10.00%	7.60%
5	Transferase (PC00220)	662	9.60%	7.30%
6	Enzyme modulator (PC00095)	634	9.20%	7.00%
7	Signaling molecule (PC00207)	481	7.00%	5.30%
8	Transporter (PC00227)	457	6.60%	5.00%
9	Cytoskeletal protein (PC00085)	402	5.80%	4.40%
10	Kinase (PC00137)	316	4.60%	3.50%
Pathway5
1	Gonadotropin releasing hormone receptor pathway (P06664)	159	2.30%	4.80%
2	Wnt signaling pathway (P00057)	156	2.30%	4.70%
3	Inflammation mediated by chemokine and cytokine signaling pathway (P00031)	134	1.90%	4.00%
4	Integrin signalling pathway (P00034)	116	1.70%	3.50%
5	Angiogenesis (P00005)	98	1.40%	3.00%
6	EGF receptor signaling pathway (P00018)	84	1.20%	2.50%
7	Cadherin signaling pathway (P00012)	80	1.20%	2.40%
8	Huntington disease (P00029)	77	1.10%	2.30%
9	TGF-beta signaling pathway (P00052)	73	1.10%	2.20%
10	PDGF signaling pathway (P00047)	74	1.10%	2.20%
KEGG
hsa01100 metabolic pathways (434)
hsa05200 pathways in cancer (189)
hsa04151 PI3K-Akt signaling pathway (162)
hsa04010 MAPK signaling pathway (137)
hsa05205 proteoglycans in cancer (134)
hsa05166 HTLV-I infection (123)
hsa04510 Focal adhesion (116)
hsa04810 regulation of actin cytoskeleton (115)
hsa05202 transcriptional misregulation in cancer (100)
hsa04144 endocytosis (100)

¹Total genes n = 6893, total function hits n = 8282;

²Total genes n = 6893, total process hits n = 15740;

³Total genes n = 6893, total component hits n = 965;

⁴Total genes n = 6893, total protein classess n = 9050;

⁵Total genes n = 6893, total pathway hits n = 3313. Contrib: Contribution; PI3K: Phosphatidylinositol-3-kinase; MAPK: Mitogen-activated protein kinase; HTLV: Human T-lymphotropic virus 1; EGF: Epidermal growth factor; PDGF: Platelet-derived growth factor; TGF-β: Transforming growth factor beta.