RNA结合蛋白RBMS3通过下调Twist1抑制乳腺癌侵袭和转移

RBMS3, located at 3p24-p23, belongs to the c-Myc gene single-strand binding protein (MSSP) family. Loss of regions on the chromosome 3 short arm(3p) is a kind of the most frequent genetic alterations in various tumors, suggesting that chromosome 3p may have important tumor suppressing effect. In our previous study, RBMS3 was lower expressed in breast cancer tissues and could inhibit tumor cell invasion and metastasis in breast cancer cells. RNA sequence showed that Twist1 expression was downregulated by RBMS3 in breast cancer cell. Additionally, RNA immunoprecipitation (RIP) was followed to identify that RBMS3 could directly bind to Twist1 mRNA. To date, the mechanism by which RBMS3 regulates biological function of breast cancer is unknown. Therefore, we put forward a research hypothesis: RBMS3 downregulates the Twist1 transcript by directly binding to regions in the 3′-untranslated region of Twist1 mRNA to suppress breast cancer invasion and metastasis. This study is aimed to confirm the role of RBMS3 in breast cancer from cells level, tissues level, animals level and molecules level; to explore whether RBMS3 could regulate Twist1 expression; to identify the specific binding sites of RBMS3 in Twist1 transcript, to uncover the new mechanism by which RBMS3 inhibits breast cancer invasion and metastasis. Our study identifies a novel mechanism in which RNA binding protein regulate the breast cancer invasion and metastasis via post-transcriptional regulation of RNA binding protein, which may provide a new insight into the diagnosis and treatment for breast cancer.

RBMS3是一种RNA结合蛋白，位于3p24-p23，且在多个肿瘤类型中皆发现3号染色体短臂的多区域缺失，表明这个部位对肿瘤的发生有重大意义。前期研究中发现RBMS3在乳腺癌组织中低表达，能抑制乳腺癌细胞的增殖、侵袭和远处转移功能。通过RNA基因测序分析发现，过表达RBMS3下调Twist1的表达；另外运用RNA免疫共沉淀实验发现RBMS3可以直接结合Twist1的mRNA。据此我们提出科研假说：RBMS3可以通过结合Twist1的mRNA相关位点抑制Twist1的表达，进而抑制乳腺癌的侵袭和转移。目前尚未见关于RBMS3调节乳腺癌生物学功能的作用机制研究，本课题拟从细胞、组织、动物、分子等多层次明确RBMS3在乳腺癌中的作用，重点探讨RBMS3对Twist1的调节作用，明确RBMS3与Twist1的具体结合位点，揭示RBMS3抑制乳腺癌侵袭和转移的新机制，为乳腺癌的诊断和治疗提供新思路。

研究基础：在前期研究中发现，RBMS3 通过抑制乳腺癌细胞的增殖、迁移侵袭和转移功能来发挥抑癌作用。且通过转录组基因测序分析发现乳腺癌细胞中过表达RBMS3能够下调Twist1的表达水平。RNA免疫共沉淀证实，RBMS3可以与Twist1的mRNA直接结合。 主要研究内容：首先采用荧光素酶报告基因实验明确RBMS3与Twist1的mRNA直接结合位点。采用Western blot法、qRT-PCR法在蛋白和mRNA水平明确RBMS3的Twist1调节作用，进一步对RBMS3调节Twist1的表达进行深入的机制研究。其次在已构建好的RBMS3过表达及其对照的SUM-1315和MDA-MB-231细胞中，转染过表达Twist1及其对照的慢病毒，验证转染成功后，分别进行相应的功能学实验，明确RBMS3对Twist1诱导的乳腺癌细胞迁移和侵袭功能的影响。为全面探究RBMS3在乳腺癌中的抑癌作用，继续进行RBMS3对细胞的生长和增殖影响的研究。我们采用平板克隆实验、EDU实验、流氏分析实验来探究RBMS3对细胞增殖的影响，证明了RBMS3在乳腺癌的增殖中起着抑制作用，进而深入研究其中存在的机制。不仅如此，本项目围绕乳腺癌进行了拓展研究，主要研究对象为KIAA1429，是一种重要的RNA结合蛋白，本项目明确KIAA1429对乳腺癌细胞侵袭，迁移功能的影响及其调节的分子机制。重要结果：首先RBMS3可直接与Twist1 mRNA的3’端非翻译区的AU富含区结合，进一步实验表明，RBMS3可通过AKT通路调节Twist1及下游靶基因MMP2的表达水平。其次RBMS3可逆转Twist1诱导的乳腺癌细胞侵袭和转移。不仅如此，RBMS3能够通过促进p21mRNA的稳定性来抑制乳腺癌细胞的增殖和生长。最后本项目拓展研究阐明了KIAA1429显著促进乳腺癌细胞的迁移和侵袭作用及其内在机制。 科学意义：这些调控机制将给对进一步认识和治疗乳腺癌提供新的思路和方向。