着丝粒蛋白T在乳腺癌中表达及其启动子区变异的研究  

Down-expression of CENP-T and variation of its promoter region in BRCA

作　　者：毕佳欣 王晓雪[1] 赵若晗[1] 韩旭[1] 宋洁[1] BI Jiaxin;WANG Xiaoxue;ZHAO Ruohan;HAN Xu;SONG Jie(Department of Biology,Mudanjiang Medical College,Mudanjiang 157011,Heilongjiang,China)

机构地区：[1]牡丹江医学院生物学教研室,黑龙江牡丹江157011

出　　处：《癌变．畸变．突变》2022年第5期327-334,共8页Carcinogenesis,Teratogenesis & Mutagenesis

基　　金：国家自然科学基金(81171467);黑龙江省高校基本科研业务费项目(2019-KYYWF-1008);牡丹江医学院博士启动基金项目(2021-MYBSKY-018);大学生创新创业项目(200710229011)。

摘　　要：目的:研究着丝粒蛋白T(CENP-T)在乳腺癌中的表达及生物学功能,并分析其基因启动子序列突变及甲基化程度。方法:用免疫组化法检测26对乳腺癌组织及癌旁正常乳腺组织中CENP-T蛋白的表达;同时提取组织基因组DNA,测序分析CENP-T启动子区突变,及亚硫酸氢盐修饰后测序检测其启动子区CpG岛甲基化水平。另采用Western blot法检测MCF10A、MCF7和MDAMB-231细胞系中CENP-T蛋白的表达。构建CENP-T siRNA瞬时转染的MCF7细胞系,Western blot法评价干扰的效果。敲减CENP-T的表达后,采用MTT法和平板集落形成实验检测MCF7细胞增殖能力,流式细胞术检测细胞周期分布及细胞凋亡的变化。结果:乳腺癌组织中CENP-T蛋白表达水平低于癌旁正常乳腺组织,差异具有统计学意义(P<0.01)。乳腺癌组织中CENP-T启动子区检测出C1417T、C1545T、C1628G共3种突变,且启动子区甲基化水平增加49.5%。细胞系中敲减CENP-T基因表达后,MCF7细胞的增殖活性增强(P<0.05),集落形成能力增强(P<0.01),G2/M期细胞所占比例明显增加(P<0.01),细胞凋亡率下降(P<0.01)。结论:CENP-T蛋白水平表达下调促进乳腺癌细胞增殖且与启动子区序列突变及甲基化水平增加相关。此结果提示CENP-T表达下调与乳腺癌风险相关。OBJECTIVE:To explore expressions and biological functions of CENP-T in breast cancer,and to analyze the mutation and methylation status in gene promoter sequences.METHODS:Expressions of the CENP-T protein in 26 pairs of breast cancer tissues and adjacent normal breast tissues were detected by immunohistochemical method.Tissue genomic DNA was extracted and sequenced to analyze mutation of CENP-T in the promoter region and methylation levels of CpG islands in the promoter region after bisulfite modification.Western blot was used to detect CENP-T protein expression in three cell lines:MCF10A,MCF7and MDA-MB-231.The MCF7 cell line was constructed by CENP-T siRNA transient transfection,and effects of the interference were evaluated using Western blot.After the knockdown,expressions of CENP-T,and proliferation ability were examined using the MTT and the plate colony formation assays.Cell cycle distribution and apoptosis were testified by flow cytometry.RESULTS:CENP-T protein expressions in the breast cancer group was significantly lower than that in the adjacent normal breast group(P<0.01).Three mutations of C1417T,C1545T and C1628G were detected in the CENT-P promoter region of breast cancer tissues,and the methylation levels were increased by 49.5%.After CENP-T knockdown,proliferation activities of the MCF7cells were enhanced(P<0.05),colony formation abilities were enhanced(P<0.01),proportions of cells in the G2/M phase were significantly increased(P<0.01),and apoptosis rates were decreased(P<0.01).CONCLUSION:The down-regulation of CENP-T protein levels promoted proliferations of breast cancer cells which were associated with promoter region mutations and increased methylations.These results indicate that the downregulation of CENP-T expression was related to breast cancer risk.

关 键 词：着丝粒蛋白T 乳腺癌 启动子区 细胞增殖 

分 类 号：R73-3[医药卫生—肿瘤]