HIC-1基因在乳腺癌的表达及其对细胞增殖和凋亡的影响

The expression of HIC-1 gene and its effects on cell proliferation and apoptosis in breast cancer

目的:探讨癌高甲基化1(hypermethylated in cancer 1,HIC-1)基因在乳腺癌组织中的表达,以及恢复HIC-1基因表达对乳腺癌细胞增殖活力和凋亡的影响。方法:应用免疫组化方法测定乳腺癌组织芯片中80例癌组织的HIC-1蛋白表达,并分析其与临床病理的关系。应用甲基化特异性PCR法检测乳腺癌MDA-MB-231细胞中HIC-1基因启动子区域甲基化与基因表达的关系,5-氮杂-2′-脱氧胞苷(5-aza-2′-deoxycytidine,5-aza-CdR)抑制甲基化后细胞HIC-1的表达水平变化。应用CCK-8方法和流式细胞仪测定恢复细胞HIC-1表达对MDA-MB-231细胞增殖和凋亡的影响。结果:正常乳腺上皮组织HIC-1免疫组化染色呈强阳性,平均染色积分9.00±0,乳腺癌组织HIC-1染色明显降低,平均染色积分为4.58±1.22(P<0.001)。HIC-1的蛋白表达与病人年龄、发生部位以及是否出现淋巴结转移无关,但与肿瘤的组织学类型相关(P<0.05)。MDA-MB-231细胞HIC-1基因启动子区域呈完全甲基化,用5μM的5-aza-CdR处理后可抑制HIC-1基因启动子甲基化、恢复HIC-1表达,并呈现浓度依赖性。恢复HIC-1基因表达抑制MDA-MB-231细胞增殖活性,促进MDA-MB-231细胞凋亡(P<0.05)。结论:乳腺癌组织HIC蛋白表达和癌细胞HIC-1基因表达明显降低,去甲基化药物可恢复肿瘤细胞内HIC-1基因表达,从而达到抑制肿瘤细胞增殖并促进细胞凋亡的目的。

Objective To explore the expression of hypermethylated in cancerl （HIC-I） in breast cancer tissues, and its effects on cell proliferation and apoptosis by restoring HIC-1 gene expression in breast cancer cell line. Methods The protein expression of HIC-1 was detected by immunohistochemistry on tissue microarray of 80 cases with breast cancer. The relationship between the clinical-pathological parameters and the protein expression level was analyzed. The methylation degree of HIC-1 gene promoter was analyzed by methylation-specific PCR method on breast cancer cell line MDA-MB-231. P^T-PCR was used to examine mRNA expression of HIC-1 before and after 5-aza-2＇-deoxyeytidine （5-aza-CdR） treatment. Cell counting kit （CCK-8） and flow cytometry were used to assay the cell proliferation and apoptosis of MDA-MB-231 cells after restoring HIC-1 expression. Results Normal breast epithelial tissue showed strong staining for HIC-1 with an average score of 9.00 ：tO by immunohistochemistry, whereas breast cancer tissue showed weak staining for HIC-1 with an average score of 4_58＋1.22 （P〈 0.001）. The expression of HIC-1 protein was not related with age, location of the tumor or lymph node metastasis. However, it closely related to tumor histological type （P〈0.05）. The promoter region of HIC-1 gene was completely methylated in MDA-MB-231 cells. The methylation could be reversed by 5 IxM of 5-aza-CdR treatment, and subsequently, up-regulating HIC-1 gene expression in a concentration-dependent manner. Increase of HIC-1 gene expression inhibited the cell proliferation and promoted cell apoptosis of MDA-MB-231 cancer cell（P〈0.05）. Conclusions The expression of HIC-1 in breast cancer tissue and gene of cell line was decreased. HIC-1 gene expression in tumor ceils can be restored by demethylation drug 5-aza-CdlL Elevated HIC-1 expression could inhibit cell proliferation and induce cell apoptosis in MDA-MB-231 cancer cell.