Promoter Hypermethylation May Be an Important Mechanism of the Transcriptional Inactivation

Promoter hypermethylation may be an important mechanism of the transcriptional inactivation of ARRDC3, GATA5 and ELP3in invasive ductal breast carcinoma

Da Wang1,Peng-Na Yang3, Jin Chen1,Xian-Yao Zhou1,Qiu-Jun Liu1,Hong-Jiang Li2*, Chang-Long Li1*

Department of Biochemistry and Molecular Biology, School of Preclinical and Forensic Medicine, Sichuan University, Chengdu, China
Department of Thyroid and Breast Surgery, West China Hospital, Sichuan University, Chengdu, China
Key Laboratory of Bio-Resources and Eco-Environment,Ministry of Education, College of Life Sciences,Sichuan University, Chengdu, China.

D. Wang, J. Chen, X.-Y. Zhou,Q.-J. Liu, C.-L. Li*

Department of Biochemistry and Molecular Biology, School of Preclinical and Forensic Medicine, Sichuan University, 3-17 Renmin South Road, Chengdu, Sichuan 610041, People’s Republic of China, e-mail:

Telephone: +86-28-85403867, telefax: +86-28-85401102

H.-J. Li*

Department of Thyroid and Breast Surgery, West China Hospital, Sichuan University, 3-17 Renmin South Road, Chengdu, Sichuan 610041, People’s Republic of China,e-mail:

P.-N. Yang

Key Laboratory of Bio-Resources and Eco-Environment, Ministry of Education, College of Life Sciences, Sichuan University,No.24 South Section 1, Yihuan Road, Chengdu, Sichuan, 610065, People's Republic of China.

Supplementary Information

Supplementary Tables 1-4

Supplementary Fig. 1

Supplementary Table 1 Patients’ clinical information

Characteristics / N / %
Age (years)(52)
≤49 / 31 / 59.6
＞ 49 / 21 / 40.4
Location(52)
Left / 27 / 51.9
Right / 25 / 48.1
tumor size (mm)(52)
≤20 / 21 / 40.4
20-50 / 24 / 46.2
≥50 / 7 / 13.5
Grade (50)
Ⅰ / 3 / 6.0
Ⅱ / 22 / 44.0
Ⅲ / 25 / 50.0
pN (51)
Positive / 26 / 51.0
Negative / 25 / 49.0
ER status(52)
Positive / 39 / 75.0
Negative / 13 / 25.0
PR status(52)
Positive / 35 / 67.3
Negative / 17 / 32.7
Ki67 status(51)
Positive / 38 / 74.5
Negative / 13 / 25.5
HER-2 status(52)
Positive / 9 / 17.3
Equivocal / 15 / 28.8
Negative / 28 / 53.8
TNM staging(52)
Stage Ⅰ / 10 / 19.2
Stage Ⅱ / 29 / 55.8
Stage Ⅲ / 9 / 17.3
Stage Ⅳ / 4 / 7.7
CK5/6 status(51)
Positive / 6 / 11.8
Negative / 45 / 88.2
P63 status(32)
Positive / 5 / 15.6
Negative / 27 / 84.4

Numbers in parentheses are numbers of evaluated cases among 52 IDCs.N, number of evaluated cases; pN, lymph node status; ER,estrogen receptor;PR,progesterone receptor;HER-2,human epidermal growth factor receptor-2;TNM,tumor-node–metastasis;CK5/6, cytokeratin 5/6.

Supplementary Table 2Primer sequences and conditions for methylation-specific PCR

Gene / Primers / annealing
temperature(°C) / product
size(bp)
ARRDC3 M / F: 5’-GGTAGTTGGTAAAACGTTGTAGTGTC-3’ / 57.3 / 174
R: 5’-CTCGACGTACTATCTAAAACCCGT-3’
ARRDC3 U / F: 5’-TAGTTGGTAAAATGTTGTAGTGTTGG-3’ / 59.2 / 174
R: 5’-CCCTCAACATACTATCTAAAACCCAT-3’
ELP3 M / F: 5’-GTAGTTTAGGGGTTTTACGGGTC-3’ / 54.8 / 122
R: 5’-ATCGTAAAATATCGATCAATATATCGTA-3’
ELP3 U / F: 5’-GTAGTTTAGGGGTTTTATGGGTTGT-3’ / 53.6 / 122
R: 5’-ATCATAAAATATCAATCAATATATCATA-3’
GATA5 M / F: 5’-GTTTTATCGTCGCGTGTTTATC-3’ / 56.0 / 148
R: 5’-AAAAACTTCTCCAAATTTATACGAA-3’
GATA5 U / F: 5’-GGGTTTTATTGTTGTGTGTTTATTG-3’ / 56.0 / 150
R: 5’-AAAAACTTCTCCAAATTTATACAAA-3’
PAX6 M / F: 5’-ATGTAGGCGAGCGTTAGAGTTC-3’ / 57.3 / 182
R: 5’-TTACTTTATACAAAACAACGCCGA-3’
PAX6 U / F: 5’-GGAGATGTAGGTGAGTGTTAGAGTTT-3’ / 59.0 / 186
R: 5’-TTACTTTATACAAAACAACACCAAA-3’

ARRDC3,Arrestin domain-containing 3; ELP3, Elongator protein 3; GATA5,GATA binding protein 5;PAX6, paired box 6;M, methylation; U, unmethylation;F, forward; R, reverse.

