外周血白细胞<i>SLC45A4</i>和<i>AHSP</i>基因甲基化与乳腺癌关系的病例对照研究

付庆臻; 李大鹏; 符金铭; 刘宇鹏; 孙鸿儒; 张磊; 张显玉; 庞达; 赵亚双

doi:10.16462/j.cnki.zhjbkz.2024.01.009

外周血白细胞SLC45A4和AHSP基因甲基化与乳腺癌关系的病例对照研究

doi: 10.16462/j.cnki.zhjbkz.2024.01.009

1.
哈尔滨医科大学公共卫生学院流行病学教研室，哈尔滨 150081
2.
哈尔滨医科大学附属肿瘤医院乳腺外科，哈尔滨 150081

基金项目:

国家自然科学基金 81172743

黑龙江省自然科学基金 ZD2021H001

详细信息

通讯作者:
赵亚双，E-mail: zhao_yashuang@263.net

中图分类号: R181.3;R737.9
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出版历程
- 收稿日期: 2023-03-22
- 修回日期: 2023-09-27
- 网络出版日期: 2024-02-05
- 刊出日期: 2024-01-10

Methylation of SLC45A4 and AHSP gene in peripheral blood leukocytes and the risks of breast cancer: a case-control study

1.
Department of Epidemiology, School of Public Health, Harbin Medical University, Harbin 150081, China
2.
Department of Breast Surgery, Harbin Medical University Cancer Hospital, Harbin 150081, China

Funds:

National Natural Science Foundation of China 81172743

Heilongjiang Provincial Natural Science Foundation of China ZD2021H001

More Information

Corresponding author: ZHAO Yashuang, E-mail: zhao_yashuang@263.net

摘要

摘要: 目的探究外周血白细胞溶质载体家族45成员4(solute carrier family 45 member 4，SLC45A4)和α血红蛋白稳定蛋白(α hemoglobin stabilizing protein，AHSP)基因甲基化与乳腺癌发病的关系。方法通过差异甲基化分析等方法在GSE51032、GSE104942和GSE89093数据集中筛选与乳腺癌发病潜在相关的基因。采用病例对照研究，纳入545例乳腺癌患者和524例非乳腺癌对照作为研究对象。使用MethylTarget靶向测序检测目标基因甲基化并分析其与乳腺癌的关系。结果本研究共筛选出4个基因，控制混杂因素后，SLC45A4和AHSP高甲基化与乳腺癌关系的OR_adj分别为0.218(95% CI：0.158~0.299)和0.535(95% CI：0.384~0.741)。在不同亚组中SLC45A4高甲基化与乳腺癌的关联仍有统计学意义，OR_adj最高为0.427(95% CI：0.270~0.679)，最低为0.153(95% CI：0.085~0.274)，而AHSP高甲基化仅在管腔A型(Luminal A)、管腔B型(Luminal B)、雌激素受体阳性(estrogen receptor +, ER+)和≤60岁年龄组中与乳腺癌的关联有统计学意义。异质核糖核酸蛋白C(heterogeneous nuclear ribonucleoproteins C，HNRNPC)甲基化与乳腺癌的关系与公共数据结果相反，OR_adj为0.747(95% CI：0.569~0.980)，锌指蛋白425(zinc finger protein 425，ZNF425)甲基化与乳腺癌的关联无统计学意义(P=0.158)。结论外周血白细胞SLC45A4和AHSP基因高甲基化可能是乳腺癌发病的保护因素。
- 乳腺癌 /
- 甲基化 /
- 溶质载体家族45成员4 /
- α血红蛋白稳定蛋白
Abstract: Objective To investigate the relationship between peripheral blood leukocyte solute carrier family 45 member 4 (SLC45A4) and alpha hemoglobin stabilizing protein (AHSP) gene methylation and the risk of breast cancer. Methods Datasets GSE51032, GSE104942 and GSE89093 were used to identify genes that potentially associated with the risk of breast cancer through differential methylation analysis and other methods. A case-control study with 545 breast cancer patients and 524 cancer-free controls was carried out to validate the correlation between the methylation levels of the screened genes and breast cancer susceptibility. Results In this study, four genes were selected. After controlling for potential confounding factors, hypermethylation of SLC45A4 and AHSP was associated with lower breast cancer risk (OR_adj=0.218, 95% CI: 0.158-0.299; OR_adj=0.535, 95% CI: 0.384-0.741, respectively). The association of SLC45A4 hypermethylation with breast cancer remained statistically significant in different subgroups. The maximum value of OR_adj was 0.427(95% CI: 0.270-0.679) and the minimum value was 0.153 (95% CI: 0.085-0.274). However, the association of AHSP hypermethylation with breast cancer risk was statistically significant only in Luminal A, Luminal B subtypes, estrogen receptor + (ER+), and ≤60 age group. The association of heterogeneous nuclear ribonucleoproteins C (HNRNPC) methylation with the risk of breast cancer in our case-control study was inconsistent to the results of public datasets (OR_adj=0.747, 95% CI: 0.569-0.980). There was no statistically significant association between zinc finger protein 425 (ZNF425) methylation and breast cancer risk (P=0.158). Conclusions Hypermethylation of SLC45A4 and AHSP in peripheral blood leukocytes may serve as protective factors for breast cancer.
- Breast cancer /
- DNA methylation /
- Solute carrier family 45 member 4 /
- α hemoglobin stabilizing protein

