Department of Nuclear Medicine, Peking Union Medical College Hospital, Beijing, 100730, China.
Department of Radionuclide Treatment center, Beijing Nuclear Industry Hospital, Beijing, 100045, China.
Mol Cell Endocrinol. 2020 Jan 15;500:110634. doi: 10.1016/j.mce.2019.110634. Epub 2019 Oct 31.
Thyroid cancer is the most common malignant tumor with relatively high incidence and mortality in endocrine system. Research about thyroid cancer-related targets is the basis for the diagnosis of thyroid cancer and the development of new drugs. However, the predictive value of long non-coding RNA (lncRNA) for the diagnosis and prognosis of thyroid cancer is still in the preliminary stage of exploration. Thus, we for the first time investigated the effects and associated regulatory mechanism of lncRNA Forkhead box D3 antisense RNA 1 (FOXD3-AS1) in thyroid cancer in vitro and in vivo.
Quantitative real-time polymerase chain reaction (qRT-PCR) was used to measure the expression of lncRNA FOXD3-AS1 and miR-296-5p. Cell proliferation was detected through colony formation assay. Cell cycle was analyzed through flow cytometry. Cell mobility was valued through transwell invasion assay and wound healing assay. Western blotting was used to examine the expression of proteins related to cell proliferation and cell migration and TGF-β1/Smads signaling pathway. Luciferase reporter assay was used to verify the targeting relationship between FOXD3-AS1 and miR-296-5p. Tumor xenograft model was established and immunohistochemistry (IHC) was used to examine the expression of Ki67 and VEGF.
We found that the expression of lncRNA FOXD3-AS1was upregulated and it had negative correlation with the level of miR-296-5p in thyroid cancer tissues and cells. LncRNA FOXD3-AS1 knockdown effectively suppressed cell proliferation and cell invasion in vitro. Further study revealed that miR-296-5p was a target of lncRNA FOXD3-AS1 and FOXD3-AS1 exerted anti-tumor effect through up-regulating miR-296-5p. Moreover, we found that FOXD3-AS1 knockdown suppressed the aggressive biological behaviors of thyroid cancer through inactivating the TGF-β1/Smads signaling pathway. Subsequently, the in vivo experiments further verified that the FOXD3-AS1/miR-296-5p axis exerted obvious anti-tumor effect through inhibiting tumor growth and metastasis and the TGF-β1/Smads signaling pathway was also inactivated in vivo by the inhibition of FOXD3-AS1.
Inhibition of LncRNA FOXD3-AS1 suppresses the aggressive biological behaviors of thyroid cancer via elevating miR-296-5p and inactivating TGF-β1/Smads signaling pathway.
甲状腺癌是内分泌系统中发病率和死亡率相对较高的最常见恶性肿瘤。甲状腺癌相关靶点的研究是甲状腺癌诊断和新药开发的基础。然而，长链非编码 RNA（lncRNA）在甲状腺癌诊断和预后中的预测价值仍处于探索的初步阶段。因此，我们首次在体外和体内研究了 lncRNA 叉头框 D3 反义 RNA 1（FOXD3-AS1）在甲状腺癌中的作用及相关调控机制。
采用实时定量聚合酶链反应（qRT-PCR）检测 lncRNA FOXD3-AS1 和 miR-296-5p 的表达。通过集落形成实验检测细胞增殖。通过流式细胞术分析细胞周期。通过 Transwell 侵袭实验和划痕愈合实验评估细胞迁移能力。Western blot 用于检测与细胞增殖和细胞迁移及 TGF-β1/Smads 信号通路相关的蛋白表达。荧光素酶报告实验用于验证 FOXD3-AS1 与 miR-296-5p 的靶向关系。建立肿瘤异种移植模型，并用免疫组织化学（IHC）检测 Ki67 和 VEGF 的表达。
我们发现 lncRNA FOXD3-AS1 在甲状腺癌组织和细胞中的表达上调，且与 miR-296-5p 的水平呈负相关。lncRNA FOXD3-AS1 敲低可有效抑制甲状腺癌细胞的体外增殖和侵袭。进一步研究表明，miR-296-5p 是 lncRNA FOXD3-AS1 的靶基因，FOXD3-AS1 通过上调 miR-296-5p 发挥抗肿瘤作用。此外，我们发现 FOXD3-AS1 敲低通过抑制 TGF-β1/Smads 信号通路抑制甲状腺癌的侵袭性生物学行为。随后，体内实验进一步证实，通过抑制 FOXD3-AS1，FOXD3-AS1/miR-296-5p 轴通过抑制肿瘤生长和转移发挥明显的抗肿瘤作用，体内 TGF-β1/Smads 信号通路也被抑制。
抑制 lncRNA FOXD3-AS1 通过上调 miR-296-5p 并抑制 TGF-β1/Smads 信号通路抑制甲状腺癌细胞的侵袭性生物学行为。
