Methylation profile of the promoter CpG islands of 14 “drug-resistance” genes in hepatocellular carcinoma

Table 1 The “drug-resistance” genes containing the promoter CpG island

Genes	Location relative to transcription start site	Genes	Location relative to transcription start site
ABCC4	-487 to +466	GSTP1	-391 to +91
ABCC1	-356 to +406	hMLH1	-72 to +393
ABCC13	+138 to +386	KIAA1337	-553 to +413
ABCC3	-96 to +358	MAPK14	-371 to +319
ABCB11	+1 255 to +1 565	OCLN	-9 to +967
ABCC5	-219 to +87	POLB	-209 to +34
ABCC6	-229 to +89	RAF1	-466 to +399
ABCC8	-185 to +531	RALBP1	-901 to +325
ABCG2	-741 to -17	RRM2	-718 to +426
ATF2	-418 to +545	SKP2	-663 to +182
B2M	-170 to +83	SLC19A1	-719 to +268
BCAR3	-328 to +126	SLC22A1L	-265 to +158
BCL2	-7 to +573	SPF45	-547 to +92
BIRC4	-527 to -235	SRI	-251 to +257
CAT	+35 to +395	SSA2	-195 to +152
CFTR	-515 to -55	TOP2A	-123 to +506
DCK	-267 to +155	TOP2B	-805 to +393
ERBB2	+173 to +534	TP53	+130 to +337

Table 2 The “drug resistance” genes in this study

Resistance mechanism	Genes and ref.	Function
Plasma membrane	ABCG2	Belongs to the abc transporter family, appears to play a major role in the multidrug resistance
transporters/pump		phenotype of MCF-7 breast cancer cell line. When over-expressed, the transfected cells become
decrease or increase to		resistant to mitoxantrone, daunorubicin and doxorubicin, display diminished intracellular
concentration		accumulation reduce intracellular drug of daunorubicin, and manifest an
		ATP-dependent increase in the efflux of rhodamine
	CFTR	Belongs to the abc transporter family. MRP subfamily, involved in the transport of chloride ions
	RALBP1	Activates specifically hydrolysis of GTP bound to RAC1 and CDC42. Mediates ATP-dependent
		transport of S-(2,4-dinitrophenyl)-glutathione (DNP-SG) and doxorubicin(DOX) and is the major
		ATP-dependent transporter of glutathioneconjugates of electrophiles (GS-E) and DOX in
		erythrocytes. Can catalyze transport of glutathione conjugates and xenobiotics, and may
		contribute to the multidrug resistance phenomenon
Transcription factor	ATF2	Belongs to the bZIP family, ATF subfamily, binding the cAMP response element (CRE)
and/or tumor suppressor		(consensus sequence: 5’GTGACGT(A/C)(A/G)-3') as a transcription activator
	TP53	Acts as a tumor suppressor in many tumor types, involved in cell cycle regulation
		as a transcription activator that acts to negatively regulate cell division by controlling
		a set of genes required for this process
The enzymes involved in	CAT	Belonging to the catalase family ,serves to protect cells from the toxic effects of
drug activation or inactivation		hydrogen peroxide
metabolism abnormality	GSTP1	Belongs to the GST superfamily. Pi family; Conjugation of reduced glutathione to
		a wide number of exogenous and endogenous hydrophobic electrophiles
	TOP2B	Control of topological states of DNA by ATP-dependent breakage, passage and
		rejoining of double-stranded DNA
	DCK	Required for the phosphorylation of several deoxyribonucleosides and certain
		nucleoside analogs widely employed as antiviral and chemotherapeutic agents
Cell signaling alteration and	RAF1	Belongs to the Ser/Thr family of protein kinases. RAF subfamily. Involved in
cell cycle checkpoints alteration		the transduction of mitogenic signals from the cell membrane to the nucleus as a
		part of the Ras-dependent signaling pathway from receptors to the nucleus
	SKP2	Involved in regulation of G1/S transition as substrate recognition component of the
		SCF (SKP1-CUL1-F- box protein) E3 ubiquitin ligase complex which mediates the
		ubiquitination and subsequent proteasomal degradation of target proteins involved in
		cell cycle progression, signal transduction and transcription
Unclear mechanisms	B2M	Beta-2-microglobulin is the beta-chain of major histocompatibility complex class I
		molecules, Containing 1 immunoglobulin-like domain
	OCLN	May play a role in the formation and regulation of the tight junction (TJ)
		paracellular permeability barrier
	SPF45	Involved in nuclear mRNA splicing, via spliceosome

Note: All the information in this table was obtained from the web site of the OMIM section of the locus link (http://www.ncbi.nlm. nih.gov/LocusLink/) and GeneCards(http://bioinfo.weizmann.ac.il/cards/).

