吉林大学学报(医学版)  2017, Vol. 43 Issue (05): 1009-1014

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于秀艳, 张晓伟, 野丽莉, 李铤, 刘晓峰, 吴雪峰
YU Xiuyan, ZHANG Xiaowei, YE Lili, LI Ting, LIU Xiaofeng, WU Xuefeng
hMAM联合MMP-9和C-erbB2 mRNA表达检测在乳腺癌外周血微转移诊断中的应用
Application of detection of expressions of hMAM combined with MMP-9 and C-erbB2 mRNA in peripheral blood in diagnosis of micrometastases of breast cancer
吉林大学学报(医学版), 2017, 43(05): 1009-1014
Journal of Jilin University (Medicine Edition), 2017, 43(05): 1009-1014
10.13481/j.1671-587x.20170528

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收稿日期: 2017-02-19
引用本文 0
于秀艳, 张晓伟, 野丽莉, 李铤, 刘晓峰, 吴雪峰. hMAM联合MMP-9和C-erbB2 mRNA表达检测在乳腺癌外周血微转移诊断中的应用[J]. 吉林大学学报(医学版), 2017, 43(05): 1009-1014.
YU Xiuyan, ZHANG Xiaowei, YE Lili, LI Ting, LIU Xiaofeng, WU Xuefeng. Application of detection of expressions of hMAM combined with MMP-9 and C-erbB2 mRNA in peripheral blood in diagnosis of micrometastases of breast cancer[J]. Journal of Jilin University (Medicine Edition), 2017, 43(05): 1009-1014.
hMAM联合MMP-9和C-erbB2 mRNA表达检测在乳腺癌外周血微转移诊断中的应用
于秀艳 , 张晓伟 , 野丽莉 , 李铤 , 刘晓峰 , 吴雪峰     
吉林省肿瘤医院检验科, 吉林 长春 130012
收稿日期: 2017-02-19
基金项目: 吉林省卫计委项目资助课题（20142021）
作者简介: 于秀艳(1986-), 女, 吉林省长春市人, 主任技师, 医学硕士, 主要从事临床分子免疫学方面的研究。
通信作者: 吴雪峰, 副主任技师(Tel:0431-85871050, E-mail:22061185@qq.com)
[摘要]: 目的: 探讨生物标记物人乳腺珠蛋白（hMAM）联合基质金属蛋白酶9（MMP-9）和人表皮生长因子受体2（C-erbB2）mRNA在乳腺癌外周血微转移患者中阳性表达情况，阐明其用于诊断乳腺癌外周血微转移的临床应用价值。方法: 选择74例乳腺癌患者、21例乳腺纤维腺瘤患者和10名健康人作为研究对象，所有患者均行手术治疗，采集外周血，运用实时荧光定量PCR检测外周血hMAM、MMP-9和C-erbB2 mRAN表达水平，比较hMAM、MMP-9和C-erbB2联合检测阳性表达率，并分析不同临床病理特征患者之间hMAM、MMP-9和C-erbB2联合检测的差异。结果: MMP-9、C-erbB2 mRNA阳性表达率在有无淋巴结转移患者间比较差异有统计学意义（χ2=6.450，P < 0.05；χ2=5.636，P < 0.05），hMAM mRNA阳性表达率在HER-2阳性与阴性患者间比较差异有统计学意义（χ2=5.804，P < 0.05）。乳腺癌患者hMAM及联合MMP-9和C-erbB2 mRNA阳性表达率分别为37.84%（28/74）、59.46%（44/74）和48.65%（36/74），三者联合阳性表达率为64.86%（48/74），均高于健康对照组（χ2=5.676，P < 0.05；χ2=3.102，P > 0.05；χ2=5.339，P < 0.05；χ2=2.310，P > 0.05）、乳腺纤维腺瘤组（χ2=8.438，P < 0.01；χ2=4.491，P < 0.05；χ2=7.982，P < 0.01；χ2=4.844，P < 0.05）和非乳腺癌组（对照组+乳腺纤维腺瘤组）（χ2=13.093，P < 0.01；χ2=6.471，P < 0.05；χ2=11.837，P < 0.01；χ2=6.103，P < 0.05）。与Ⅰ+Ⅱ期比较，Ⅲ+Ⅳ期乳腺患者hMAM mRNA单独及联合阳性表达率均增加，其中hMAM mRNA单独及联合C-erbB2 mRNA阳性表达率比较差异有统计学意义（χ2=5.157，P < 0.05；χ2=4.912，P < 0.05）。结论: hMAM诊断乳腺癌微转移的阳性率较低，联合MMP-9和C-erbB2 mRNA检测可以提高诊断阳性率，对于早期乳腺癌微转移诊断具有一定临床应用价值。
关键词: 乳腺肿瘤    微转移    人乳腺珠蛋白    基质金属蛋白酶9    人表皮生长因子受体2    
Application of detection of expressions of hMAM combined with MMP-9 and C-erbB2 mRNA in peripheral blood in diagnosis of micrometastases of breast cancer
YU Xiuyan, ZHANG Xiaowei, YE Lili, LI Ting, LIU Xiaofeng, WU Xuefeng     
Department of Clinical Laboratory, Jilin Provincial Tumor Hospital, Changchun 130012, China