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图 1 MethylTarget靶向测序2步PCR体系和反应条件

PCR, 聚合酶链式反应。

Figure 1. The two-step PCR panel and reaction conditions of MethylTarget sequencing

PCR, polymerase chain reaction.

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表 1 GEO数据集中基因甲基化差异及与乳腺癌的关系

Table 1. Gene methylation differences and association with breast cancer in the GEO datasets

亚组Subgroup	Δβ^①	P值value	OR值value (95% CI) ^②	P值value
GSE51032
DNMBP(cg02732134)	-0.052	< 0.001	0.114(0.065~0.188)	< 0.001
NA(cg03252499)	0.069	< 0.001	3.948(2.663~5.959)	< 0.001
SLC45A4(cg07437919)	-0.064	< 0.001	0.313(0.221~0.442)	< 0.001
AHSP(cg14387505)	-0.057	< 0.001	0.358(0.253~0.504)	< 0.001
HNRNPC(cg22872033)	0.055	< 0.001	2.441(1.722~3.482)	< 0.001
ZNF425(cg25009327)	-0.055	< 0.001	0.279(0.196~0.394)	< 0.001
GSE104942
DNMBP(cg02732134)	-0.012	0.155	0.571(0.322~1.001)	0.052
NA(cg03252499)	0.020	0.040	2.302(1.283~4.166)	0.005
SLC45A4(cg07437919)	-0.024	0.003	0.288(0.158~0.515)	< 0.001
AHSP(cg14387505)	-0.026	0.021	0.465(0.220~0.966)	0.041
HNRNPC(cg22872033)	0.012	0.155	1.805(1.034~3.183)	0.039
ZNF425(cg25009327)	-0.019	< 0.001	0.250(0.112~0.516)	< 0.001
GSE89093
DNMBP(cg02732134)	-0.033	0.146	0.194(0.065~0.535)	0.002
NA(cg03252499)	0.027	0.431	2.429(0.884~6.807)	0.086
SLC45A4(cg07437919)	-0.012	0.599	0.481(0.176~1.268)	0.144
AHSP(cg14387505)	-0.015	0.439	0.445(0.159~1.182)	0.110
HNRNPC(cg22872033)	0.029	0.203	2.729(1.028~7.561)	0.047
ZNF425(cg25009327)	-0.012	0.698	0.407(0.142~1.098)	0.083
注：①Δβ=β_病例-β_对照；②OR为甲基化水平每增加1个s与乳腺癌的关系。 Note: ①Δβ=β_case-β_control; ②OR is the association between each 1 s deviation increase in methylation level and breast cancer.

