FOXP3在激素受体阳性乳腺癌中的表达及其对MCF-7细胞侵袭和迁移的影响

FOXP3 expression in the hormone receptor positive breast cancer and its effect on invasion and migration of MCF-7 cell

目的探讨转录因子FOXP3在激素受体阳性乳腺癌组织中的表达及其对MCF-7细胞侵袭和迁移的影响。方法收集2010年9月-2011年10月河北医科大学第四医院乳腺中心收治的66例激素受体阳性乳腺癌患者的石蜡标本,采用免疫组化检测组织中FOXP3的表达情况,分析FOXP3表达与临床病理特征的关系;采用Kaplan-Meier法分析FOXP3表达与激素受体阳性乳腺癌预后的关系。采用qRT-PCR检测5种乳腺癌细胞系(MDA-MB-231、MDA-MB-453、MDA-MB-474、BR-3和MCF-7)中FOXP3mRNA的相对表达量,选取FOXP3表达量最高的MCF-7细胞系进行基因沉默。设置si-FOXP3转染组(转染si-FOXP3)、si-NC转染组(转染si-NC)与空白对照组(不做处理),采用Western blotting检测FOXP3蛋白的表达,划痕实验和Transwell实验检测细胞迁移和侵袭能力。结果FOXP3蛋白在激素受体阳性乳腺癌细胞质和细胞核中均有表达,细胞核FOXP3阳性表达率为40.9%(27/66),且在无淋巴结转移、Ki-67低表达、Luminal A型及TNM分期Ⅰ期乳腺癌组织中阳性表达率较高(P<0.05);细胞质FOXP3阳性表达率为54.6%(36/66)。Kaplan-Meier生存分析显示,细胞核FOXP3阳性组总生存率明显高于阴性组[96.30%(26/27)vs.82.05%(32/39),P=0.042],而细胞质FOXP3阳性组与阴性组总生存率差异无统计学意义[88.89%(32/36)vs.86.67%(26/30),P=0.715]。qRT-PCR检测结果显示,MCF-7细胞系FOXP3 mRNA相对表达量最高(P<0.05);沉默FOXP3后,MCF-7细胞的迁移和侵袭能力均较空白对照组明显增强(P<0.05)。结论FOXP3在激素受体阳性乳腺癌中的预后意义与表达部位相关,细胞核FOXP3的表达可提高总生存率,但细胞质FOXP3的意义尚不明确。FOXP3在乳腺癌MCF-7细胞系中发挥抑制细胞迁移和侵袭的抑癌作用,可作为激素受体阳性乳腺癌的潜在分子标志物。

Objective To investigate the expression of FOXP3 in hormone receptor-positive breast cancer tissues and its effect on invasion and migration of MCF-7 cells.Methods Formalin-fixed,paraffin-embedded blocks from 66 hormone receptor-positive breast cancer patients who received surgery at Breast Center of the Fourth Hospital of Hebei Medical University from September 2010 to October 2011 were used in this study.The protein expression level of FOXP3 was detected by immunohistochemistry.The relationship between the FOXP3 expression levels and the clinicopathologic features was analyzed.Kaplan-Meier analysis was used to assess the overall survival rate,according to the presence or absence of FOXP3 expression.The expression levels of FOXP3 mRNA in breast cancer cell lines(MDA-MB-231,MDA-MB-453,MDA-MB-474,BR-3,and MCF-7)were detected by qRT-PCR.The MCF-7 cell line,with the highest expression level of FOXP3,was selected for the following gene silencing experiment.The si-FOXP3 transfection group(transfected with si-FOXP3),si-NC transfection group(transfected with siNC),and blank control group(without treatment)were set.The expression of FOXP3 protein was detected by Western blotting,and the migration and invasion ability of cells was detected by scratch test and Transwell test.Results The FOXP3 exhibited a heterogeneous subcellular location in tumor cells.The positive expression ratio of FOXP3 in the nucleus was 40.90%(27/66),associated with negative lymph node metastasis,lower Ki-67 index,Luminal A typing,and TNM I stage(P<0.05);positive expression ratio of FOXP3 in the cytoplasm was 54.6%(36/66).Kaplan-Meier analysis indicated that nuclear FOXP3 expression correlated with better overall sur vival[96.30%(26/27)vs.82.05%(32/39),P=0.042],but the cytoplasmic FOXP3 was not significantly associated with overall survival[88.89%(32/36)vs.86.67%(26/30),P=0.715].qRT-PCR results showed that the MCF-7 cell line has the highest expression level of FOXP3 mRNA in five breast cancer cell lines above(P<0.05).Thus,we performed the gene silencing experiment in the MCF-7 cell line.Compared with control group,knockdown of FOXP3 significantly enhanced the migration and invasion of MCF-7 cells(P<0.05).Conclusion In hormone receptor-positive breast cancer,the prognostic significance of FOXP3 expression is relevant to the different subcellular localization of FOXP3.Nuclear FOXP3 suggested an improved overall survival,whereas cytoplasmic FOXP3 was not significantly relevant to survival.FOXP3 may reduce the migration and invasion of MCF-7 cells,and it may be a prognostic marker for breast cancer.