[Abstract]: Objective: To explore the positive expressions of biological markers human mammaglobin(hMAM)combined with matrix metallopeptidase 9(MMP-9) and human epidermal growth factor receptor 2(C-erbB2) mRNA in peripheral blood of the breast cancer patients with micrometastases, and to clarify its clinical application value in diagnosis of the micrometastases in peripheral blood of the breast cancer patients. Methods: A total of 74 patients with breast cancer, 21 patients with breast fibroadenoma and 10 healthy controls were selected as the subjects. All the patients received surgical treatment and the peripheral blood was collected.The mRNA expression levels of hMAM, MMP-9 and C-erbB2 in peripheral blood were measured by the real-time fluorescent quantitative PCR.The positive expression rates of detection of hMAM, MMP-9 and C-erbB2 were compared, and the differences in detection of hMAM combined with MMP-9 and C-erbB2 between the patients with different clinicopathologic features were analyzed. Results: In the breast cancer patients with lymph node metastasis, the differences of positive expression rates ofMMP-9 and C-erbB2 mRNA were significant(χ2=6.450, P < 0.05;χ2=5.636, P < 0.05), and the difference of positive expression rate of hMAM mRNA was sigificant between HER-2 positive and negative patients(χ2=5.804, P < 0.05).The positive expression rates ofindividualhMAM and combined with MMP-9 and C-erbB2 were 37.84%(28/74), 59.46%(44/74) and 48.65%(36/74) in the breast cancer patients, the combined postive expression rate of these three kinds of markers was 64.86%(48/74), which were higher than those in healthy controls group(χ2=5.676, P < 0.05;χ2=3.102, P>0.05;χ2=5.339, P < 0.05;χ2=2.310, P>0.05), fibroadenoma ofbreast group(χ2=8.438, P < 0.01; χ2=4.491, P < 0.05; χ2=7.982, P < 0.01; χ2=4.844, P < 0.05) and non-breast cancer group(healthy controls group+ breast fibroadenoma group)(χ2=13.093, P < 0.01; χ2=6.471, P < 0.05; χ2=11.837, P < 0.01;χ2=6.103, P < 0.05).The positive expression rates ofindividualhMAM and the joint detection in the breast cancer patients at stage Ⅲ+Ⅳ were higher than those in the patients at stageⅠ+Ⅱ; the positive expression rates of individualhMAM and combined with C-erbB2 were statistically significant(χ2=5.157, P < 0.05; χ2=4.912, P < 0.05). Conclusion: hMAM has a low positive rate in the diagnosis of micrometastases in the breast cancer patients, while hMAM combined with MMP-9 and C-erbB2 detectioncould improve the positive rates.which presents some clinical application value for the early diagnosis of breast cancer micrometastases.
Key words: breast neoplasms     micrometastases     human mammaglobin     matrix metallopeptidase 9     human epidermal growth factor receptor 2    

