lncRNAAP000472.2通过激活TGF-β信号通路促进甲状腺乳头状癌转移作用机制研究

Recently, the incidence of thyroid papillary carcinoma (PTC) increases dramatically. Despite the good prognosis, parts of PTCs are more vulnerable to local and distant metastasis, while the specific mechanism is still unclear. In previous experiments, we have successfully screened and cultured a high metastatic PTC cell line IHH4-M, and found lncRNA AP000472.2 may be associated with the invasion and metastasis of PTC by using the lncRNA Array and clinic samples. Besides, with the gene transfection and RNA interference technology, we confirmed that the overexpression/lost expression of AP000472 would obviously promote/inhibit the invasion and migration ability of PTC cell lines in vitro. Herein, in this study, we will focuse on downstream target genes and signaling pathways of AP000472 through the lncRNA-mRNA co-expression network, RT-PCR, Western Blot and lentivirus technologies. Finally, nude mice model will be used to validate the influence of AP000472 on invasion and migration ability of PTC cell in vivo. our research can reveal the mechanism of AP000472.2 in PTC cells and provide a new biomarker drug intervention target for early diagnosis and treatment of high-risk PTC patients.

近年来甲状腺乳头状癌（PTC）发病率迅速上升，部分PTC恶性程度高，易发生局部及远处转移，但其中的具体机制尚不清楚。我们在前期实验中成功筛选培育了高转移PTC细胞株IHH4-M，利用lncRNA芯片检测技术初步筛查发现lncRNA AP000472.2很可能与PTC侵袭转移相关，并通过慢病毒转染及干扰技术证实AP000472.2导入或沉默后可明显促进或抑制PTC细胞株体外侵袭和迁移能力。在此基础上，本项目拟进一步通过lncRNA-mRNA共表达网络，结合RT-PCR、Western Blot及慢病毒转染和干扰技术，筛选AP000472.2下游靶基因及信号通路，最后通过裸鼠转移瘤模型验证AP000472.2在体内对PTC细胞侵袭转移能力的影响。本研究对揭示AP000472.2在PTC侵袭转移中的机制具有重要意义，将为甲状腺乳头状癌高危患者的早期诊治提供新的预警标记物或药物干预靶点。

近年来甲状腺乳头状癌（PTC）发病率迅速上升，部分PTC恶性程度高，易发生局部及远处转移，但其中的具体机制尚不清楚。本项目首先利用transwell侵袭实验及单克隆细胞培养技术不断重复筛选出高转移甲状腺乳头状癌IHH4细胞亚株IHH4-M，通过裸鼠转移瘤模型验证高转移IHH4-M细胞亚株高侵袭与转移能力。进而利用高通量lncRNA芯片筛选甲状腺乳头状癌转移相关lncRNA，构建甲状腺乳头状癌转移相关lncRNA差异表达谱，筛选出候选研究目标lncRNA AP000472.2，并通过慢病毒过表达及干扰实验，证实lncRNA AP000472.2导入或沉默后可明显促进或抑制PTC细胞株体外增殖、侵袭和迁移能力。其次，通过RNA-pull down实验和双荧光素酶报告实验验证lncRNA AP000472.2通过海绵吸附作用内源性竞争miRNA-1197，进而阻断了miRNA-1197对SMAD3基因mRNA的降解作用，上调SMAD3基因表达，激活TGF-β信号通路促进甲状腺乳头状癌转移的相关机制。最后，通过裸鼠成瘤实验，进一步证实lncRNA AP000472.2在体内也能促进甲状腺乳头状癌细胞成瘤能力。综上所述，本项目为lncRNA AP000472.2促进甲状腺乳头状癌增殖及转移的相关机制奠定了一定基础，有望为甲状腺乳头状癌高危患者的早期诊治提供新的预警标记物或药物干预靶点，为甲状腺癌患者分层诊断及精准治疗提供理论基础与实验依据。项目资助发表相关论文7篇。