Supplementary Table3qPCR primers for target gene examined

Primer name / Sequence(5’→3’) / Annealing
temperature(C) / product
size(bp)
ARRDC3-F / GACTATGTATCACCTCTTC / 54.6 / 154
ARRDC3-R / CTTCCTCCTCTTATCTCT
ELP3-F / AAGGTAAGAGTTCAAGTTCA / 51.0 / 98
ELP3-R / AAGAGGCAGTTCTACAAG
GATA5-F / CATCACAGACTTACGCACTT / 61.4 / 125
GATA5-R / ACAGGGCTACCAGACAAT
PAX6-3F / TTCTCGTTCAATATCTACTGTT / 58.3 / 140
PAX6-3R / CAACTTAGGAAGGTGTCTC
GAPDH-1F / GAGTCAACGGATTTGGTCGT / 60.6 / 238
GAPDH-1R / TTGATTTTGGAGGGATCTCG

F, forward; R, reverse; 3 of 3F or 3R, transcript variant 3; 1 of 1F or 1R, transcript variant 1

Supplementary Table 4Correlationsbetween mRNA expression changes and clinicopathological features

ARRDC3 / ELP3 / GATA5 / PAX6
Characteristics / N / Median / P / N / Median / P / N / Median / P / N / Median / P
Age (years)
≤49 / 26 / -2.69 / 26 / -1.68 / 14 / -3.45 / 25 / -2.15
＞ 49 / 14 / -2.08 / 0.461 / 14 / -3.86 / 0.148 / 6 / -7.04 / 0.083 / 10 / -2.03 / 0.770
Location
Left / 21 / -2.84 / 21 / -1.98 / 12 / -4.82 / 16 / -3.57
Right / 19 / -1.80 / 0.323 / 19 / -1.33 / 0.597 / 8 / -4.53 / 1.000 / 19 / -1.03 / 0.105
tumor size (mm)
≤20 / 14 / -1.73 / 14 / -1.70 / 6 / -4.03 / 11 / -2.14
20-50 / 20 / -2.79 / 20 / -1.92 / 11 / -4.93 / 18 / -2.02
≥50 / 6 / -4.63 / 0.592 / 6 / -7.01 / 0.311 / 3 / -6.80 / 0.700 / 6 / -5.35 / 0.416
Grade
Ⅰ / 2 / -1.86 / 2 / 0.26 / 1 / -4.49 / 1 / 2.47
Ⅱ / 16 / -2.68 / 16 / -1.53 / 8 / -2.60 / 15 / -2.14
Ⅲ / 20 / -2.74 / 0.631 / 20 / -2.36 / 0.362 / 10 / -4.99 / 0.701 / 17 / -2.35 / 0.492
pN
Positive / 18 / -3.00 / 18 / -1.92 / 9 / -4.56 / 15 / -2.34
Negative / 21 / -2.30 / 0.297 / 21 / -1.66 / 0.573 / 11 / -4.49 / 0.790 / 19 / -2.14 / 0.615
ER status
Positive / 31 / -2.30 / 31 / -1.74 / 16 / -4.74 / 28 / -2.39
Negative / 9 / -3.53 / 0.293 / 9 / -5.08 / 0.149 / 4 / -1.31 / 0.299 / 7 / 1.28 / 0.232
PR status
Positive / 28 / -2.44 / 28 / -1.92 / 14 / -4.52 / 26 / -2.58
Negative / 12 / -2.96 / 0.443 / 12 / -2.43 / 0.637 / 6 / -3.26 / 0.869 / 9 / 1.28 / 0.105
Ki67 status
Positive / 30 / -2.63 / 30 / -2.35 / 16 / -4.74 / 26 / -2.55
Negative / 9 / -2.23 / 0.947 / 9 / -1.15 / 0.083 / 4 / -1.74 / 0.299 / 8 / -2.07 / 0.543
HER-2 status
Positive / 6 / -3.86 / 6 / -1.57 / 3 / -11.0 / 5 / -1.04
Equivocal / 14 / -1.88 / 14 / -2.66 / 5 / -3.33 / 12 / -0.42
Negative / 20 / -2.35 / 0.489 / 20 / -1.70 / 0.771 / 12 / -4.99 / 0.405 / 18 / -3.30 / 0.174
TNM staging
Stage Ⅰ / 7 / -1.82 / 7 / -1.67 / 3 / 1.02 / 5 / -2.13
Stage Ⅱ / 23 / -2.67 / 23 / -2.05 / 11 / -6.31 / 20 / -2.20
Stage Ⅲ / 7 / -4.19 / 7 / -1.98 / 3 / -1.14 / 7 / -3.88
Stage Ⅳ / 3 / -1.62 / 0.859 / 3 / 1.66 / 0.685 / 3 / -3.57 / 0.076 / 3 / 1.06 / 0.865
CK5/6 status
Positive / 5 / -6.68 / 5 / -14.0 / 2 / 0.15 / 5 / -1.32
Negative / 34 / -2.27 / 0.275 / 34 / -1.92 / 0.257 / 18 / -4.74 / 0.059 / 29 / -2.35 / 0.543
P63 status
Positive / 5 / -2.40 / 5 / -1.40 / 2 / -7.21 / 4 / -3.18
Negative / 24 / -2.12 / 0.908 / 24 / -2.35 / 0.773 / 12 / -3.45 / 0.201 / 22 / -2.39 / 1.000

N, number of evaluated cases among 40 IDCs; P-values correspond to Mann-Whitney U or Kruskal-Wallis H test.

Supplementary Fig. 1Promoter region of ARRDC3 (a), ELP3 (b), GATA5 (c) and PAX6 (d) analyzed by MSP.For each region, CpG sites and islands around the transcription start site (TSS)predicted by MethPrimer are shown, and the location of the primers for the methylated and the unmethylated condition is given in the picture.