人乳腺珠蛋白(human mammaglobin，hMAM)是1996年Watson等在乳腺癌组织中发现的一种具有高特异性的分泌性球蛋白[1-2]。有研究[3]表明：hMAM对于乳腺癌外周血微转移诊断具有重要临床意义。外周血循环中的肿瘤细胞是导致乳腺癌血行转移与复发的重要原因之一，监测外周血中的肿瘤细胞对早期诊断乳腺癌微转移具有重要意义。在国内外研究中，外周血hMAM mRNA及多种生物标记物联合检测已广泛应用于乳腺癌早期诊断，有常规逆转录PCR法[3-4]、巢式PCR[5]和实时荧光定量PCR[6-7]。hMAM与基质金属蛋白酶9(matrix metallopeptidase 9，MMP-9) 和人表皮生长因子受体2(C-erbB2) mRNA联合检测尚未见相关报道。本研究应用实时荧光定量PCR联合检测乳腺癌患者外周血中hMAM、MMP-9及C-erbB2 mRNA的表达，探讨其在诊断乳腺癌外周血微转移的临床应用价值。

1 资料与方法 1.1 一般资料

选择2015年3—12月在吉林省肿瘤医院乳腺治疗中心行手术治疗、术后经病理确诊且病历完整的患者作为研究对象。乳腺癌组74例，患者均为女性，年龄28~79岁，中位年龄49.2岁。乳腺癌分子分型依据2013年StGallen乳腺癌会议国际专家共识，组织学类型按WHO乳腺肿瘤组织学标准分类：浸润性导管癌61例，浸润性小叶癌13例。按照UICC 2003年制订的乳腺癌TNM分期标准：Ⅰ期11例，Ⅱ期17例，Ⅲ期29例，Ⅳ期17例；雌激素受体(ER)阳性19例，孕激素受体(PR)阳性21例，人表皮生长因子受体2(HER2) 阳性18例。乳腺纤维腺瘤组21例，均经术后病理确诊，均为女性，年龄22~75岁，中位年龄47.6岁；健康体检对照组10名，均为女性，年龄24~71岁，中位年龄48.3岁，经胸片和B超检查未发现乳腺相关性疾病。乳腺癌组、乳腺纤维腺瘤组和健康对照组研究对象年龄等一般情况比较差异无统计学意义(P＞0.05)，具有可比性。

1.2 入组标准及排除标准

乳腺癌入组患者结合临床症状、体征，均经病理学确诊为乳腺癌，术前未接受过放、化疗或生物治疗等干预措施，均具有完整的病历资料。排除存在严重心脏及肝肾疾病、自身免疫性疾病、严重并发症、长期嗜酒等情况患者。

1.3 主要试剂和仪器

人外周血淋巴细胞分离液(天津市灏洋生物制品科技公司)，RNA提取试剂盒(上海Omega公司)，逆转录试剂盒和SYBR Green Realtime PCR试剂盒(日本Takara公司)，hMAM、MMP-9、C-erbB2和β-actin基因引物(表 1)由宝生物工程(大连)有限公司设计及合成。罗氏LC 480Ⅱ荧光定量PCR仪, 罗氏诊断产品(上海)公司。

表 1 PCR引物序列 Table 1 Primer sequences of PCR
GenePrimer(5′-3′)Product
size (bp)
hMAMF: ACTCTGAGCAATGTTGAGGTGTTT
R: GCAATCCGTAGTTGGTTTCTCAC		129
MMP-9F: TGGGCTGCTGCTTTGCT
R: GCCTGTCGGTGAGATTGGTT		87
C-erbB2F: GACGAGACAGAGTACCATGCAGA
R: TCACACCATAACTCCACACATCAC		115
β-actinF: TGAGCGGGCTACAGCTT
R: TCCTTAATGTCACGCACGATTT		301
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1.4 标本采集

