miR-9-5p竞争性调节LncRNA SNHG7通过BRAF逆转甲状腺癌碘耐受的机制研究

The effectively combined differentiated thyroid cancer treatment is doing radioactive iodine-131 (131I) after subtotal hysterectomy. However, there has been about 10% to 15% of DTC patients experienced radioactive iodine tolerance (RAIR) at first 131I treatment or first effective treatment. In the event of RAIR, the therapeutic effect will decrease significantly. Therefore, its radioactive iodine tolerance mechanism and how to reverse its iodine tolerance has become hot spot of the current research. At present, studies have shown that BRAF gene mutations play an important role in the mechanism of DTC. Yet, there is no relevant research on what regulates mutation of BRAF gene. Our preliminary bioinformatics research showed that the differential expression of miR-9-5p may be associated with BRAF , and it can be applied to the 3-terminal UTR region of downstream target gene BRAF, resulting in a decrease in synthesis, and there is a high prediction in both genes. Further, the StarBase database analysis showed that SNHG7 was most closely related to miR-9-5p.So,we put forward the following hypothesis depending on that the competitive regulation of miR-9-5p on LncRNA SNHG7 does the influence on BRAF reversing thyroid iodine-tolerance .The aim of the study is to investigate the biological behavior of LncRNA SNHG7 and miR-9-5p on thyroid cancer cells through clinical tissues, cells and to establish a model of thyroid cancer in mice.Even more, it will validate the interaction between LncRNA SNHG7 and miR-9-5p, and confirm whether LncRNA SNHG7 & miR-9-5p play an important role in the development of the thyroid cancer animal model or not, which will definite the specific molecular mechanisms of LncRNA SNHG7 and miR-9-5p in thyroid cancer tolerance and reverse the differentiation, in order to provide an objective basis for the experiment in preventing thyroid cancer clinical progress.

目前约有10%-15%的分化型甲状腺癌(DTC)在首次131I治疗或首次有效治疗后再次行131I治疗时出现碘耐受（RAIR）。出现RAIR，其疗效将显著下降，故碘耐受机制研究及如何逆转碘耐受成为研究重点及热点。近来研究认为BRAF基因突变在DTC发生机制中起重要作用，而BRAF突变受什么因子调控,目前尚未明确。我们前期研究显示miR-9-5p差异性表达与BRAF可能存在潜在联系，且可作用于下游靶基因BRAF的3端UTR区导致其合成减少。StarBase数据库分析SNHG7与miR-9-5p相关性最密切。我们提出以下假说：miR-9-5p通过竞争性调节LncRNA SNHG7影响BRAF表达，最终起到逆转甲状腺癌碘耐受的作用。本项目拟通过临床组织、细胞及小鼠甲状腺癌模型，研究LncRNA SNHG7及miR-9-5p在甲状腺癌发生发展中的作用，以明确其在甲状腺癌碘耐受及逆转分化方面的机制。

甲状腺癌（TC）是世界范围内最普遍的内分泌癌，特别是过去十年中发病率迅速上升。甲状腺乳头状癌（PTC）是TC中最主要的形式，占所有TC病例的85%。PTC绝大多数患者预后良好，但少数患者在首次131I治疗时或首次有效治疗后再次行131I治疗时会出现放射性碘耐受，给131I治疗带来困难，此类患者预后差。因此，进行放射性碘耐受机制研究并开发潜在的预防和治疗靶点具有重要临床意义。本研究项目通过对PTC临床样本的转录组测序及qPCR法确证，证明在碘耐受PTC中lncRNA SNHG7被异常上调及lncRNA CASC2和赖氨酸甲基转移酶2B（KMT2B）的表达被沉默。随后通过生物信息学分析、双荧光素酶报告基因分析和RNA pull down分析，明确了lncRNA SNHG7是miR-9-5p的ceRNA。在体外细胞水平上通过对癌细胞增殖、迁移、侵袭、克隆形成能力的测定，及细胞凋亡率和耐药相关核心通路蛋白因子活化状态的检测，以及通过在体小鼠荷瘤实验，确证了lncRNA SNHG7作为miR-9-5p的ceRNA促进DPP4的表达及BRAF的活化，激活Erk1/2/Mek信号通路和PI3K/Akt信号通路，赋予PTC细胞对131I及顺铂治疗的耐药性；lncRNA CASC2被抑制后引起TC细胞EMT水平上调；和赖氨酸甲基转移酶2B（KMT2B）的表达沉默，下调了泛素连接蛋白和解旋酶SHPRH，从而促进Src激酶家族成员FYN的稳定并增强131I抵抗的分子机制。为以上述因子为靶点的靶向治疗策略的开发奠定了坚实的理论基础。