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ADAM17-siRNA对MCF-7人乳腺癌细胞增殖的影响
Effects of ADAM17-siRNA on the Proliferation of MCF-7 Breast Cancer Cell
【作者】 路延芹；
【作者基本信息】 河北联合大学 ， 外科学， 2011， 硕士
【摘要】 目的:乳腺癌是女性最常见的恶性肿瘤之一,近年来其发病率逐年上升。其具体发生机制至今尚未完全清楚,目前公认乳腺癌的发生和发展是一个复杂、多步骤的过程,与许多基因和蛋白的表达相关,包括原癌基因的激活、抑癌基因的突变和丢失等。近年来,对乳腺癌发生、发展中的基因改变和功能分析一直是该领域研究的热点。解聚素-金属蛋白酶(a disintegrin and metalloproteinases,ADAMs)是近年来发现的一类具有多种功能的细胞膜表面糖蛋白家族,大约有800-1200个氨基酸残基组成,通过影响表皮细胞生长因子受体(pidermal Growth Factor Receptor,EGFR )信号转导等多种途径在肿瘤靶向治疗中发挥重要作用。解聚素-金属蛋白酶17(a disintegrin and metalloproteinase 17,ADAM17)是ADAM蛋白家族成员之一,又名肿瘤坏死因子转化酶(Tumor necrosis factor converting enzyme, TACE),已发现其在多种肿瘤中高表达,对肿瘤的发生、发展起重要作用。我们课题组前期的实验结果也显示ADAM17在乳腺纤维腺瘤中呈低表达,而在乳腺癌中呈高表达。RNA干扰(RNA interference,RNAi)技术是近几年来出现的新的基因沉默技术,相较于反义寡核苷酸技术具有高效、特异、低毒和发挥效应快等优点,在基因阻断方面逐渐替代传统的反义技术。我们通过RNAi使ADAM17基因沉默,观察ADAM17 mRNA表达水平的改变及其对MCF-7人乳腺癌细胞增殖的影响,从而揭示ADAM17与肿瘤细胞增殖的关系,为乳腺癌靶基因治疗提供一些理论及实验依据。方法:本实验以MCF-7人乳腺癌细胞株作为研究对象,观察ADAM17-siRNA转染MCF-7人乳腺癌细胞后对其增殖的影响;利用脂质体介导ADAM17- siRNA转染MCF-7细胞,采用实时荧光聚合酶链反应(Realtime polymerase chain reaction,RT-PCR)检测ADAM17在mRNA转录水平的表达水平;通过绘制细胞生长曲线法、MTT法检测细胞增殖活性;以流式细胞仪检测细胞周期。结果:1. RT-PCR结果显示利用脂质体介导ADAM17-siRNA转染MCF-7细胞后,转染组ADAM17 mRNA表达水平较对照组显著下调,差异具有统计学意义(p<0.05)。2. MTT法及细胞生长曲线显示转染ADAM17-siRNA组的MCF-7细胞增殖能力较对照组显著降低,差异具有统计学意义(p<0.05)。3.流式细胞术检测结果显示,转染组MCF-7细胞生长出现抑制,停滞在G0/Gl期,较对照组显著增加,差异具有统计学意义(p<0.05)。结论:通过脂质体转染法将ADAM17-siRNA转染入MCF-7人乳腺癌细胞后,MCF-7细胞ADAM17 mRNA表达水平明显下调,细胞停滞于G0/G1期,细胞增殖能力明显下降。提示ADAM17在乳腺癌细胞异常增殖中起到重要作用,有可能成为乳腺癌基因治疗的侯选基因,具有潜在的临床应用前景。
【Abstract】 ObjectiveBreast tumor is the most common malignant tumor in female, its incidence is increasing in recent years. For now, its pathogenic mechanism is not clear but it is considered that it results from multiple actions among many factors in coordination with many genes and protein high expression in breast cancer, including oncogene active and antioncogene defective. In recent years, More and more researchers have attracted much attention in the field of genetic changes and function analysis in the development and prognosis of breast cancer. It has been recently discovered that A Disintegrin And Metalloproteinase is of the same category of multifunctional cell membranecell protein family, which is made up of 800 to 1200 amino acid. ADAM, as a new target, may play a valuable role in tumor target therapy by affecting transduction of EGRF signal. ADAM17, TACE, is one of the members of ADAMs, which has been observed high expression in multiple tumors, and played a significant role in tumor genesis and development by regulating cell cycle. Our published reports further confirmed that ADAM17 protein was observed low expression in fibroadenoma, but high expression in breast cancer tissue.RNAi (RNA interference) technology has been commonly used in the approach of silencing gene expression recently. Compared with antisense oligonucleotides, RNA interference technology has following advantages: higher efficiency, better specificity, lower toxicity, quicker effect, as well as more convenient manipulation. So it will gradually substitute traditional antisense technology in gene block.The study was designed to investigate the effect of ADAM17-si- RNA on the growth and proliferation of MCF-7 breast cancer cell line by making ADAM17 gene silence with siRNA. The research is expected to reveal the relationship between ADAM17 and cell cycle regulation and establish the theory basis for target gene therapy.MethodsRNAi technology was used to observe its inhibitory effect on the growth of human breast cancer MCF-7 cells. siRNA targeting ADAM17 was transfected into MCF-7 cells mediated by oligofectamine. ADAM17 mRNA expression were detected by RT-PCR after transfection. The phenotypic change of MCF-7 cells including proliferation ability and cell cycle after RNAi trasfection was studied by MTT assay and flow cytometry.ResultsRT-PCR revealed significantly lowered ADAM17 expression at mRNA level in transfected MCF-7 cells. MCF-7 cells exhibited a significantly lower growth rate after transfection as shown by cell growth curve and MTT assay and flow cytometry analysis.ConclusionsUsing RNAi technology,siRNA targeting ADAM17 mediated by oligofectamine efficiently knocks down the expression of ADAM17 in human breast cancer MCF-7 cells, results in decrease of cell proliferation activity, arrests cell cycle. RNAi is a new gene silencing technology with high efficiency, ADAM17 can be the candidate genes for gene therapy of breast cancer, which will have potential application prospects in clinics.