XB130通过竞争性内源RNA促进非小细胞肺癌进展的分子机制研究

Lung cancer is one of the most common malignant tumors worldwide, of which non-small-cell lung cancer (NSCLC) accounts for 80%. Since NSCLC presents more progressive, invasive and metastatic characteristics, it is thus vital for scientists to understand the molecular mechanism of NSCLC progression, which will provide new strategies for lung cancer early diagnosis and treatment. The competing endogenous RNA (ceRNA）represents a new way of gene regulation, and has been proved to be closely related to the development of cancer. The ceRNA hypothesis posits that specific RNAs can function as sinks for pools of active miRNAs, functionally liberating other transcripts targeted by that set of miRNAs. XB130 is a newly identified multifunctional adaptor protein. As a tumor promoter, XB130 has been found to enhance cell proliferation, metastasis, and resistance to cell death in various kinds of cancer cells. However, its role in lung carcinogenesis remains largely unknown. Recently, we demonstrated that XB130 3’UTR could bind to miRNA-30a, 30c, 203 and 219. In addition, XB130 3’UTR could promote the proliferation and migration of NSCLC cells, which indicated that XB130 mRNA might function as a ceRNA to promote NSCLC progression. Therefore, in this project, we aim to get a more comprehensive view of the XB130 ceRNA function in NSCLC progression. First, we will further test the coordinate regulation between XB130 3’UTR and miRNAs above using RNA immunoprecipitation, Real time PCR and Western blotting. Then we will transfect NSCLC cells with an allelic series of XB130 recombinant expression constructs to have a better understanding of XB130 ceRNA activity in NSCLC cells. In addition, we will study the effects of XB130 ceRNA activity on tumorigenesis and metastasis in vivo through examining subcutaneous transplantation tumor and lung metastases formations on nude mice. In order to further understand the molecular mechanism of NSCLC progression mediated by XB130 ceRNA, we will screen and confirm XB130 ceRNA target genes by RNA sequencing technology and experiments in vitro and in vivo. Then, we will investigate the influences of XB130 ceRNA on the cell signaling pathways in which XB130 ceRNA target genes participate. An important corollary of the ceRNA hypothesis is the coordinate regulation between the ceRNA and its target genes, such that upregulation of the ceRNA should lead to higher expression of the target genes and vice versa. Thus, we will finally use NSCLC gene expression data from NSCLC patient samples to assess the coordinate regulation between XB130 ceRNA and its target genes. The data obtained from this research project will contribute to new insights into the molecular mechanism of XB130 promoting NSCLC progression, leading to the development of novel diagnostic and therapeutic strategies.

非小细胞肺癌（NSCLC）是目前临床死亡率最高的肿瘤，其发病机制有待研究。竞争性内源RNA（ceRNA）是基因调控的一个新层面，与癌症发生密切相关。XB130作为一个多功能接头蛋白，是一个重要的癌症介导因子，其ceRNA功能及其在肺癌中的作用研究甚少。我们前期研究发现XB130 3'UTR能与多个miRNA结合，并能调控NSCLC细胞的增殖、凋亡和迁移，这暗示XB130 mRNA可作为ceRNA调节肺癌进展。本项目拟通过研究XB130 mRNA对NSCLC细胞增殖、迁移和浸润等能力以及对裸鼠移植瘤及肺转移灶形成的影响，全面了解XB130 mRNA的ceRNA活性。同时通过RNA测序等技术以及细胞和裸鼠实验筛选确定XB130 ceRNA靶基因，深入研究XB130 ceRNA对相关信号通路的影响，从一个全新的层面阐明XB130促进肺癌进展的分子机制，为寻找肺癌早期诊断和治疗新方法提供理论依据。

非小细胞肺癌（NSCLC）是目前临床死亡率最高的肿瘤，其发病机制有待研究。XB130作为一个潜在的癌症诊断和治疗靶点，其作为竞争性内源RNA（ceRNA）的功能及其在肺癌中的作用研究甚少。因此为了解XB130在NSCLC进展过程中的作用，本项目首先检测了XB130 3’UTR与预测miRNA之间的靶向调控关系，然后对其ceRNA功能进行验证，但结果与预期不符，因此我们调整了研究计划，对XB130的表达调控机制进行了不同层次的研究。结果显示XB130沉默能够显著抑制NSCLC细胞的增殖、迁移、侵袭和上皮间质转化（EMT）；XB130 3’UTR与miRNA-30家族成员之间不存在靶向关系，但miRNA-30家族成员过表达均可抑制XB130的表达，进而调控肺癌细胞的EMT；而miRNA-203、219以及4782-3p则可通过与XB130 3’UTR结合来抑制XB130的表达，进而调控肺癌细胞的增殖、迁移、侵袭以及EMT。此外，分段克隆和双荧光素酶报告体系研究显示XB130 3’UTR中113-230、503-660和970-1053片段可通过调控mRNA稳定性和翻译而显著上调或下调报告基因表达；然后通过RNA-Protein Pull Down、质谱等实验进行筛选，我们发现3个候选RNA结合蛋白（hnRNPR、hnRNPAB和hnRNPC）可能分别与XB130 3’UTR 113-230或503-660片段结合而上调XB130表达。以上研究表明XB130表达失调可能在NSCLC进展过程中发挥重要作用，而XB130 3’UTR在调控XB130表达方面扮演重要角色；除了miRNA靶向结合途径，RNA结合蛋白也参与了该调控过程，该研究为后续深入阐明NSCLC中XB130转录后表达调控机制以及促进XB130 在恶性肿瘤诊断和治疗中的应用奠定了坚实的基础。