Identification of methylation profile of HOX genes in extrahepatic cholangiocarcinoma

Table 1 Primer sequences, fragments and annealing temperature used for bisulfite-polymerase chain reaction and reverse transcription-polymerase chain reaction

Genes	Primer 5'→3'	Annealing temperature (°C)	Size (bp)
BSP
HOXA2	L: TGTTTTAATAGAATTTATGTGGTTGG	56	190
	R: ATAACTACCCTCTACCTCCCCC
HOXA5	L: GTTTTGGAGAAATATTATATAAAAGTTATT	50	145
	R: CAATTAAAAAAATAAATCCTACCC
HOXA11	L: TGAGTATAAGTATGTTGTATGGGGG	60	148
	R: TTATAACCACCTCAAAAAAAACAAC
HOXB4	L: TAGAGGTGAGGTAGAATAGGAGGGT	60	204
	R: ACCCAACACCAAAATTTACATAAAA
HOXD13	L: GTGGGTTTAGTTAGGTTTGGGT	60	239
	R: TCTAACCCTCTCTCCCTCTATAAAC
RT-PCR
HOXA2	F: GTCACTCTTTGAGCAAGCCC	59	345
	R: TAGGCCAGCTCCACAGTTCT
HOXA5	F: ACTCCGGCAGGTACGGCTACG	62	259
	R: -CCGCTGGAGTTGCTTAGGGAG
HOXA11	F: TGCCAAGTTGTACTTACTACGTC	61	181
	R: GTTGGAGGAGTAGGAGTATGTA
HOXB4	F: GTGCAAAGAGCCCGTCGTCTA CC	65	161
	R: CGTGTCAGGTAGCGGTTGTAGTG
HOXD13	F: TGCTCCTCTTCTGCCGTTGT	55	467
	R: CCTGTGGCTGGTCCTTGGT

BSP: Bisulfite-polymerase chain reaction; RT-PCR: Reverse transcription-polymerase chain reaction.

Table 2 Methylated HOX genes identified by methyl-DNA immunoprecipitation microarray

No.	Genes	Description	CpG location	Strand	Position of gene
1	HOXA2	HomeoboxA2	chr7:27109706-27110004	-	Promoter
2	HOXA5	HomeoboxA5	chr7:27149138-27152087	-	Promoter
3	HOXA11	HomeoboxA11	chr7:27191575-27192154	-	Promoter
4	HOXB2	HomeoboxB2	chr17:43982786-43983443	-	Promoter
5	HOXB4	HomeoboxB4	chr17:44010214-44010603	-	Promoter
6	HOXB5	HomeoboxB5	chr17:44025521-44026457	-	Promoter
7	HOXC10	HomeoboxC10	chr12:52664963-52666369	+	Promoter
8	HOXD1	HomeoboxD1	chr2:176761203-176762596	+	Promoter
9	HOXD9	HomeoboxD9	chr2:176694670-176696537	+	Promoter
10	HOXD12	HomeoboxD12	chr2:176672308-176673755	+	Promoter
11	HOXD13	HomeoboxD13	chr2:176665300-176666525	+	Promoter

Table 3 HOXA2, HOXA5, HOXA11, HOXB4, and HOXD13 methylation status of 30 cholangiocarcinoma patients and 9 normal controls n (%)

Genes	Age (n = 39)			Normal (n = 9)	Patients (n = 30)			P value1
	< 60 yr (n = 11)	> 60 yr (n = 28)	P value		Tumor stageI/II (n = 3)	Tumor stage III/IV (n = 27)	P value
HOXA2	4 (7.69)	6 (15.39)	0.424	7 (77.78)	1 (33.33)	2 (7.41)	0.511	< 0.001
HOXA5	3 (7.69)	7 (17.95)	1.0	9 (100)	1 (33.33)	0 (0)	0.206	< 0.001
HOXA11	2 (5.13)	7 (17.95)	1.0	9 (100)	1 (33.33)	0 (0)	0.206	< 0.001
HOXB 4	3 (7.69)	5 (12.82)	0.663	6 (66.67)	2 (66.67)	0 (0)	0.007	0.001
HOXD13	4 (7.69)	6 (15.39)	0.424	8 (88.89)	1 (33.33)	1 (3.70)	0.193	< 0.001

Fisher exact test was performed to identify differences in median expression values within the tissue samples.

¹Fisher test between normal controls and cholangiocarcinoma patients.