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表 2 病例组和对照组的基本信息

Table 2. Basic information of the cases and controls

特征Features	病例组 ^② Case group ^②	对照组 ^② Control group ^②	χ²值 value	P值 value
年龄组/岁Age group/years	52(46.0, 58.0)	50(44.0, 60.0)	-0.591	0.555
≤60	428(82.5)	386(77.2)	4.395	0.036
>60	91(17.5)	114(22.8)
BMI/(kg·m^-2) ^①	23.4(21.5, 25.7)	23.6(21.5, 26.0)	0.063	0.949
< 18.5	15(2.9)	22(4.4)	-0.005	0.996
18.5~ < 24.0	274(52.8)	252(50.4)
≥24.0	230(44.3)	226(45.2)
居住地Location			0.741	0.389
城市City	99(19.1)	85(17.0)
农村Rutal	420(80.9)	415(83.0)
乳腺癌家族史Family history of breast cancer			23.969	< 0.001
无No	478(92.1)	493(98.6)
有Yes	41(7.9)	7(1.4)
其他恶性肿瘤家族史Family history of other cancers			25.108	< 0.001
无No	378(72.8)	428(85.6)
有Yes	141(27.2)	72(14.4)
月经初潮年龄/岁 ^① Age at menarche/years ^①			-0.055	0.956
≥13	428(82.5)	413(82.6)
12~＜13	89(17.1)	85(17.0)
< 12	2(0.4)	2(0.4)
钼靶检查Mammography			8.935	0.003
否No	453(87.3)	402(80.4)
是Yes	66(12.7)	98(19.6)
良性乳腺疾病史History of benign breast disease			52.082	< 0.001
无No	345(66.5)	429(85.8)
有Yes	174(33.5)	71(14.2)
妇科手术史History of gynecological surgery			0.137	0.711
无No	318(61.3)	312(62.4)
有Yes	201(38.7)	188(37.6)
绝经Status of menopause			0.732	0.392
否No	228(43.9)	233(46.6)
是Yes	291(56.1)	267(53.4)
母乳喂养Breast feeding			0.382	0.536
否No	57(11.0)	49(9.8)
是Yes	462(89.0)	451(90.2)
避孕药使用Usage of contraception pill			4.139	0.042
否No	454(87.5)	457(91.4)
是Yes	65(12.5)	43(8.6)
激素类药物使用Usage of estrogen			1.652	0.199
否No	498(96.0)	487(97.4)
是Yes	21(4.0)	13(2.6)
活产次数Number of live birth			8.506	0.004
≤1	336(64.7)	279(55.8)
>1	183(35.3)	221(44.2)
流产次数Number of abortion			14.242	< 0.001
≤1	391(75.3)	424(84.8)
>1	128(24.7)	76(15.2)
乳腺癌一级亲属人数First-degree relatives with a history of breast cancer			16.618	< 0.001
0	490(94.4)	495(99.0)
≥1	29(5.6)	5(1.0)
吸烟Smoking			0.053	0.819
否No	449(86.5)	435(87.0)
是Yes	70(13.5)	65(13.0)
饮酒Drinking			0.042	0.838
否No	465(89.6)	446(89.2)
是Yes	54(10.4)	54(10.8)
注：①采用秩和检验; ②以M(P₂₅, P₇₅) 或人数(占比/%)表示。 Note: ① Rank-sum test; ② M(P₂₅, P₇₅) or number of people (proportion/%).

