基于转录组学研究肿瘤转移中骨桥蛋白对肿瘤细胞释放外泌体的调控

Tumor-derived exosomes are documented as key mediators in tumor cell-microenvironment communication through signal molecule they carried, including various proteins, mRNAs and miRNAs. They play a crucial role in regulation of immune response, tumor growth, progression and metastasis. However, the study of exosomes RNA expression profiles is still in early stages. Osteopontin (OPN) is overexpressed in multiple tumor tissues and is associated with tumorigenesis or metastasis in numerous human cancers, such as liver and breast cancer. OPN promotes tumor cell migration via the regulation of multiple signaling pathways and activation of metastasis-related gene expression. However, the influence of OPN on tumor-derived exosomes in cancer metastasis remains unlear. In present study, we isolate exosomes from engineered breast cancer cells and hepatoma carcinoma (HCC) cells with high or low OPN expression. The total RNAs of these exosomes are extracted and profiled using transcriptome sequencing technology (RNA-seq). Cancer metastasis associated candidate mRNAs and non-coding RNAs (ncRNAs) are screenined through bioinformatic analysis and verified by qRT-PCR. The influence of the verified candidate RNAs on cell migration and invasion are validated using transwell assay and tube formation assay. The object of this study is to find key factors contribute to OPN mediated tumor metastais in the microenvironment and clarify new mechanism involving the promotion of cell migration by the interaction between internal and external environmental factors. This study may provie new insight into the development of antitumor drugs targeting to components of the tumor microenvironment and into the diagnosis and treatment of malignant tumors.

肿瘤外泌体(Exosomes)携带多种蛋白质、mRNAs、miRNAs等信号分子，介导肿瘤细胞与环境细胞间通讯，在诱导肿瘤存活、增殖、转移等方面发挥重要作用。但从转录组学角度研究外泌体还在起步阶段。骨桥蛋白(OPN)在许多肿瘤如肝癌、乳腺癌组织高表达，可调控许多信号通路或转移相关基因促进肿瘤细胞迁移。然而，OPN是否可以通过调控微环境中外泌体信号分子促进肿瘤转移尚不清楚。本项目建立高表达和低表达OPN的肝癌和乳腺癌细胞模型，分离其释放的外泌体，利用转录组测序技术研究OPN对外泌体整体转录本的影响。通过生物信息学分析筛选出与肿瘤转移相关的mRNA和非编码RNA (ncRNA)，并以qRT-PCR及转移功能实验加以验证。发现肿瘤微环境中与OPN促肿瘤转移相关的关键基因，阐明内外环境因素相互作用影响肿瘤细胞迁移的新机制。为认识肿瘤发生、发展规律，以及恶性肿瘤的诊断治疗及药物开发提供新思路。

肿瘤外泌体(Exosomes)携带多种蛋白质、mRNAs、miRNAs等信号分子，介导肿瘤细胞与环境细胞间通讯，在诱导肿瘤存活、增殖、转移等方面发挥重要作用。骨桥蛋白(OPN)在许多肿瘤如肝癌、乳腺癌组织高表达，可调控许多信号通路或转移相关基因促进肿瘤细胞迁移。然而，OPN是否可以通过调控微环境中外泌体信号分子促进肿瘤转移尚不清楚。本项目以高表达OPN的肝癌和乳腺癌细胞为模型，并分离其释放的外泌体，利用转录组测序技术及生物信息学分析筛选出与肿瘤转移相关的非编码RNA (ncRNA)，并以qRT-PCR及转移功能实验加以验证。成功建立了高表达和低表达OPN的肝癌和乳腺癌细胞模型，并优化了肿瘤细胞培养上清中外泌体分离方法。测序技术及生物信息学分析结果显示转染OPN的肝癌细胞外泌体中miRNA表达有显著差异的79个，上调36个，下调43个；转染OPN的乳腺癌细胞外泌体中miRNA表达有显著差异的32个，上调15个，下调17个。采用miRanda和RNAhybrid两个软件对差异表达miRNA的靶基因进行预测，以两个软件预测结果的交集作为最终结果。对差异表达miRNA的靶基因（候选靶基因）GO（Gene Ontology）富集分析和KEGG富集分析，阐明实验中样本差异在基因功能上的体现和显著性富集的Pathway。通过qRT-PCR技术对测序结果进行验证及其靶基因的表达情况进行检测，筛选出可能参与转移调控的miRNA及其靶基因，并通过功能实验加以验证。发现在肝癌细胞中OPN可能通过调控外泌体miR-4660及其靶基因SGALS3BP的表达从而促进肝癌细胞迁移; 乳腺癌细胞中，OPN可能通过外泌体miR-615-3p调控转移相关基因TMUB1和TIMP2的表达从而促进乳腺癌细胞迁移。本项目从转录组学(Transcriptomics)角度，以乳腺癌和肝癌细胞为模型，发现肿瘤微环境中与OPN促肿瘤转移相关的关键基因，阐明内外环境因素相互作用影响肿瘤细胞迁移的新机制。为以肿瘤微环境中的成分作为靶点筛选抗肿瘤药物提供思路，对认识肿瘤发生、发展规律，以及肿瘤诊断治疗都具有重要意义。