受试者于清晨空腹采取EDTA抗凝及非抗凝静脉血各3 mL，抗凝血用淋巴细胞分离液分离单个核细胞，提取总RNA用DEPC处理的无菌EP管保存，冻于-80℃待检测。

1.5 实时荧光定量PCR

按RNA提取试剂盒说明书提取单个核细胞RNA，NanoDrop 2000C测定RNA含量。取1 μg RNA进行逆转录反应合成cDNA，－80℃保存。总反应体系为25 μL：SYBR Premix Ex Taq Ⅱ 12.5 μL、上下游引物(10 μmol·L-1)各0.5 μL、cDNA 2 μL, 最后加入去离子水9.5 μL。混合后，进行扩增，反应条件为：95℃、30 s, 95℃、5 s, 62℃、30 s, 40个循环。每个样本的每个待测项目均检测3次，取平均值, 记录各样品的Ct值，采用2－ΔΔCt方法计算目的基因的相对表达量[8]，以健康对照组为对照。计算公式：标记物基因ΔCt＝标记物基因Ct－β-actin Ct；ΔΔCt＝乳腺癌或乳腺纤维腺瘤患者ΔCt－健康对照组ΔCt；肿瘤标记物基因的相对表达量为2-ΔΔCt，测定值2-ΔΔCt＞2为表达阳性，hMAM联合MMP-9和C-erbB2检测中任意一个指标阳性即为阳性表达。

1.6 统计学分析

采用SPSS 17.0统计软件进行统计学分析。乳腺癌组患者外周血中hMAM、MMP-9和C-erbB2 mRNA表达水平以x±s表示，计算采用描述性统计分析及正态性分析，资料均符合正态性分布; 计数资料组间阳性率比较采用χ2检验。检验水准α=0.05。

2 结果 2.1 实时荧光定量PCR标准曲线

以浓度为2、2×101、2×102、2×103、2×104和2×105拷贝数·μL-1标准品对数组为横坐标，以扩增CT值为纵坐标绘制标准曲线，线性方程和相关系数：β-actin：y = -3.624 9x + 42.575，R2 = 0.987 8；MMP-9：y = -3.287 1x + 43.643，R2 =0.986 4；C-erbB2：y =-3.964 3x + 41.203，R2 = 0.996 2；hMAM：y =-3.262 9x + 44.953，R2 = 0.989 7。结果显示其线性良好(图 1A)。

CO:Concentration of starting templates. 图 1 β-actin、MMP-9、C-erbB2和hMAM mRNA PCR标准曲线(A)和扩增曲线(B) Figure 1 PCR standard curves (A) and amplification curves (B) of β-actin (control), MMP-9, C-erbB2 and hMAM mRNA
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2.2 乳腺癌患者外周血中MMP-9、C-erbB2和hMAM mRNA表达水平

MMP-9、C-erbB2和hMAM mRNA在乳腺癌患者外周血中均有表达(图 1B)，其表达水平分别为1.91±1.24、5.63±3.18和8.24±2.72。

2.3 乳腺癌患者外周血中MMP-9、C-erbB2和hMAM mRNA表达阳性率与病理特征的关系

随着TNM分期恶性程度增加，乳腺癌患者外周血中MMP-9、C-erbB2和hMAM mRNA阳性表达率增加，但阳性表达率比较差异无统计学意义(P＞0.05)；MMP-9、C-erbB2 mRNA阳性表达率在有无淋巴结转移患者间比较差异有统计学意义(P＜0.05)，而hMAM mRNA阳性表达率在有无淋巴结转移患者间比较差异无统计学意义(P＞0.05)；三者在不同患者年龄、肿瘤大小、病理类型、ER和PR患者间比较差异无统计学意义(P＞0.05)；其中在HER-2阳性患者中MMP-9 mRNA阳性表达率较HER-2阴性患者高，但差异无统计学意义(P＞0.05)，而hMAM mRNA阳性表达率比较差异有统计学意义(P＜0.05)。见表 2。