Table 3 The primers for MSP analysis

GenBank No.	Gene symbol		Sense 5’-3’	Antisense 5’-3’	Position	Size(bp)
NT_016354	ABCG2	m	TGTCGCGTTGAGTCGTTA	AACGTCCCCGATACTTCG	-698 to -464	235
		U	TGTGTTTTGTTGTGTTGAGTTGT	TCACTCTAATTCATTCCATTCAATC	-591 to -457	135
NT_005403	ATF2	m	GGGTCGGAATAACGAACG	ATCACCTCGAATACTCCTAACG	230 to 375	146
		U	GGAGGGGTTGGAATAATGAAT	CCCATTTCCCATCACCTCAA	220 to 379	160
NT_010194	B2M	m	ATTTGGTATTGCGTCGTTG	ACGAAACGAAACATCTCGAC	-128 to 23	151
		U	TTTTTAATTTGGTATTGTGTTGTTG	AACTCACACTAAATAACCTCCAAAC	-134 to 83	217
NT_009237	CAT	m	AGTAGCGGGTCGCGTAG	AACCACCCGAACCTATCG	118 to 372	255
		U	GGAAGGAGTAGTGGGTTGTGT	AACCACCCAAACCTATCACA	112 to 372	261
NT_007933	CFTR	m	AGAGGTCGCGATTGTCGTT	CGACTTTCTCCACCCACTACG	-316 to -114	203
		U	TTAAAGAGAGGTTGTGATTGTTGTT	TCCTTCACTCCCTCACCA	-322 to -174	149
NT_006216	DCK	m	TATACGCGCGGTTTCGT	CGCCGACGAATATCGAA	-168 to 27	195
		U	TTTTTTGTTATATGTGTGGTTTTGT	TACCCCTCAACCCTCACC	-176 to -75	102
NT_078018	OCLN	m	TGCGTTCGTTAGGTGAGC	CGAATCCCAACTCGAAAACG	538 to 753	216
		U	GTTAGGTGTGTTTGTTAGGTGAGT	CACACCTCTCTAATTCCCACA	532 to 772	241
NT_022517	RAF1	m	TCGGTCGTTTTGGAAGTC	CCCTAAAACGCGAAACG	-72 to 180	252
		U	GGTTTGGTTGTTTTGGAAGTT	CACCAAATATAACCACCTCCCACT	-2 to 183	185
NT_010859	RALBP1	m	GGGTAAGTCGTTCGTTTTCG	CCTCTCCGCTCAAACGACT	-495 to -215	281
		U	GTTAGTATTATATTGGGGTAAGTTGTTTG	CCCTTCATCCCCAAACTCA	-510 to -371	140
NT_006576	SKP2	m	GTCGTAGCGTCGTTCGTT	CTACAACCCGCTCTACTTCG	-198 to 10	208
		U	TTTTTTAGTTAGTTGTAGTGTTGTTTGTT	ACCCACTCTACTTCACAACCAC	-209 to 5	214
NT_077569	SPF45	m	AGTGTCGTTCGGTTTCGTT	CCTCGAAAACTCCGACTACG	-216 to -6	211
		U	GTGGAGTGTTGTTTGGTTTTGT	AACTTACATCTAACACCTCCCAAA	-220 to -67	154
NT_022517	TOP2B	m	TGGGTTTCGTCGTTTCGT	CCGCGCTAAACCCGAAC	-241 to -66	176
		U	GGTTGTTGGGTTTTGTTGTTTT	TTCTCCTCAACCACCACACTAA	-254 to -60	195
NT_010718	TP53	m	CGGAGTCGAGAGTTCGTG	CCGAAAACACTTTACGTTCG	157 to 279	123
		U	GTTGAAAATATATGGAGTTGAGAGTTT	CTTTCCACAACAATAACACACTTC	190 to 291	102
NT_033903	GSTP1	m	GCGATTTCGGGGATTTTA	ACGACGACGAAACTCCAA	-183 to 16	199
		U	GTTGGGGATTTGGGAAAG	TATAAAAATAATCCCACCCCACT	-230 to -28	203

Table 4 The early stage-specific change in methylation

Gene	C/M	N/M	P
GSTpi	24/30	17/30	0.052
CFTR	23/30	16/30	0.058