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表 3 病例组和对照组的基本信息

Table 3. Basic information of the cases and controls

	亚组Subgroup	β截断值分组 ^①Group of βcut off value ^①	病例组[人数(占比/%)]Case group[Number of people(proportion/%)]	对照组[人数(占比/%)]Control group[Number of people(proportion/%)]	OR值value (95% CI)	P值value	OR_adj值value(95% CI) ^②	P值value
SLC45A4	总体Overall
		hypo	223(43.3)	77(15.6)	1.000	< 0.001	1.000	< 0.001
		hyper	292(56.7)	418(84.4)	0.241(0.178~0.324)		0.218(0.158~0.299)
	年龄组/岁Age group/years
	≤60	hypo	187(44.0)	57(14.9)	1.000	< 0.001	1.000	< 0.001
		hyper	238(56.0)	326(85.1)	0.223(0.157~0.311)		0.186(0.128~0.267)
	>60	hypo	36(40.0)	20(17.9)	1.000	< 0.001	1.000	0.008
		hyper	54(60.0)	92(82.1)	0.326(0.169~0.614)		0.386(0.188~0.799)
	ER分组ER subgroup
	ER-	hypo	64(52.5)	77(15.6)	1.000	< 0.001	1.000	< 0.001
		hyper	58(47.5)	418(84.4)	0.167(0.108~0.256)		0.160(0.100~0.255)
	ER+	hypo	111(33.9)	77(15.6)	1.000	< 0.001	1.000	< 0.001
		hyper	216(66.1)	418(84.4)	0.358(0.256~0.500)		0.308(0.214~0.440)
	分子分型分组Molecular types of subgroup
	Luminal A	hypo	65(40.6)	77(15.6)	1.000	< 0.001	1.000	< 0.001
		hyper	95(59.4)	418(84.4)	0.269(0.181~0.401)		0.236(0.153~0.361)
	Luminal B	hypo	46(27.5)	77(15.6)	1.000	0.001	1.000	< 0.001
		hyper	121(72.5)	418(84.4)	0.485(0.320~0.739)		0.427(0.270~0.679)
	HER-2	hypo	35(51.5)	77(15.6)	1.000	< 0.001	1.000	< 0.001
		hyper	33(48.5)	418(84.4)	0.174(0.101~0.296)		0.153(0.085~0.274)
	Basal_like	hypo	29(53.7)	77(15.6)	1.000	< 0.001	1.000	< 0.001
		hyper	25(46.3)	418(84.4)	0.159(0.088~0.285)		0.156(0.081~0.297)
AHSP	总体Overall
		hypo	322(77.6)	294(65.2)	1.000	< 0.001	1.000	< 0.001
		hyper	93(22.4)	157(34.8)	0.541(0.399~0.730)		0.535(0.384~0.741)
	年龄组/岁Age group/years
	≤60	hypo	255(75.2)	213(61.7)	1.000	0.001	1.000	0.001
		hyper	84(24.8)	132(38.3)	0.532(0.382~0.737)		0.545(0.381~0.776)
	>60	hypo	67(88.2)	81(76.4)	1.000	0.049	1.000	0.243
		hyper	9(11.8)	25(23.6)	0.435(0.181~0.966)		0.574(0.216~1.414)
	ER分组ER subgroup
	ER-	hypo	59(68.6)	294(65.2)	1.000	0.541	1.000	0.562
		hyper	27(31.4)	157(34.8)	0.857(0.516~1.393)		0.855(0.497~1.440)
	ER+	hypo	233(79.3)	294(65.2)	1.000	< 0.001	1.000	< 0.001
		hyper	61(20.7)	157(34.8)	0.490(0.346~0.687)		0.452(0.309~0.654)
	分子分型分组Molecular types of subgroup
	Luminal A	hypo	118(83.7)	294(65.2)	1.000	< 0.001	1.000	< 0.001
		hyper	23(16.3)	157(34.8)	0.365(0.220~0.584)		0.337(0.197~0.554)
	Luminal B	hypo	115(75.2)	294(65.2)	1.000	0.023	1.000	0.015
		hyper	38(24.8)	157(34.8)	0.619(0.405~0.930)		0.561(0.348~0.886)
	HER-2	hypo	35(71.4)	294(65.2)	1.000	0.383	1.000	0.316
