LncRNA-RP11-159K7.2作为新的喉癌标志物及其调控DNA甲基化的机制研究

Laryngeal carcinoma is a malignant tumour with high mortality in the advanced stage, which seriously threaten human health. It is urgent to search for reliable molecular markers of laryngeal carcinoma and elucidate the molecular mechanisms of the disease. Non-coding RNA plays a significant regulatory role in carcinogenesis process. Our research group has screened out lncRNA-RP11-159K7.2 from substantial miRNAs, lncRNAs and circRNAs which are significantly higher expressed in tumour tissue and blood of laryngeal carcinoma. Besides, we proved that it is involved in the regulation of DNA methylation in laryngeal carcinoma for the first time. However, it is not clear whether RP11-159K7.2 can be used as a reliable molecular biomarker, and the regulation mechanism of DNA methylation which RP11-159K7.2 involved in still needs further study. In order to clarify this problem, we intend to detect the expression of RP11-159K7.2 in large sample of laryngeal carcinoma tissues, serum and exosome, and observe its relationship with the metastasis, recurrence and prognosis of the malignancy. Using gene transfection, CRISPR/Cas9, invasion experiment, luciferase experiment, RIP, pulldown and other methods, we will reveal the role of RP11-159K7.2 in laryngeal carcinoma, and its regulation mechanisms of DNA methylation from a new perspective. This project is expected to provide a new target and strategy for early warning, prognosis evaluation and treatment of laryngeal carcinoma.

喉癌是严重威胁人类健康的疾病，尤其是晚期喉癌死亡率仍较高，因此，寻找喉癌可靠的分子marker及阐明其分子机制迫在眉睫。非编码RNA在肿瘤进程中起到重要的调控作用。本课题组的前期研究在miRNA、lncRNA和circRNA中筛选出喉癌组织和血液中显著高表达的lncRNA-RP11-159K7.2，并首次证明其参与了喉癌的DNA甲基化调控。但它能否作为喉癌可靠的分子marker，以及它调控DNA甲基化的机制尚不清楚。为阐明这一科学问题，我们将检测大样本喉癌组织、血清及外泌体中RP11-159K7.2的表达，观察其与复发、转移和预后的关系。进一步运用基因转染，CRISPR/Cas9，侵袭实验，荧光素酶，RIP，pulldown等方法，从新的视角揭示RP11-159K7.2在喉癌中的作用及其调控DNA甲基化的机制。本项目可望为喉癌预警、预后评估及治疗提供新的靶点和策略。

研究背景:喉癌(LSCC)是头颈部最常见的恶性肿瘤之一，其晚期患者生存率仍较差。寻找特异、有效的LSCC肿瘤标志物和潜在的治疗靶点，以及揭示喉癌发展机制已成为喉癌研究的主要方向。课题组前期发现lRP11-159K7.2在喉癌中表达显著升高，但其作用和机制不明。本课题进一步探究RP11-159K7.2在LSCC发生发展中的作用机制及意义。研究内容: (1)检测RP11-159K7.2在225例LSCC组织及细胞系中的表达。分析RP11-159K7.2表达水平与患者临床病理特征相关性及预后的关系。(2)敲除LSCC细胞内RP11-159K7.2使其表达下调，检测LSCC细胞的增殖和侵袭能力变化。(3) 采用MeDIP-chip技术检测LSCC细胞基因组启动子的总体甲基化水平。(4)检测miR-206是否特异性结合RP11-159K7.2和DNMT3A。亚硫酸氢盐测序评估LSCC细胞中已鉴定的CpG位点的原始甲基化状态。重要结果:RP11-159K7.2在LSCC组织和细胞中的表达显著增高，与LSCC患者的预后密切相关。LSCC细胞下调后，其细胞的增殖和侵袭能力均下降。成瘤实验显示敲除RP11-159K7.2后小鼠肿瘤体积与对照组相比明显较小，重量较轻。RP11-159K7.2的下调显著影响LSCC细胞的甲基化水平。miR-206为RP11-159K7.2下游靶标，miR-206特异性结合RP11-159K7.2及DNMT3A。RP11-159K7.2敲除后小鼠肿瘤中DNMT3A的表达显著下降。CpG位点的高甲基化状态抑制LSCC细胞中miR-206的表达，表明miR-206和DNMT3A之间存在负反馈抑制作用。结论及科学意义: (1) RP11-159K7.2在LSCC组织及细胞中的表达水平明显增高，并与LSCC患者临床病理学特征及预后相关，提示RP11-159K7.2在LSCC中扮演癌基因的功能，可作为喉癌候选的分子标记物。(2) 体外细胞功能学实验和体内裸鼠荷瘤实验证实下调RP11-159K7.2后LSCC细胞增殖和侵袭能力均下降。预示着RP11-159K7.2可作为LSCC潜在的治疗干预靶点。(3) LRP11-159K7.2通过与miR-206结合调节DNMT3A的表达，进而调控LSCC整体DNA甲基化水平，从而促进了喉癌的侵袭发展，丰富了喉癌的发展机制。