长链非编码RNA在钌配合物抗骨肉瘤中的作用及其机制研究

Ruthenium complexes are one of the most prospecting anti-cancer drugs. In our preliminary experiments, we have synthesized a number of ruthenium complexes and found that they could bind DNA to effectively induce apoptosis of osteosarcoma cells, but the underlying mechanism is unclear. The newly discovered long non-coding RNAs (lncRNAs) are an important group of regulatory factors of gene expression and are crucially implicated in tumor initiation and development. In this project, we will test our central hypothesis that lncRNAs play important role in the anti-cancer effects of ruthenium complexes. Three specific aims have been proposed: Aim 1: to identify candidate lncRNAs that mediate the anti-osteosarcoma effects of ruthenium complexes; Aim 2: to evaluate the clinical significance of candidate lncRNAs in the diagnosis, prognosis and therapy of osteosarcoma; Aim 3: to elucidate the mechanisms by which candidate lncRNAs modulate the malignant behaviors of osteosarcoma cells.To achieve these aims, we will establish osteosarcoma cell lines and xenografts in nude mice as in vitro and in vivo model, respectively. Based on preliminary lncRNAs expression profiling on osteosarcoma cells treated by ruthenium complexes, we will select several candidate lincRNAs for further analysis. First, we will detect the expression of candidate lncRNAs in osteosarcoma cell lines by quantitative RT-PCR. Next, we will apply a variety of techniques such as siRNA transfection, lentivirus transduction, flow cytometry, gene chip, chromatin immunoprecipitation, and RNA pull-down to explore the role of candidate lncRNAs in the modulation of the biological behaviors of osteosarcoma cells, including cell proliferation, cell cycle progression and apoptosis. Especially, we will focus on epigenetic modulation by candidate lncRNAs to characterize the mechanism of action of candidate lncRNAs. In addition, we will employ nude mice and clinical osteosarcoma specimens to confirm the results of in vitro experiments and validate the significance of candidate lncRNAs in the diagnosis and treatment of osteosarcoma. In particular, we will analyze the correlations of the expression levels of candidate lncRNAs with the clinicopathologic parameters of osteosarcoma, such as patient age, patient sex, tumor size, stage, metastasis and recurrence of the disease. It is expected that the completion of this project will help elucidate the mechanism by which ruthenium complexes achieve anti-osteosarcoma effects, and identify potential therapeutic targets and molecular biomarkers of osteosarcoma. Most significantly, this project will reveal lincRNA expression profiling as a new approach to investigate the mechanism of action of ruthenium complexes in different biological contexts, and will promote the further development and utilization of ruthenium complexes.

钌配合物是最具开发前景的抗癌药物之一，本课题组前期合成了一批新型钌配合物，能结合DNA诱导骨肉瘤细胞凋亡，但机制不明。长链非编码RNA(lncRNA)是基因表达的重要调控因子，与肿瘤的发生发展密切相关。前期实验表明钌配合物作用骨肉瘤细胞后能诱导细胞内部分lncRNA表达显著变化，因此本课题组推断lncRNA在钌配合物抗骨肉瘤中起重要作用。本项目应用siRNA、慢病毒感染等技术建立研究体系，体外筛选出钌配合物抗骨肉瘤过程中具有重要功能的几种lncRNA，并采用裸鼠模型和临床标本验证体外实验结果，探讨候选lncRNA在骨肉瘤诊断和治疗中的意义，进一步在骨肉瘤细胞中应用基因表达谱芯片、Chip-PCR等技术从表观遗传学方面探讨其作用机制。本项目将阐明lncRNA在钌配合物抗骨肉瘤中的作用和机制，揭示骨肉瘤诊断治疗新靶点，并开辟了钌配合物作用机制研究新思路，为其进一步开发利用奠定理论和实验基础。

钌配合物是最具开发前景的抗癌药物之一，但机制不明。长链非编码RNA(lncRNA)是基因表达的重要调控因子，与肿瘤的发生发展密切相关。本项目前期实验发现钌配合物作用骨肉瘤细胞后能诱导部分lncRNA表达显著变化，拟探讨lncRNA在钌配合物抗骨肉瘤中的作用及机制。本课题组合成的钌配合物能诱导骨肉瘤细胞凋亡，使细胞周期阻滞。但与经典抗骨肉瘤药物顺铂相比，未有更高药效性，拟进一步完善药物结构式，合成药效更高，毒性更小的钌配合物，再进一步探讨其作用机制。经研究组讨论本项目的研究内容调整为：.1、筛选在骨肉瘤发生发展中的特异性LncRNA，探究其作用和机制。2、研发新型钌配合物，研究其抗肿瘤活性和基础抗肿瘤机制。. 本项目首先利用lncRNA芯片筛查骨肉瘤组织及瘤旁组织中差异表达的lncRNA，根据lncRNA和mRNA表达谱，综合GO分析、CNC分析等，筛选出4条候选lncRNA进行深入研究；因工作量的原因，我们首先选取SATB2-AS1作为研究对象，SATB2-AS1为SATB2 mRNA的天然反义转录物，SATB2能促进成骨细胞分化和骨形成，促进骨肉瘤的生成，推断SATB2-AS1可能在骨肉瘤发生发展中起重要调控作用，这也为我们研究进一步研究SATB2-AS1的作用机制提供了思路。 .本课题组首先利用荧光定量RT-PCR验证SATB2-AS1在骨肉瘤及瘤旁组织中的表达；并扩大临床标本量，证实SATB2-AS1在骨肉瘤组织中异常高表达，并在有远处转移骨肉瘤组织中的表达量增高。构建SATB2-AS1慢病毒过表达和干扰载体，感染至骨肉瘤细胞株，检测生长曲线、周期、凋亡，细胞迁移能力和细胞侵袭能力等功能。并构建裸鼠荷瘤模型，体内验证SATB2-AS1的功能。最后验证SATB2-AS1和靶SATB2表达的相关性等，探索具体作用机制。.同时新合成5种钌配合物，对多种肿瘤细胞进行细胞毒性实验，实验证明[Ru(dmp)2(NMIP)](ClO4)2对骨肉瘤具有较高的药效性，进行后续凋亡、周期、活性氧检测、凋亡蛋白检测等实验，验证钌配合物的初步抗肿瘤机制。.本项目合成了一批新型的钌配合物，证实其对各种肿瘤细胞具有较好的药效性和低毒性；明确了SATB2-AS1在骨肉瘤发生发展中的作用及初步作用机制，为其进一步开发利用奠定理论和实验基础。