PDGF-D稳定沉默后对乳腺癌细胞SK-BR-3生物学功能的影响  

Study on the effect of PDGF-D shRNA on proliferation and invasion of breast cancer SK-BR-3 cells

作　　者：李红昌[1] 冯雯[1] 陈亚峰[1] 梅怡[1] 蔡含[1] 蒋一鸣[1] 陈腾[1] 殷佩浩[1] 奉典旭[1] 

机构地区：[1]上海中医药大学附属普陀医院普外科,上海200062

出　　处：《临床和实验医学杂志》2016年第3期203-210,共8页Journal of Clinical and Experimental Medicine

基　　金：上海市普陀区科委科研项目(2011PTKW007);上海中医药大学"创新团队发展计划"

摘　　要：目的通过慢病毒载体沉默乳腺癌细胞系SK-BR-3中血小板衍生生长因子D(PDGF-D)基因的表达,探讨PDGF-D基因沉默后对乳腺癌细胞增殖、凋亡及迁移的调控作用。方法应用RT-PCR及蛋白质印迹方法检测5种乳腺癌细胞系中PDGF-D的表达状况;设计人PDGF-D基因的shRNA慢病毒载体,在293T细胞中包装病毒并感染PDGF-D高表达的乳腺癌细胞系,运用蛋白质免疫印迹的方法鉴定其对PDGF-D基因的沉默效果;PDGF-D基因沉默后通过细胞增殖实验和软琼脂克隆形成实验检测其对细胞增殖能力的影响,并应用流式细胞技术检测细胞凋亡;运用Transwell迁移实验检测细胞的体外侵袭迁移能力。结果 PDGF-D在5种细胞系中存在差异性表达,其中具有低转移潜能的HT-29和SK-BR-3细胞与具有高转移潜能的LOVO、RKO和SW620细胞相比,具有更高的PDGFD表达水平(t=3.880,P<0.05)。选取低转移细胞株中PDGF-D表达相对较高的SK-BR-3作为研究对象,构建PDGF-D shRNA慢病毒载体沉默SK-BR-3细胞中PDGF-D的表达,蛋白质免疫印迹结果提示,PDGF-D稳定沉默的SK-BR-3细胞系构建成功;在稳定沉默PDGF-D基因后,MTT细胞增殖实验和软琼脂克隆形成实验表明SK-BR-3细胞增殖能力增强(F=75.23,P<0.001);流式细胞技术检测显示PDGF-D基因沉默后可抑制SK-BR-3细胞凋亡(F=84.44,P<0.001);细胞小室实验结果显示,PDGF-D沉默后SK-BR-3迁移能力增强(F=155.9,P<0.001)。结论 PDGF-D基因可能与乳腺癌的细胞增殖、凋亡与转移有关,进而参与了乳腺癌的发生、发展。Objective The shRNA lentiviral vector was constructed to silence PDGF-D expression in BrCa cells,in order to explore the role of PDGF-D in proliferation,apoptosis and migration of BRCA cells. Methods RT-PCR and Western-blot were used to detect PDGF-D expression profiles in 5 different BrCa cell lines,in order to confirm the target cell line. In vitro,the lentiviral vector was constructed to silence PDGF-D expression in SK-BR-3 cells. Then,the MTT and soft agar assay were used to detect cell proliferation. Additionally,cell apoptosis and cell migration after knockdown of PDGF-D were measured by the method of flow cytometry and transwell assay respectively. Results The expression of PDGF-D in protein and mRNA level in different cell lines of breast cancer were detected,among which the highly metastatic MDA-MB-231,SK-BR-3 cell lines showed higher PDGF-D expression than that in low metastatic MCF7 cell lines（ t = 3. 880,P〈0. 05）.After PDGF-D gene silencing by the lentivirus vector,the ability of growth and colony formation had been significantly decreased（ F = 75. 23,P〈0. 001）,together with an induction of apoptosis effect on SK-BR-3 cells（ F = 84. 44,P〈0. 001）. Moreover,SK-BR-3 cells with silenced PDGF-D diminished significantly aggressive potential in migration and invasion than other tested cells（ F = 155. 9,P〈0. 001）. Conclusion PDGF-D may be closely associated with the proliferation,apoptosis as well as migration of BRCA cells,which indicate that it plays a pivotal role in the development and progression of BRCA.

关 键 词：乳腺癌 血小板衍生生长因子D 细胞增殖 迁移 

分 类 号：R737.9[医药卫生—肿瘤]