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
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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.
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.
引文
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[4]Bonnet D,Dick J E.Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell[J].Nature Med,1997,3:730-737.
[5]Scheel C,Eaton E N,Li S H,et al.Paracrine and autocrine signals induce and maintain mesenchymal and stem cell states in the breast[J].Cell,2011,145:926-940.
[6]Wing L Y,Chuang P C,Wu M H,et al.Expression and mitogenic effect offibroblast growth factor-9 in human endometriotic implant is regulated by aberrant production of estrogen[J].J Clin Endocrinol Metab,2003,88:5547-5554.
[7]Al-Hajj M,Wicha M S,Benito-Hernandez A,et al.Prospective identification of tumorigenic breast cancer cells[J].Proc Natl Acad Sci USA,2003,100:3983-3988.
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[9]Ponti D,Costa A,Zaffaroni N,et al.In vitro propagation of breast tumorigenic cancer cells with stem/progenitor cell properties[J].Cancer Res,2005,65:5506-5511.
[10]Velasco-Velázquez M A,Homsi N,De La Fuente M,et al.Breast cancer stem cells[J].Int J Biochem Cell Biol,2012,44:573-577.
[11]Smalley M,Piggott L,Clarkson R.Breast cancer stem cells:obstacles to therapy[J].Cancer Lett,2013,338:57-62.
[12]Vermeulen L,de Sousa e Melo F,Richel D J,et al.The developing cancer stem-cell model:clinical challenges and opportunities[J].Lancet Oncol,2012,13:e83-e89.
[13]Iliopoulos D,Hirsch H A,Struhl K.Metformin decreases the dose of chemotherapy for prolonging tumor remission in mouse xenografts involving multiple cancer cell types[J].Cancer Res,2011,71:3196-3201.
[14]Fillmore C M,Gupta P B,Rudnick J A.Estrogen expands breast cancer stem-like cells through paracrine FGF/Tbx3 signaling[J].Proc Natl Acad Sci USA,2010,107:21737-21742.
[15]Dontu G,El-Ashry D,Wicha M S.Breast cancer,stem/progenitor cells and the estrogen receptor[J].Trends Endocrinol Metab,2004,15:193-197.
[16]Dontu G,Abdallah W M,Foley J M,et al.In vitro propagation and transcriptional profiling of human mammary stem/progenitor cells[J].Genes,2003,17:1253-1270.
[17]Baselga J,Campone M,Piccart M,Everolimus in postmenopausal hormone-receptor-positive advanced breast cancer[J].N Engl J Med,2012:366:520-529.