微囊泡介导的分泌型非编码RNA调控喉癌细胞上皮-间质转化的分子机制研究

Microvesicles (MVs) as an important mediator between different cells, carrying the non-coding RNA (ncRNAs), are closely related with tumor invasion and metastasis. In our previous works, microvesicles were detected in the serum of the patients suffering from laryngeal squamous cell carcinomas (LSCC). In addition, RT-PCR showed that long-chain ncRNA such as HOTAIR and miRNAs such as miR-21 were differentially expressed in the MVs; ncRNAs expression were not only correlated with the clinical parameters of LSCC, but closely associated with invasion and apoptosis of laryngeal cancer Hep-2 cell line in vitro and in vivo experiments. Epithelial to mesenchymal transition (EMT) facilitates tissue remodelling during embryonic development and is viewed as an essential early step in tumor invasion and metastasis, but the mechanism is unclear. Based on our previous research and the research of others,we proposed that ncRNA secreted from MVs may regulate EMT in LSCC. We would (1) find the correlation between MVs,ncRNA and EMT-related molecules and clinical data of LSCC patients, aiming to find any important tumor marker.(2)find the relationships between MVs and ncRNA by in vivo and in vitro experiments utilizing the techniques of molecular cloning,reporter gene,qRT-PCR and so on;(3) demonstrate how ncRNAs secreted from MVs mediate signaling networks of EMT in LSCC and its effect on the proliferation, migration and apoptosis of the tumor cells.(4) a single unit (oligonucleotide, ODN) that is capable of simultaneously expressing one or more target genes will be engineered to investigate the possibility and effectiveness of the inhibition for invasion and metastasis of LSCC. The main objectives of this research are to demonstrate the mechanisms of EMT regulated by ncRNA secreted from MVs and the impact of ncRNA on tumorigenesis of LSCC, scrutinize the efficiency of a single sequence targeting multi genes in suppressing the cancer invasive properies, which will be essential for developing improved treatment plans for LSCC.

微囊泡（MVs）作为细胞间信息的重要介质，运载非编码RNA(ncRNAs),与肿瘤的侵袭和转移密切相关。我们前期发现，喉癌病人血清含有丰富的MVs，其中长链ncRNA如HOTAIR等和微小RNA如miR-21等有着明显的差异表达；体内外实验表明：ncRNA的表达与喉癌的侵袭及凋亡密切相关。一般认为，上皮间质转化（EMT）能够促进肿瘤的侵袭和转移，但机制尚不清楚。我们基于前期研究和国内外研究现状，提出"微囊泡能够分泌ncRNA并诱发EMT促进喉癌侵袭和转移"的假说。拟采用分子克隆、报告基因、定量PCR等技术从体内外水平研究MVs与ncRNA的关系。揭示在MVs介导的分泌型ncRNA调控的信号网络中，EMT发生的分子机制，筛查预测喉癌侵袭转移的肿瘤标记物。并针对性地设计相应反义或拟核苷酸制剂，探讨抑制喉癌侵袭转移的可能性及有效性。该课题将完善喉癌侵袭转移的认识，为建立新的分子治疗策略提供基础。

喉癌是头颈部常见的恶性肿瘤之一，近年来综合治疗的不断进展下，如何提高患者的生存率仍然是需要攻克的课题，如何有效地抑制喉癌侵袭、转移和诱导凋亡是研究的关键问题。微囊泡（MVs）作为细胞间信息的重要介质，运载非编码RNA(ncRNAs),与肿瘤的侵袭和转移密切相关，大量的研究表明ncRNA与喉癌的发生发展密切相关。本项目在执行期间从四方面进行了相关研究：1、首先发现miR-101、203、205可以抑制喉癌细胞增殖和侵袭以及诱导其凋亡，并对其作用机制进行了相关研究，发现部分相应的靶基因，其次进行了联合miRNAs调控喉癌发生发展的实验研究，喉癌的治疗是综合治疗的过程，本项目发现miRNA在喉癌放疗中可以起到增敏剂的作用；2、本项目成功提取了微囊泡，并通过电镜以及Western进行了证实，并研究其中 HOTAIL和microRNA-21与喉癌的关系；3、竞争性内源RNA调控网络的基础是在RNA 组学的基础上构建的庞大的调控网络，我们进行了炎性基因相关的ceRNA网络对喉癌预后生物标记的识别。通过公共可用GEO数据库，构建ceRNA网络，然后进行相关内容的网络分析；4、在现代生物－心理－社会医学模式下，患者的生存质量（quality of life，QOL）受到越来越多的关注，对喉癌患者心理因素产生的躯体改变所引起的症状进行相关分析，并得到较有效的解决办法。依托本基金参与资助的研究工作所取得的相关数据，共发表相关SCI文章11篇，该课题的结果可以为此研究领域提供全新信息，为喉癌生物靶向治疗提供另一条途径，为建立新的分子治疗策略提供基础，并且本项目中所涉及的临床研究成果可以进一步在临床中起到应用价值。