雌激素受体α短发夹RNA慢病毒载体的构建及其对乳腺癌干细胞的影响  

Construction and Identification of Lentiviral Interference Vector of ERα Gene and its Effect on Breast Cancer Stem Cells

作　　者：杜佩云[1,2] 程龙[2] 姜丽娜[2] 陈立涵[2] 林佳佳[2] 叶棋浓[2] 苏金为[1] 

机构地区：[1]福建农林大学生命科学学院,福建福州350002 [2]军事医学科学院生物工程研究所,北京100850

出　　处：《生物技术通讯》2015年第3期325-328,共4页Letters in Biotechnology

基　　金：国家自然科学基金(31200565);北京市自然科学基金(5132027)

摘　　要：目的:构建特异抑制雌激素受体α(ERα)基因的短发夹RNA(sh RNA)慢病毒表达载体,检测其对ERα的干扰效果,并探讨其对MCF7细胞中乳腺癌干细胞含量的影响。方法:以人的ERα核酸序列为靶标,根据sh RNA设计原则设计并化学合成特异的sh RNA序列,经退火处理后与p SIH-H1质粒连接,转化大肠杆菌DH5α后挑取单克隆并测序;将该克隆进行病毒包装并感染乳腺癌MCF7细胞,用嘌呤霉素进行筛选,最终得到稳定克隆;收取细胞,用Western印迹和q RT-PCR检测ERα在m RNA及蛋白水平上的变化,流式细胞术检测敲低ERα能否影响MCF7乳腺癌干细胞的含量。结果:经测序分析表明目标sh RNA序列成功插入载体,q RT-PCR检测稳定克隆,发现该sh RNA能有效抑制目的基因的m RNA转录水平,同时Western印迹检测发现内源性ERα量明显下降;流式细胞术检测发现ERα敲低的MCF7稳定克隆中雌激素诱导的乳腺癌干细胞含量上升的现象被明显抑制。结论:设计并构建的抑制ERα的sh RNA能够有效抑制ERα的表达,并下调MCF7细胞中乳腺癌干细胞的含量,为研究ERα在乳腺癌干细胞中的功能及机制奠定了实验基础。Objective： To construct a sh RNA interference vector of estrogen receptor α（ERα） gene,and to de-tect its effect on breast cancer stem cells（BCSCs）.Methods： Based on human ERα gene sequence,the specificsh RNA oligonucleotide sequences were designed by Invitrogen online software.The synthesized sequences were an-nealed to form double-strand oligonucleotides and cloned into interference vector plasmid p SIH-H1.DNA sequenc-ing confirmed that this sh RNA had successfully been cloned into the p SIH-H1 vector.The sh RNA interferencevector was then cotransfected into 293 T cells with the lentivirus packing vectors.MCF7 cells were infected withthe virus and selected by puromycin.MCF7 cells stably expressing ERα sh RNA were detected by real-time quanti-fication PCR and Western blot.Finally,the effect of inhibition of ERα on BCSCs were detected by flow cytome-try.Results： ERα sh RNA was successfully cloned into the p SIH-H1 vector.q RT-PCR and Western blot showedthat the sh RNA could suppress the expression of ERα.Knockdown of ERα could inhibit estrogen induced BCSCs.Conclusion： The constructed ERα sh RNA interference vector can effectively inhibit the expression of ERα and es-trogen induced BCSCs,which provided a basis for studying the function and mechanisms of ERα in BCSCs.

关 键 词：雌激素受体Α 肿瘤干细胞 短发夹RNA 流式细胞术 

分 类 号：Q78[生物学—分子生物学]