BRCA2对乳腺癌癌症干细胞的影响及其机制研究

目的 探讨乳腺癌易感基因2(breast cancer suscepbility gene 2, BRCA2)对上皮型乳腺癌癌症干细胞的影响及从Notch信号角度分析其机制。方法 利用本实验室前期建立的反转录病毒干扰载体pMSCVneo/eGFP-U6构建BRCA2的载体, 合成慢病毒颗粒, 转染上皮型乳腺癌细胞系MCF7细胞。筛选出单克隆细胞后, 利用Western blot法检测MCF7细胞中干扰BRCA2的效果。球形成试验分析干扰BRCA2后对MCF7上皮型乳腺癌干细胞的影响。克隆形成试验分析BRCA2下调后对MCF7上皮型乳腺癌干细胞增殖能力的影响。Western blot法检测乳腺癌癌症干细胞自我更新因子Notch1和Notch4在干扰BRCA2的上皮型乳腺癌中的影响。结果 干扰BRCA2的反转录病毒载体和对照载体测序正确, 可以合成反转录病毒。Western blot法发现干扰BRCA2的上皮型乳腺癌细胞系MCF7细胞中BRCA2的表达较对照细胞显著下调。球形成试验结果显示干扰BRCA2的上皮型乳腺癌细胞系MCF7细胞球形成数量较对照细胞显著增加。克隆形成试验结果显示干扰BRCA2的上皮型乳腺癌细胞系MCF7细胞克隆形成能力较对照细胞显著增加。Western blot法检测结果显示干扰BRCA2的上皮型乳腺癌细胞系MCF7细胞中Notch1的表达较对照细胞显著上调, 未在对照和干扰BRCA2的MCF7细胞中检测到Notch4的表达。结论 BRCA2沉默的上皮型乳腺癌细胞系MCF7细胞可通过上调Notch1表达使其形成癌症干细胞的能力增加。

Influence of BRCA2 on Breast Cancer Stem Cells and the Underlying Mechanism

Objective To explore the influence of breast cancer suscepbility gene 2 (BRCA2) on cancer stem cells of epithelial breast cancer and the underlying mechanisms especially the related to Notch signaling. Methods Using our previously established retrovirus-based RNAi vectorpMSCVneo/eGFP-U6, we synthesized the interfering vector and control vector to silence the BRCA2 in MCF7 cells. After single-cloned cell being selected, Western blot was performed to detect the silencing effect. Sphere forming assay was used to measure the potential alteration of cancer stem cells after silencing BRCA2. Clone forming assay was done to observe the alteration of proliferation ability when BRCA2 was downregulated.Western blot of Notch1 and Notch4 were carried out to analyze the potential mechanism of the alteration of cancer stem cells after silencing BRCA2 in epithelial breast cancer. Results The vector-containing mock and BRCA2-silencing sequence were confirmed correct.Compared with mock cells, downregulation of BRCA2 was obvious in BRCA2-silenced MCF7 cells. The number of cancer stem cells increased in BRCA2-silenced MCF7 cells; the proliferation ability elevated in BRCA2-silenced MCF7 cells, and Notch1 expression was upregulated in BRCA2-silenced MCF7 cells.No Notch4 expression was found in BRCA2-silenced MCF7 cells. Conclusion The potentiality to form cancer stem cells was increased in MCF7 cells when the BRCA2 gene was silenced, and Notch1(but not Notch4) upregulation may underline this alteration.