Understanding Vitamin D resistance using Nonsense Mediated Decay arrays

Vitamin D is the strongest known natural anti-proliferative. A large number of studies in a wide spectrum of cancers, including epidemiological, in vitro and animal models, demonstrate that the active form of Vitamin D has anti-cancer benefits, affecting both progression and metastasis. Alike the role in calcium Vitamin D regulation, its anti-proliferative effect is thought to function through the Vitamin D receptor (VDR), although convincing evidence is lacking. Notwithstanding, separation of the calcemic and the anti-proliferative activity of Vitamin D analogues has been a major obstacle in developing new drugs for the treatment of cancer. The work presented attempts to unveil the molecular mechanism behind the anti-proliferative action of Vitamin D using genomic tools. For that purpose four independently developed Vitamin D sensitive/resistant MCF7 cell line pairs were collected. These unique biological replicates enabled us, both to increase the power of our study and to omit the use of Vitamin D. We deem this omission crucial since in the presence of Vitamin D only downstream genes involved in proliferation and cell cycle would be identified rather than causal resistance genes. The use of a variety of genomic techniques including expression, NMD and oligo CGH arrays reveal in the resistant cell lines the 11q13-14 as a region of DNA copy number loss and an altered expression of EGFR signaling pathway genes. Surprisingly, no genes known from calcium Vitamin D regulation were identified, nor did the VDR silencing by RNAi induce resistance to the sensitive cell lines. Keywords: Expression microarray, NMD array, cell type comparision Several MCF7 breast tumor cell lines independently derived from the human breast cancer cell line, both resistant and sensitive to Vitamin D, were used. The pairs of cell lines (parental-resistant) were developed in different laboratories and using different methodologies; while some cell lines acquired resistance by long exposure to the physiological concentration of Vitamin D (100nM), others were induced to resistant by being exposed to increasing amounts of Vitamin D. A total of 4 microarray expression experiments were carried out. In all microarray experiments RNA from the Vitamin D resistant cell line treated with Emetine was hybrizided agains RNA from the respsective sensitive/parental cell line treated with emetine.

维生素D是目前已知活性最强的天然抗增殖物质。目前已有大量针对多种癌症的研究——涵盖流行病学调查、体外实验与动物模型——证实，活性形式的维生素D具有抗癌益处，可影响肿瘤的进展与转移过程。与维生素D在钙稳态调节中的作用类似，其抗增殖效应被认为是通过维生素D受体（Vitamin D receptor, VDR）介导的，但目前仍缺乏确凿的实验证据。尽管如此，分离维生素D类似物的高钙血症活性与抗增殖活性，一直是开发新型抗癌药物的核心障碍。本研究旨在借助基因组学工具，揭示维生素D抗增殖作用背后的分子机制。为此，我们收集了4组独立构建的维生素D敏感/耐药MCF7细胞系对。这些独特的生物学重复样本，既提升了本研究的统计效力，又使我们得以规避直接使用维生素D进行处理。我们认为这一实验设计的调整至关重要：若直接暴露于维生素D，仅能识别参与细胞增殖与细胞周期调控的下游基因，而非介导耐药产生的因果基因。通过表达芯片、无义介导的mRNA降解（Nonsense-mediated mRNA decay, NMD）芯片及寡核苷酸比较基因组杂交（oligo CGH）等多种基因组学技术，我们在耐药细胞系中发现，11q13-14区域存在DNA拷贝数缺失，且EGFR信号通路基因的表达发生显著改变。令人意外的是，我们未发现任何已知参与维生素D钙调节的基因，且通过RNA干扰（RNAi）沉默VDR也未能诱导敏感细胞系产生耐药性。
关键词：表达微阵列、NMD芯片、细胞系比较
本研究使用了多株独立从人乳腺癌MCF7细胞系衍生而来的维生素D敏感与耐药细胞系。这些（亲本-耐药）细胞系对由不同实验室采用不同方法构建：部分细胞系通过长期暴露于生理浓度（100nM）的维生素D获得耐药性，其余则通过逐步升高维生素D浓度的方式诱导产生耐药。本研究共开展了4组微阵列表达实验。所有微阵列实验中，均将经依米丁处理的维生素D耐药细胞系的RNA，与经依米丁处理的对应敏感/亲本细胞系的RNA进行杂交。