启动子DNA甲基化修饰的长链非编码RNA调控乳腺癌转移的研究

Epigenetic modification such as DNA methylation has important effect on the regulation of gene expression. long noncoding RNA (LncRNA), as a new mechanism for Epigenetic modification, play an essential role in the development of malignant tumor. The upstream regulatory mechanism of the expression of LncRNA and its role in metastasis of breast cancer have been hotspots of epigenetic research. It is still unclear whether there is LncRNA which is regulated by DNA methylation of the gene promoter in breast and regulate the metastatic characteristics. Basing on our previous research about LncRNA on the development of malignant tumor, our recently preliminary experiments indicates that LncRNA-Uc003lxs.1 is differently expressing in various breast cancer cell, and the expression level of LncRNA is closely related to the DNA methylation of the gene promoter, knockdown the LncRNA could inhibit the breast cancer cell migration and invasion. By treating breast cancer cell with 5-AZA,we try to confirm the LncRNA regulated by promoter methylation is a key factor in breast cancer progress of metastasis through LncRNA chip, qRT-PCR, RNA interfence, migration, invasion and animal expression. The study will also clarify the LncRNA-associated protein take part in the signal transduction pathway regulating breast cancer metastasis. The study hereby will illustrate the mechanism of breast cancer metastasis regulated by LncRNA promoter methylation that enriches the theory of regulating LncRNA expression. which then provides a new idea for studying the molecular mechanism of cancer metastasis and new target for the treatment of breast cancer.

DNA甲基化等表观遗传修饰机制对基因表达具有重要的调控作用，长链非编码RNA（LncRNA）作为表观遗传修饰的新机制，密切影响恶性肿瘤的发生发展。LncRNA如何参与乳腺癌转移的调控、其本身表达的上游调控机制等目前尚不明确。我们前期研究发现LncRNA对乳腺癌等肿瘤的重要作用，进一步预实验表明，乳腺癌中LncRNA-Uc003lxs.1的表达与启动子DNA 甲基化状态密切相关，并调控乳腺癌细胞侵袭和迁移。本课题采用去甲基化药物5-Aza建立乳腺癌细胞去甲基化模型，通过LncRNA芯片、qRT-PCR、RNA干扰、RNA沉降技术、动物实验等体内外实验，筛选启动子受甲基化修饰的功能性LncRNA,研究其作用机制及功能网络。本研究将阐明启动子DNA甲基化修饰的LncRNA调控乳腺癌转移的分子机制，阐明新的LncRNA及相关蛋白参与的调控乳腺癌转移的信号转导通路，为乳腺癌的诊治提供新的靶点和思路。

转移是乳腺癌患者的主要死亡原因，而三阴性乳腺癌是乳腺癌中预后最差的一个亚型。表观遗传包括长非编码RNA（lncRNA）和甲基化等能调节乳腺癌的发生发展，但具体机制尚不明确，因此乳腺癌我们构建了乳腺癌的高转移模型和去甲基化的三阴性乳腺癌细胞模型，对这两个模型高通量测序，筛选出参与调控三阴性乳腺癌转移并且受到甲基化调控的lncRNA，Uc003xsl.1和AFAP1-AS1，我们在体外发现Uc003xsl.1在高转移的细胞中低度甲基化，高表达，受到受DNMT1甲基化修饰， Uc003xsl.1可促进乳腺癌的增殖、迁移和侵袭，进一步机制研究，Uc003xsl.1可以作为诱饵分子，竞争结合NF-κB的抑制因子NKRF，与NKRF蛋白C端R3H功能结构域结合后，占据了NKRF与IL8启动子上负性反应元件（NRE）的结合位点，阻碍了NKRF与NRE的结合，促进IL8的转录，进而促进下游炎性白诱导的三阴乳腺癌细胞转移。AFAP1-AS1主要存在细胞浆中，可以促进三阴性乳腺癌细胞增殖，迁移侵袭能力等增强，可以促进肿瘤细胞干性及间质上皮转化（EMT）能力的增强，在裸鼠体内证实AFAP1-AS1可以促进三阴性乳腺癌增殖、转移。通过BSP克隆测序实验及CHIP实验表明AFAP1-AS1的启动子区域受甲基化调控，主要受DNA甲基转移酶 3B（DNMT 3B）调控。通过RNA Pull Down、RNA IP、Western Blot等实验表明AFAP1-AS1可以与β-catenin结合，激活Wnt/β-catenin信号通路，从而导致三阴性乳腺癌增殖、转移；进一步深入机制研究表明AFAP1-AS1主要通过招募E3泛素化连接酶Smurf2降解β-catenin降解复合物中的Axin, Axin被降解后导致GSK3β磷酸化减弱，从而使β-catenin磷酸化减弱进而在胞浆中积聚，激活Wnt/β-catenin通路的作用，并主要影响下游靶基因CCND2的表达进而促进肿瘤的增殖和转移。