lncRNA Loc554202通过调控染色质状态促进脊索瘤 发生和进展的机制研究

Chordoma is a rare primary malignant bone tumor. So far there is a lack of effective treatment. Globally, less research on the basic biology of chordoma occurrence and progression has seriously affected the development of clinical treatment of chordoma. In recent years, people gradually realized that long-chain non-coding RNA (lncRNA) plays a key role in tumorigenesis and progression. Our previous studies showed that lncRNA LOC554202 is positively correlated with proliferation and invasion of chordoma cells and interacts with EZH2 in PRC2, a chromatin-modified complex. Therefore, we speculate that lncRNA LOC554202 may act as a molecular scaffold to collect PRC2 and LSD1, thereby affecting chromatin status, which subsequently promotes the occurrence and progression of chordoma. Therefore, this study intends to conduct a comprehensive and exhaustive study on how lncRNA LOC554202 acts as a scaffold molecule for the regulation of chromatin modification at molecular, cellular and clinical levels through RNA / protein interaction techniques, chromatin immunoprecipitation, DNA microarrays. Eventually, we aim to clarify that the relocation of proteins PRC2 and LSD1 on the genome-wide promoter regulates the occurrence and progression of chordoma by influencing the closure and opening of a series of tumor-associated genes. Through this study, we hope to elucidate biomarkers related to prognosis and prognosis of chordoma, and to provide potential therapeutic targets for drug therapy of chordoma.

脊索瘤是一种少见的恶性骨肿瘤，目前缺少有效的治疗手段。近年来，人们逐渐认识到长链非编码RNA(lncRNA)在肿瘤发生和进展中扮演关键作用。我们之前的研究显示，lncRNA LOC554202与脊索瘤细胞的增殖与侵袭呈正相关性，且与染色质修饰复合物体LSD1和PRC2中的EZH2存在相互作用。因此，我们推测lncRNA LOC554202可能作为分子支架募集PRC2和LSD1，进而影响染色质状态，从而促进脊索瘤的发生和进展。因此，本研究拟通过RNA/蛋白相互作用技术、染色质免疫共沉淀、DNA芯片等技术手段，在分子、细胞和临床样本三个层次研究lncRNA LOC554202如何作为支架分子调控染色质修饰蛋白PRC2和LSD1在全基因组启动子上的再定位，进而调控脊索瘤的发生和进展。希望通过本研究阐明脊索瘤诊断和预后相关的生物标记分子，并为脊索瘤的药物治疗提供潜在治疗靶标。

脊索瘤虽是低度恶性肿瘤，具有易复发、侵袭性强等特点。对该疾病从分子水平进行研究，找到新的分子治疗靶点，延长患者生存期具有重要的意义。本研究通过RNA免疫共沉淀技术发现 LOC554202 能够与 PRC2 和 LSD1 特异性结合；通过敲降和过表LOC554202明确LOC554202是EZH2和LSD1发生相互作用的必须分，且LOC554202表达能够影响EZH2在H3K27me3 启动子占位的影响；在细胞增殖和克隆形成实验中，明确LOC554202 介导的脊索瘤增殖和侵袭依赖于 PRC2 或 LSD1。进一步通过构建EZH2稳定敲降及过表达和Y641F突变体的脊索瘤细胞系，在EZH2稳定过表达和Y641F突变体的脊索瘤细胞系中进行生物学功能实验，发现EZH2能够促进脊索瘤的发生发展，并通过使用EZH2的功能抑制剂发现其能抑制脊索瘤的进展，同时为进一步研究其相关分子机制，我们通过使用RNA-Seq分析其下游靶基因富集所在通路，发现其富集在TGFβ通路，在进一步RT-qPCR及WB的验证中得到证实。研究发现LOC554202能够上调GSK-3β的表达，通过探索发现使用糖原合成激酶GSK-3β能够促进脊索瘤的增殖和侵袭，且GSK-3β通过调节P21的表达来促进脊索瘤的进展。因此，本研究从LOC554202调控脊索瘤染色质状态，进而探索其相关基因EZH2和P21在调控脊索瘤增殖和侵袭能力的生物学机制研究，为脊索瘤的治疗提供潜在的分子靶点。