表 2 不同病理特征乳腺癌患者外周血中MMP-9、C-erbB2和hMAM mRNA阳性表达率 Table 2 Positive expression rates of MMP-9, C-erbB2 and hMAM mRNA in peripheral blood of breast cancer patients with different clinicopathological characteristics
[n(η/%)]
Characteristic nMMP-9 mRNAPC-erbB2 mRNAPhMAM mRNAP
Age(year)0.8980.7920.825
 ≤ 504223 (54.76)13 (30.95)16 (38.10)
 ＞503218 (56.25)9 (28.13)13 (40.63)
Tumor size(d/cm)0.6320.7230.093
 ≤22813 (46.43)11 (39.29)9 (32.14)
 ＞24624 (52.17)20 (43.48)24 (52.17)
TNM stage0.5790.0850.085
 Ⅰ114 (36.36)3 (27.27)2 (18.18)
 Ⅱ179 (52.94)6 (35.29)4 (23.53)
 Ⅲ291 (58.62)15 (51.72)12 (41.38)
 Ⅳ1712 (70.59)12 (70.59)10 (58.82)
LM0.0110.0180.150
 Yes3926 (66.67)23 (58.97)21 (53.85)
 No3513 (37.14)11 (31.43)13 (37.14)
ER0.7030.9350.941
 Negtive5532 (58.18)15 (27.27)14 (25.45)
 Positive1912 (63.16)5 (26.32)5 (26.32)
PR0.9660.6950.817
 Negtive5330 (56.60)15 (28.30)14 (26.42)
 Positive2112 (57.14)5 (23.81)5 (23.81)
HER-20.8830.0000.016
 Negtive5630 (53.57)0 (0.00)14 (25.00)
 Positive1810 (55.56)16 (88.89)10 (55.56)
Pathological type0.7320.8330.551
 IDC6136 (59.02)17 (27.87)14 (22.95)
 ILC137 (53.85)4 (30.77)4 (30.77)
 LM: Lymphatic metastasis; ER: Restrogen receptor; PR: Progesterone receptor; IDC: Infitrating ductal carcinoma; ILC: Infiltrating lobular carcinoma.
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2.4 各组受试者外周血中hMAM及联合MMP-9和C-erbB2 mRNA阳性表达率

hMAM mRNA在健康人中未检测到，乳腺纤维腺瘤组仅检测到1例阳性表达。与健康对照组和乳腺纤维腺瘤组比较，乳腺癌组患者hMAM mRNA不论是单独还是联合表达阳性率均明显增加；乳腺癌组患者hMAM mRNA阳性表达率与健康对照组和乳腺纤维腺瘤组比较均增加(P＜0.05)，hMAM与MMP-9联合阳性表达率从37.84%增加到59.46%，与乳腺纤维腺瘤组比较差异有统计学意义(P＜0.05)；与C-erbB2联合检测阳性表达率从37.84%增加到48.65%，与健康对照组和乳腺纤维腺瘤组比较差异均有统计学意义(P＜0.05)；而在乳腺癌患者中3种基因联合阳性表达率更高，达到64.86%，与乳腺纤维腺瘤组比较差异有统计学意义(P＜0.05)。与非乳腺癌组(健康对照组+乳腺纤维腺瘤组)比较，乳腺癌组患者hMAM mRNA单独和联合表达阳性率差异均有统计学意义(P＜0.05)。见表 3。

表 3 各组受试者hMAM及联合MMP-9和C-erbB2 mRNA检测阳性表达率 Table 3 Positive expression rates of detection of hMAM and combined with MMP-9, C-erbB2 mRNA in patients in various groups
[n(η/%)]
GroupnhMAMMMP-9 +hMAMC-erbB2+hMAMMMP-9 +
C-erbB2+ hMAM
Healthy control100 (0.00)*a3 (30.00)1 (10.00)*b4 (40.00)
Breast fibroadenoma211 (4.76)**c7 (33.33)*d3 (14.29)**e8 (38.10)*f
Breast cancer7428 (37.84)△A44 (59.46)△△B36 (48.65)△△C48 (64.86)△D
 aχ2 =5.676, bχ2= 5.339, cχ2 = 8.438, dχ2 = 4.491, eχ2 = 7.982, fχ2 = 4.844, *P＜0.05, ** P＜0.01 compared with breast cancer group; Aχ2 = 13.093, Bχ2 = 6.471, Cχ2= 11.837, Dχ2 = 6.103, △P＜0.05, △△P＜0.01 compared with healthy control group+ breast fibroadenoma group.
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2.5 不同分期乳腺癌患者hMAM及联合MMP-9和C-erbB2 mRNA阳性表达率

随着肿瘤临床分期的增加，hMAM mRNA单独和联合阳性表达率均逐渐增加，与Ⅰ+Ⅱ期比较，Ⅲ+Ⅳ期乳腺癌患者hMAM及联合C-erbB2 mRNA阳性表达率均增加(P＜0.05)。见表 4。

