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中华乳腺病杂志(电子版)

中华乳腺病杂志(电子版) ›› 2012, Vol. 06 ›› Issue (01) : 54 -61. doi: 10.3877/cma. j. issn.1674-0807.2012.01.008

论著

乳腺癌中脆性组氨酸三联体异常甲基化能下调其mRNA 及蛋白的表达
赵典朋1, 贾丽燕1, 刘桂玲1,†(), 李世平1, 申安1   
  1. 1.261031 山东潍坊,山东省潍坊医学院附属医院普外二科
  • 收稿日期:2011-07-12 出版日期:2012-02-01
  • 通信作者: 刘桂玲

Effect of abnormal methylation of FHIT gene in down-regulating the expression of FHIT mRNA and protein in breast cancer

Dian-peng ZHAO1, Li-yan JIA1, Gui-ling LIU1,†(), Shi-ping LI1, An SHEN1   

  1. 1.Department of General Surgery, Affiliated Hospital of Weifang Medical University, Weifang 261031, Shandong Province, China
  • Received:2011-07-12 Published:2012-02-01
  • Corresponding author: Gui-ling LIU
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赵典朋, 贾丽燕, 刘桂玲, 李世平, 申安. 乳腺癌中脆性组氨酸三联体异常甲基化能下调其mRNA 及蛋白的表达[J/OL]. 中华乳腺病杂志(电子版), 2012, 06(01): 54-61.

Dian-peng ZHAO, Li-yan JIA, Gui-ling LIU, Shi-ping LI, An SHEN. Effect of abnormal methylation of FHIT gene in down-regulating the expression of FHIT mRNA and protein in breast cancer[J/OL]. Chinese Journal of Breast Disease(Electronic Edition), 2012, 06(01): 54-61.

目的

通过检测脆性组氨酸三联体(fragile histidine triad,FHIT)基因在乳腺癌组织和正常乳腺组织中的异常甲基化及其mRNA 和蛋白表达情况,探讨FHIT基因在乳腺癌发生发展中的作用,为乳腺癌生物治疗及判断预后提供实验依据。

方法

分别应用免疫组织化学SP 法、逆转录聚合酶链式反应(reverse transcriptionpolymerase chain reaction,RT-PCR)和甲基化特异性PCR(methylation specific PCR,MSP)检测50 例乳腺癌组织和距离肿瘤5 cm 以外的正常乳腺组织中FHIT 蛋白及其mRNA的表达情况。对FHIT 表达与乳腺癌临床病理指标的关系采用卡方检验, 对FHIT 甲基化与蛋白、mRNA 表达的关系进行Spearman 相关性分析。

结果

免疫组织化学染色显示:FHIT 蛋白在乳腺组织细胞浆内呈棕黄色颗粒状分布。FHIT 在乳腺癌和正常乳腺组织中的阳性表达率分别为32.0%(16/50)和88.0%(44/50),差异有统计学意义(χ2=32.667,P=0.000);RT-PCR 结果显示:在乳腺癌和正常乳腺组织中均有FHIT mRNA 的表达。乳腺癌组织中FHIT 条带吸光度(optical density, OD)值与相应的β-actin条带的OD 值的比值(0.1081±0.0128)明显低于正常乳腺组织(0.2088±0.0137),差异有统计学意义(P＜0.05),表明乳腺癌组织中FHIT 的表达量减少;MSP扩增结果显示:乳腺癌组织和正常乳腺组织的甲基化率分别为46.0%(23/50)、12.0%(6/50),差异有统计学意义(χ2=14.036,P=0.000)。FHIT 基因甲基化率与蛋白表达、有无淋巴结转移有关(P ＜0.05),而与肿瘤大小、组织学分级及TNM 分期无关(P ＞0.05)。

结论

FHIT 基因在乳腺癌中的甲基化能下调其mRNA 及蛋白的表达。

关键词: 乳腺肿瘤, 甲基化, 脆性组氨酸三联体

Objective

To detect the aberrant methylation of fragile histidine triad(FHIT) gene and the expression of the corresponding mRNA and proteins in breast tumor tissue and normal breast tissue, explore the role of FHIT gene in the development of breast cancer and provide experimental evidence for biotherapy and prognosis.

