外泌体lncRNA SNHG9通过细胞自噬途径对甲状腺乳头状癌的调控作用及其机制研究

Thyroid cancer is the fifth common female malignancy, and thyroid papillary carcinoma (PTC) accounted for more than 90% of them. More and more studies have found that exosomes as a key vector in the regulation of tumor development and metastasis in the tumor microenvironment, targeted drugs and serum biological markers has important research value. The expression of long-chain non-coding RNA (LncRNA) in PTC exosomes was drawn in the previous study. The results showed that LncRNA SNHG9 was enriched in PTC exosomes and confirmed by real-time PCR. SNHG9 May play a role similar to the oncogene by regulating the level of autophagy in tumors and adjacent cells. This project will further explore the role of SNHG9-riched exosomes as a signaling vector which regulates the autophagy level of PTC cells by tumor cell-cell microenvironment interaction and in-depth study of its specific molecular pathway. To elucidate the above problems will help to fill the gaps in the relevant research fields, improve the understanding of the pathogenesis of PTC, and provide new ideas and new evidence for the study of the cell microenvironment in the field of malignant tumors.

甲状腺癌是女性第5位常见的恶性肿瘤，而甲状腺乳头状癌（PTC）占其中的90%以上。目前越来越多的研究发现外泌体作为关键分子载体在调节肿瘤发生发展与转移方面发挥了重要的作用，在肿瘤微环境、靶向药物及血清生物学标志方面具有重要的研究价值。课题组在前期研究中绘制了PTC细胞外泌体中的长链非编码RNA（LncRNA）表达谱，通过多条件筛选发现，LncRNA SNHG9在PTC外泌体中的富集，并初步证实了SNHG9分子可能通过调控肿瘤及邻近细胞的自噬水平发挥类似促癌基因的作用。本项目将在此基础上，进一步验证外泌体作为信号载体介导LncRNA SNHG9分子，通过肿瘤细胞-细胞微环境交互作用，调控PTC细胞自噬水平这一作用模式，并深入研究其具体分子通路。阐明以上问题，将有助于填补相关研究领域的空白，完善对PTC发病机制的认识，并为外泌体-细胞微环境在恶性肿瘤领域方面的研究提供新思路和新证据。

本研究旨在发掘细胞外泌体lncRNA参与调控PTC发生发展的分子机制的探索。利用高通量测序检测PTC外泌体与PTC细胞中lncRNA，通过生物信息学分析筛选出高表达lncRNA SNHG9，利用实时荧光定量PCR检测86对甲状腺乳头状癌与癌旁组织中SNHG9表达水平，分析其与PTC临床病理之间的关系。随后，构建mCherry-GFP-LC3自噬观察模型，探索PTC外泌体SNHG9对甲状腺上皮细胞 Nthy-ori-3自噬与凋亡的影响，利用扫描电镜观察Nthy-ori-3中自噬小体的形成，利用流式细胞技术检测PTC外泌体SNHG9对Nthy-ori-3凋亡的影响，利用Western blot检测凋亡、自噬相关蛋白的表达。利用RNA pull down技术和蛋白质谱筛选出SNHG9相互作用蛋白YBOX3，并利用RIP实验和Western blot证明SNHG9与YBOX3的相互作用关系，进一步利用Western blot探索SNHG9通过YBOX3对PTC自噬与凋亡的影响。因此LncRNA SNHG9可以作为临床上甲状腺癌的分子靶向治疗的靶点进行进一步的研究。