		hyper	14(28.6)	157(34.8)	0.749(0.380~1.405)		0.699(0.336~1.379)
	Basal_like	hypo	24(64.9)	294(65.2)	1.000	0.968	1.000	0.946
		hyper	13(35.1)	157(34.8)	1.014(0.489~2.017)		0.974(0.446~2.044)
HNRNPC	总体Overall
		hypo	315(62.5)	271(55.3)	1.000	0.258	1.000	0.158
		hyper	189(37.5)	219(44.7)	0.742(0.576~0.956)		0.747(0.569~0.980)
	年龄组/岁Age group/years
	≤60	hypo	255(61.0)	209(55.4)	1.000	0.112	1.000	0.117
		hyper	163(39.0)	168(44.6)	0.795(0.599~1.055)		0.794(0.594~1.059)
	>60	hypo	60(69.8)	62(54.9)	1.000	0.033	1.000	0.019
		hyper	26(30.2)	51(45.1)	0.527(0.289~0.945)		0.464(0.240~0.874)
	ER分组ER subgroup
	ER-	hypo	75(64.7)	271(55.3)	1.000	0.068	1.000	0.022
		hyper	41(35.3)	219(44.7)	0.676(0.441~1.025)		0.594(0.377~0.922)
	ER+	hypo	199(61.4)	271(55.3)	1.000	0.084	1.000	0.158
		hyper	125(38.6)	219(44.7)	0.777(0.583~1.034)		0.810(0.603~1.084)
	分子分型分组Molecular types of subgroup
	Luminal A	hypo	96(60.8)	271(55.3)	1.000	0.230	1.000	0.411
		hyper	62(39.2)	219(44.7)	0.799(0.553~1.149)		0.852(0.579~1.247)
	Luminal B	hypo	103(62.0)	271(55.3)	1.000	0.130	1.000	0.276
		hyper	63(38.0)	219(44.7)	0.757(0.526~1.083)		0.803(0.540~1.189)
	HER-2	hypo	43(66.2)	271(55.3)	1.000	0.099	1.000	0.045
		hyper	22(33.8)	219(44.7)	0.633(0.362~1.079)		0.549(0.299~0.975)
	Basal_like	hypo	32(62.7)	271(55.3)	1.000	0.310	1.000	0.355
		hyper	19(37.3)	219(44.7)	0.735(0.399~1.320)		0.738(0.381~1.391)
注：ER, 雌激素受体。 ① SLC45A4、AHSP和HNRNPC高低甲基化分组所依据的β截断值分别为0.961、0.743及0.025，hypo：＜对应位点的β截断值，hyper：≥对应位点的β截断值；②OR_adj为校正混杂因素后的OR，年龄亚组的校正因素为乳腺癌及其他恶性肿瘤家族史、钼靶检查、良性乳腺疾病史、避孕药使用、活产次数、流产次数及乳腺癌一级亲属人数，总体、ER和分型亚组的校正因素在年龄亚组的校正因素基础上增加年龄因素；表中数据存在缺失，缺失样本为测序深度低于100×的样本和乳腺癌分子分型缺失的样本。 Note: ER, estrogen receptor. ①The β cut-off values for the methylation levels in SLC45A4, AHSP, and HNRNPC are 0.961, 0.743, and 0.025, respectively. ′Hypo′ refers to values below the corresponding β cut-off, while ′Hyper′ denotes values that are equal to or exceed the β cut-off; ②OR_adj represents the OR adjusted for confounding factors. The age subgroup adjustments include family history of breast cancer and other cancers, mammography, history of benign breast diseases, contraceptive pill usage, number of live births, number of abortion, and the number of first-degree relatives with breast cancer. The overall, ER, and molecular type subgroups have additional adjustments for age on top of those used for the age subgroup. Some data in the table are missing. The missing samples are those with sequencing depths below 100× and those lacking molecular subtyping for breast cancer.

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