剪接蛋白SRSF2调控人结直肠癌恶性增殖的机制研究

Abnormal functions of splicing factors is recognized to contribute to aberrant splicing events, which is one of the pathological mechanisms of colorectal cancer. Recent studies showed that RNA-specific adenosine deaminase B1(ADARB1) is aberrantly spliced in glioma cells which leads to two transcripts with distinct functions. Our previous results showed that the expression of splicing factor SRSF2 is upregulated in human colorectal cancer tissues, and the proliferation is significantly inhibited when silencing the expression of SRSF2 in colorectal cancer cells. At the molecular level, SRSF2 can regulate the alternative splicing of target genes such as ADARB1. This study aims to screen the alternative splicing events regulated by SRSF2 in colorectal cancer cells through overexpression or knocking down SRSF2 in colorectal cancer cells. By analyzing the alternative splicing changes of downstream target genes such as ADARB1, we can identify the cis-acting elements in target genes that SRSF2 binds with. And the splicing regulation mechanism will be discussed, and the functions of different splicing isoforms of target genes will be well studied. Our study will explore the pathogenesis of colorectal cancer at the splicing regulatory molecular level, and provide new targets and new methods for the treatment of colorectal cancer.

剪接蛋白功能异常会导致靶基因选择性剪接异常，是恶性结直肠癌发生的病理机制之一。近年来研究发现RNA特异性腺苷脱氨酶B1（ADARB1）在神经胶质瘤细胞中异常选择性剪接，产生两种功能截然不同的转录本。我们前期的结果发现：剪接蛋白SRSF2在人结直肠癌组织样本中表达显著增加，沉默SRSF2表达可抑制结直肠癌细胞增殖及存活能力；在分子水平上，SRSF2可以调控靶基因ADARB1等的选择性剪接。本研究拟筛选SRSF2在结直肠癌细胞中调控的选择性剪接靶基因，通过基因克隆和敲减改变SRSF2在结直肠癌细胞中的功能，研究其对结直肠癌细胞恶性增殖的作用，通过分析其下游靶向调控基因ADARB1等的选择性剪接变化，识别靶基因中SRSF2结合的顺式作用元件，并探讨其剪接调控机制。我们的研究将从剪接调控分子水平探索结直肠癌发病机理，为结直肠癌的治疗提供新的药物靶点和临床治疗思路。

剪接调控因子SRSF2的异常表达可导致肿瘤发生，但目前其在结直肠癌中的分子机制尚不清楚。本研究中，我们发现与正常结直肠组织相比，剪接蛋白SRSF2在人结直肠癌（CRC）样本中高表达。SRSF2可以明显促进结直肠癌细胞在体内及体外的增殖能力。利用转录组高通量测序技术，我们筛选并确定了33个由SRSF2调节的选择性剪接事件。敲减SLMAP-L或CETN3-S剪接异构体可以显著抑制结直肠癌细胞的增殖，预示着它们在结直肠癌细胞恶性增殖中的重要作用。机制方面，体内紫外交联免疫共沉淀实验证明SRSF2蛋白的RNA识别结构域（RRM）与SLMAP或CETN3前体mRNA直接结合。SRSF2通过与SLMAP组成性25号外显子结合，激活了其选择性24号外显子的接入；而SRSF2通过与CETN3组成性6号外显子结合，促进了其选择性5号外显子的跳跃。敲减SRSF2，或其剪接靶点SLMAP-L或CETN3-S剪接异构体可导致结直肠癌细胞停滞在细胞周期的G1期。在敲减SRSF2的结直肠癌细胞中回补SLMAP-L或CETN3-S剪接异构体可以通过调控细胞周期部分恢复由于SRSF2敲减而导致的细胞增殖受抑制的现象。与正常结直肠样本相比，在结直肠癌样本中SLMAP第24号外显子接入比例显著增加，CETN3第5号外显子接入比例显著减少。我们的研究结果表明异常表达的SRSF2蛋白在剪接水平调控结直肠癌恶性增殖，并揭示了结直肠癌病人中潜在的剪接靶点。