BLACAT2通过结合SF1调控mRNA可变剪接促进膀胱癌细胞上皮-间质转化和转移

Epithelial-mesenchymal transition (EMT) is important in the process of acquiring metastatic ability of bladder cancer cells. But the molecular mechanisms of epithelial-mesenchymal transition in bladder cancer cells remain largely unexplored. Our pilot study have revealed that, overexpression of a long intergenic noncoding RNA (lincRNA) termed Bladder Cancer Associated Transcript 2 (BLACAT2) promotes EMT process of bladder cancer, and possible mechanism may be through binding splicing factor (SF1) and thus modulating massage RNA alternative splicing. In the current study, we will further verify the role of BLACAT2 overexpression in EMT and metastatic process of bladder cancer in cell line model, animal model and clinical tissues, through immunoblot and in situ hybridization, etc. We will further confirm the direct physical association of BLACAT2 with SF1 and its impact on the function of SF1 through RNA immunoprecipitation assay and protein co-immunoprecipitation assay, etc. We will further verify the role of BLACAT2 in modulating mRNA alternative splicing (eg. CD44) through functional reversal and rescue assay. This study will focus on the hypothesis that BLACAT2 promotes bladder cancer EMT and metastasis directly binds SF1 and modulating mRNA alternative splicing and study the novel mechanism of BLACAT2 and thus reveal the mechanistic nature of it in promoting bladder cancer metastasis. Success of this study will provide original scientific basis for BLACAT2 acting as novel biomarker and candidate therapeutic target for bladder cancer in the future.

上皮-间质转化（EMT）是膀胱癌获得转移能力的重要途径，但膀胱癌EMT的调控机制尚未阐明。我们的前期研究发现：长链基因间非编码RNA BLACAT2过表达促进膀胱癌细胞EMT，机制可能是通过结合剪接因子1（SF1）调控mRNA可变剪接。本课题拟进一步：应用免疫印迹、原位杂交等方法在细胞模型、动物模型和临床组织中证明BLACAT2过表达促进膀胱癌EMT和转移，应用RNA免疫沉淀、蛋白质免疫共沉淀等阐明BLACAT2与SF1的直接结合和对SF1功能的调控机制，并应用功能逆转及挽救实验证明BLACAT2对CD44等mRNA可变剪接的调控作用。本项目紧紧围绕"BLACAT2直接结合SF1进而调控mRNA可变剪接促进膀胱癌EMT和转移"这一科学假说，从BLACAT2的全新调控机制入手，揭示其促进膀胱癌转移的本质，为确立BLACAT2作为膀胱癌分子标记和治疗新靶点提供原创性的科学依据。

肌层浸润性膀胱癌非常容易发生淋巴结转移和远处转移，一旦发生转移，患者的五年生存率迅速下降。但目前膀胱癌转移的机制尚未完全阐明。本课题针对膀胱癌细胞上皮-间质转化的关键分子机制进行了深入的研究。我们首先在体外细胞模型确证BLACAT2过表达促进膀胱癌细胞上皮-间质转化；在联合免疫缺陷小鼠（NOD/SCID）皮下成瘤及尾静脉注射转移模型中证实，BLACAT2过表达后，膀胱癌细胞的侵袭性明显增强，肿瘤侵袭边缘呈现 “虫蚀状”改变，并促进肿瘤远处转移；在膀胱癌患者组织标本中证实，BLACAT2过表达与患者膀胱癌组织淋巴管密度升高及淋巴转移相关，BLACAT2过表达的患者总生存和无转移生存率均明显降低。我们通过分子机制研究发现，BLACAT2可与剪接因子SF1蛋白结合，而与SF1直接结合的关键碱基序列位于BLACAT2的5’端的400-600 nt。关键结合序列的发现，将为未来设计小分子靶向药物提供依据和靶点位置。BLACAT2过表达导致hnRNPA1、CD44、LDHA、HMGA1、ERBB2、VEGF-A均发生了外显子跳跃现象，说明BLACAT2过表达可能影响了剪接因子SF1的功能，进而对下游分子的剪接调控发生了改变。另外，我们还发现，BLACAT2通过与人类组蛋白H3K4甲基转移酶复合物的核心亚单位WD repeat domain 5（WDR5）蛋白直接结合，促进VEGF-C启动子区组蛋白H3K4三甲基化，上调VEGF-C表达。而BLACAT2-WDR5复合物精确定位到VEGF-C启动子的秘密，则是通过BLACAT2的5’端第33-66个碱基与VEGF-C启动子-540~-512碱基对、-1273~-1238碱基对分别形成RNA-DNA三聚体完成的。该研究阐明了BLACAT2促进膀胱癌转移的关键分子机制，对于指导膀胱癌的治疗和针对BLACAT2调控的下游通路开发新的分子靶向治疗药物具有重要意义。