Methods

Immunohistochemical SP method, reverse transcription polymerase chain reaction (RT-PCR)and methylation specific PCR (MSP) were used to detect the protein and mRNA expressions of FHIT in 50 cases of breast tumor tissues and adjacent normal tissues over 5 cm away from the margin of tumor. The relationship between FHIT expression and clinic-pathological parameters were analyzed by chi-square test. The correlation between FHIT methylation and the FHIT mRNA and protein expression was processed by Spearman analysis.

Results

Immunohistochemical staining showed that FHIT protein was mainly located in the cytoplasma of breast cells, being brown granules. The positive expression ratios of FHIT were 32.0% in breast tumor tissue and 88.0% in normal breast issue, respectively. The difference was significant (P ＜0.05). RT-PCR results showed that FHIT mRNA was expressed in both breast tumor tissue and normal tissue. The ratio of FHIT band optical density (OD) value to OD value of β-actin was 0.1081±0.0128 in breast tumor tissue, which was significantly lower than 0.2088±0.0137 in normal tissue (P＜0.05). It indicated that the expression of FHIT was decreased in breast tumor tissue. MSP results showed that FHIT methylation rate were 46.0%(23/50)in tumor tissue, 12.0%(6/50)in normal tissues. The difference was statistically significant (P＜0.05). FHIT methylation rate was correlated with lymph node metastasis (P ＜0.05), not related with tumor size, histological grade, TNM stage (P ＞0.05).

Conclusions

Methylation of FHIT gene in breast cancer can significantly downregulate FHIT mRNA and protein expression.

Key words: Breast neoplasms, Methylation, FHIT
表1 FHIT 蛋白表达与肿瘤临床病理特征的关系
临床病理特征 例数 FHIT 阳性率(%) χ 2值 P值
+ -
肿瘤大小 2.401 0.981
≤2cm 28 9 19 32.1
＞2mm 22 7 15 31.8
TNM分期 0.023 0.879
Ⅰ+Ⅱ 32 10 22 31.2
Ⅲ 18 6 12 33.3
组织学分级 0.678 0.410
中、高分化 38 11 27 28.9
低分化 12 5 7 41.7
淋巴结转移 4.177 0.041
有 27 12 15 44.4
无 23 4 19 17.4
图1 乳腺癌组织和正常乳腺组织中FHIT mRNA 的表达 M:标记物;1-3:正常乳腺组织;4-7:乳腺癌组织
图2 FHIT mRNA 在乳腺癌和正常乳腺组织中的表达量
图3 MSP 法检测FHIT 基因启动子甲基化 1:标记物;2:非甲基化产物阳性;3:非甲基化和甲基化产物均阳性;4:甲基化产物阳性M:甲基化引物;U:非甲基化引物
表2 乳腺癌组织与正常乳腺组织中FHIT 甲基化的表达情况
组别 总例数 FHIT甲基化 阳性率(%) χ 2值 P值
+ -
乳腺癌 50 23 27 46.0 14.036 0.000
正常乳腺组织 50 6 44 12.0
表3 FHIT 基因甲基化与肿瘤临床病理特征的关系
临床病理特征 例数 FHIT甲基化 χ 2值 P值
+ -
肿瘤直径 0.351 0.555
≤2cm 26 13 13
＞2cm 24 10 14
TNM分期 0.234 0.569
Ⅰ+Ⅱ 28 15 13
Ⅲ 22 10 12
组织学分级 4.276 0.039
中、高分化 36 14 22
低分化 14 10 4
淋巴结转移 7.065 0.008
有 30 19 11
无 20 5 15
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