表 4 不同分期乳腺癌患者hMAM及联合MMP-9和C-erbB2 mRNA阳性表达率 Table 4 Positive expression rates of hMAM and combined with MMP-9 and C-erbB2 mRNA in patients at different TNM stages
[n(η/%)]
TNM stagenhMAMhMAM+MMP-9hMAM+C-erbB2hMAM+MMP-9+C-erbB2
Ⅰ+Ⅱ286 (21.43)14 (50.00)9 (32.14)16 (57.14)
Ⅲ+Ⅳ4622 (47.83) *a30 (65.22)27 (58.70)*b32 (69.57)
 aχ2 = 5.157, bχ2 = 4.912, *P＜0.05 compared withstageⅠ+ Ⅱ.
表选项
3 讨论

hMAM在血液和骨髓中不表达，在正常乳腺上皮组织表达水平较低，而在乳腺癌组织中高表达，其他肿瘤，如直肠癌、胃癌、卵巢癌和前列腺癌等均不表达[9-12]，与CK-19、MUC-1和CEA等肿瘤标志物相比具有乳腺组织高特异性[13-14]。有研究[15]表明：hMAM与乳腺癌细胞的侵袭和转移有关。MMPs是一种促进肿瘤血管生成和降解细胞外基质最重要的酶类，而MMP-9是MMPs中相对分子质量最大的酶，主要通过降解细胞外基质和基底膜的结构蛋白Ⅳ型胶原，使肿瘤细胞具有侵袭能力，可在恶性肿瘤中高表达[16]，与乳腺癌转移和浸润有关[17-18]。C-erbB2在25%乳腺癌患者中存在过表达或基因异常扩增，控制着乳腺癌细胞的生长，在乳腺癌患者中，C-erbB2 mRNA过表达，与侵袭性表型和不良预后相关[19]。

本研究采用实时荧光定量PCR法对乳腺癌患者外周血中hMAM联合MMP-9及C-erbB2 mRNA进行检测，结果显示：hMAM mRNA阳性表达率与HER-2表达有关，与ER、PR表达及淋巴结是否无关。吴娜萍等[20]发现：hMAM mRNA阳性表达率与HER-2、ER和PR表达及淋巴结转移均无关；鲍慧铮等[7]也发现：hMAM mRNA表达阳性率与淋巴转移无关；颜蕴文等[21]发现：hMAM mRNA阳性表达与ER、HER-2表达和淋巴结是否转移有关，与PR表达无关；不同研究中hMAM mRNA阳性表达率与乳腺癌临床病理特征关系不尽相同，可能是因样本量和纳入标准存在差异而产生，应在大样本大数据前提下进行统计分析进一步证实。葛明广[22]通过实时荧光定量PCR法检测发现：外周血hMAM mRNA阳性表达率为32.1%；鲍慧铮等[7]通过实时荧光定量PCR法检测发现：外周血hMAM mRNA阳性表达率为38.6%；有文献[23-24]报道：常规逆转录PCR检测外周血hMAM mRNA阳性表达率为12%和18%，体现了实时荧光定量PCR的高敏感性特点。本研究结果显示：乳腺癌患者外周血hMAM mRNA阳性表达率[37.84%(28/74)]明显高于健康人和乳腺纤维腺瘤患者，与以往报道相符；与MMP-9及C-erbB2 mRNA联合表达，其阳性表达率也明显高于健康人及乳腺纤维腺瘤患者。本研究还发现：hMAM mRNA与C-erbB2 mRNA联合检测，乳腺癌患者与健康人及乳腺纤维腺瘤患者阳性表达率比较差异均具有统计学意义；而与MMP-9联合检测，在乳腺纤维腺瘤患者阳性表达率差异有统计学意义，在健康人中阳性表达率差异无统计学意义，这可能与本研究健康对照组入组只有10名，样本量太少有关。研究表明：随着分期增加，hMAM mRNA阳性表达率增加[25]，与Ⅰ+Ⅱ期比较，Ⅲ+Ⅳ期乳腺癌患者hMAM及联合C-erbB2 mRNA阳性表达率增加，与文献报道一致[26]。

综上所述，本研究结果显示：实时荧光定量PCR法检测乳腺癌外周血hMAM具有一定阳性率，而联合MMP-9及C-erbB2检测可以提高检测阳性率，应用于临床诊断乳腺癌微转移具有一定价值，hMAM检测的标准化问题有待进一步研究。

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