MRAK081523与MiR-98靶向调控ADAMTS8在甲状腺乳头状癌转移机制中的研究

Papillary thyroid carcinoma（PTC） is the most common malignant tumor in thyroid.The primary reason of PTC causing death is tumor metastasis. So it is very important to explore the new mechanism of PTC tumor metastasis. Our early experiments by deep sequencing have shown that ADAMTS8 was abnormal expression in lung metastases of PTC. At the same time, MiR-98 express increased abnormally in metastases by MiR array analysis, and Luciferase Reporter Assay have shown that ADAMTS8 was a target of MiR-98. By sequence alignment and RNA silencing technology , we confirmed that MRAK081523 and MiR-98 were competing endogenous RNAs. In addition, the tumor signal protein chip analysis have shown that the pathway of Ras/MEK/ERK and PI3K-Akt/PKB were abnormally activated. And we found expression of MRAK081523 in metastases was significantly decreased. In order to explore the interacting protein of ADAMTS8, co-immunoprecipitation assays was shown that EGF was a direct receptor of ADAMTS8. EGF can activate EGFR. And the pathways of Ras/MEK/ERK and PI3K-Akt/PKB are downstream signaling pathways. In this experiment, we will verify MRAK081523 and MiR-98 coregulate ADAMTS8 and the downstream pathways of Ras/MEK/ERK and PI3K-Akt/PKB at the levels of molecule, cell, animal and histopathology. Our research aims to establish that ADAMTS8 can be used as a therapertic target for invasive and metastatic PTC, and to provide a theoretical basis for developing a novel therapy in the refractory PTC of malignant metastases and improve the clinical treatment effect.

甲状腺乳头状癌转移是甲状腺癌致死的主要原因。二代测序发现ADAMTS8在肺转移灶中表达异常，micro RNA芯片分析发现MiR-98表达增高，荧光素酶报告实验验证ADAMTS8是miR-98的靶基因；lnc RNA芯片发现MRAK081523在转移灶中异常降低；通过序列比对及RNA沉默技术，确认二者是竞争性内源RNA。蛋白芯片发现肺转移灶中Ras/MEK/ERK和PI3K-Akt/PKB 通路异常活化。经免疫共沉淀发现EGF是ADAMTS8的靶蛋白，而EGFR是其特异结合受体，Ras/MEK/ERK和PI3K-Akt/PKB通路是EGFR的下游通路。本研究拟在分子，细胞，体内以及临床水平上验证MRAK081523联合miR-98作用于ADAMTS8，调控Ras/MEK/ERK和PI3K-Akt/PKB通路在PTC恶性转移中的机制，为甲状腺癌临床治疗恶性转移寻找新靶点。

长链非编码RNAs (Long noncoding RNAs, lncRNAs)是一类新型的与肿瘤发生相关的基因表达调控因子，但其在甲状腺乳头状癌(PTC)中的作用尚不清楚。采用实时荧光定量PCR (qPCR)技术检测lncRNA OIP5-AS1、miR-98和ADAMTS8在甲状腺乳头状癌组织中的表达。免疫组化(IHC)检测ADAMTS8的表达。利用RNA免疫沉淀(RIP)和荧光素酶报告基因分析来评估OIP5-AS1、miR-98和ADAMTS8之间的关系。利用MTT法、菌落形成法、transwell法和离体创面愈合法检测了OIP5-AS1、miR-98和ADAMTS8在PTC细胞中的生物学功能。在体内实验中，我们使用异种移植模型来研究OIP5-AS1和ADAMTS8对PTC的影响。在这里，我们研究了lncRNA OIP5-AS1在PTC中的作用。功能实验表明，过表达OIP5-AS1可促进PTC细胞的增殖、迁移侵袭和体内增殖。OIP5-AS1通过MEK/ERK和PI3k/Akt信号通路部分促进了PTC细胞的进展，而ADAMTS8在PTC中过表达。进一步，我们确定OIP5-AS1表达升高，并与PTC组织和细胞中的ADAMTS8水平相关。荧光素酶报告基因分析结果显示，miR-98可直接作用于ADAMTS8，而机制实验表明OIP5-AS1可作为竞争性内源性RNA (ceRNA)，并充当miR-98的海绵，可激活ADAMTS8。我们的研究结果表明，OIP5-AS1是影响PTC发展的肿瘤调节因子，对PTC的认识可以提高PTC的诊断和治疗水平。