天然反义转录物lncRNA FGF14-AS2调控FGF14抑制乳腺癌骨转移的机制研究

At present, how to prevent and control the bone metastasis of breast cancer is an urgent problem to be solved in clinic. Natural antisense transcripts (NATs) are a class of very important molecules involved in the regulation of gene expression. LncRNA FGF14-AS2 is a NAT of the first intron of FGF14 gene, and its downregulation is closely related to the metastasis of breast cancer. However, the precise roles and the underlying mechanisms of FGF14-AS2 in the metastasis of breast cancer still need to be explored further. The preliminary data of our group showed that both FGF14-AS2 and FGF14 inhibited the migration and invasion of breast cancer cells when the expression of these two genes was elevated. In addition, FGF14-AS2 overexpression increased the protein level of FGF14, but had no effects on the mRNA expression and protein stability of FGF14. More importantly, we found that the expression of PTHrP and IL-8, which are important stimulating factors for osteoclast differentiation, was decreased by FGF14-AS2 overexpression. Moreover, FGF14-AS2 was demonstrated to be located in the cytoplasm of breast cancer cells and bound with eIF4E, a eukaryotic translation initiation factor. Based on these findings, we postulate that FGF14-AS2 may regulate the expression of FGF14 at the translational level by binding with eIF4E, which further inhibits the bone metastasis of breast cancer. We will test this hypothesis by performing cell biological experiments, construction of bone metastasis model in mice as well as carrying out clinical investigations. Results of this project will not only be helpful in the elucidation of the molecular mechanisms of the bone metastasis of breast cancer further, but also provide a new target for the prevention and treatment of the bone metastasis of breast cancer.

如何预防和治疗乳腺癌骨转移是当前临床亟待解决的问题。LncRNA FGF14-AS2为FGF14基因第一个内含子的天然反义转录物（NAT），其表达下调与乳腺癌转移密切相关，但具体作用及机制尚不清楚。本课题组前期实验结果表明：过表达FGF14-AS2和FGF14均明显抑制乳腺癌细胞的迁移和侵袭，过表达FGF14-AS2上调FGF14的蛋白表达，但对其mRNA表达和蛋白稳定性均无影响；过表达FGF14-AS2还下调破骨细胞分化促进因子PTHrP和IL-8的表达；FGF14-AS2定位于细胞质并与真核翻译起始因子eIF4E相结合。由此我们推测：FGF14-AS2通过与eIF4E结合在翻译水平调控FGF14的表达，进而抑制乳腺癌骨转移。本项目拟通过细胞、动物和临床水平的研究对此假说进行验证。该项目的研究结果不仅有助于乳腺癌骨转移的分子机制的进一步阐明，也将为乳腺癌骨转移的预防和治疗提供新的靶点。

骨转移是乳腺癌常见的并发症。我们先前的研究已经证明lncRNA FGF14-AS2是乳腺癌转移的重要抑制因子。然而，FGF14-AS2是否参与乳腺癌骨转移过程的调控尚不清楚。本研究的目的在于探讨FGF14-AS2在乳腺癌骨转移中的作用和机制。通过体内和体外研究，我们发现FGF14-AS2显著抑制了乳腺癌的溶骨性骨转移。机制上来讲，FGF14-AS2一方面通过与eIF4E结合而干扰eIF4E/eIF4G复合物的形成；另一方面，FGF14-AS2通过与EIF2AK2结合而促进EIF2AK2的泛素化降解，从而抑制p38/Mnk1信号通路，进而抑制eIF4E的磷酸化。eIF4E/eIF4G复合物形成和eIF4E磷酸化的抑制导致了RUNX2的翻译水平下降，进而抑制了破骨细胞分化的关键调节因子RANKL的转录，最终抑制了破骨细胞分化和乳腺癌骨转移。另外，m6A阅读蛋白YTHDF2与FGF14-AS2的结合导致了FGF14-AS2 RNA稳定性的下降，这是FGF14-AS2在乳腺癌中表达下调的原因之一。临床水平研究发现，高水平的YTHDF2或低水平的FGF14-AS2的乳腺癌患者呈现较差的无远端转移生存期（distant metastasis-free survival, DMFS），并且在乳腺癌组织中YTHDF2和FGF14-AS2的表达水平呈显著负相关。综合以上研究结果，我们发现并证明了FGF14-AS2在乳腺癌溶骨性骨转移中起关键调节作用，是非常有前景的乳腺癌骨转移预后的生物标志物和治疗靶点。. 此外，我们还通过一系列的体外、体内实验证明了FGF14-AS2参与乳腺癌细胞的迁移、侵袭和乳腺癌转移过程。进一步地，我们发现FGF14-AS2通过ceRNA机制竞争性吸附miR-370-3p，从而上调其母基因FGF14的表达。临床水平的研究表明，与癌旁组织相比，miR-370-3p在乳腺癌组织中表达上调，而FGF14-AS2的表达下调，且miR-370-3p与FGF14-AS2的表达呈负相关，FGF14-AS2与FGF14的表达呈正相关。以上研究结果提示：FGF14-AS2/miR-370-3p/FGF14轴参与了乳腺癌转移的调控，该研究结果提供了乳腺癌治疗的新的干预靶点和方向。