LncRNA-CCAT2结合并调控BOP1诱导结直肠癌染色体不稳定性的作用研究

Chromosomal instability (CIN) plays a key role in colorectal cancer (CRC) carcinogenesis and progression, but the therapeutic strategies targeting CIN are poorly developed. Previous studies suggested that Colon Cancer-Associated Transcript 2 (CCAT2), which is a kind of long non-coding RNA (lncRNA) could promote the induction of CIN in CRC, but the molecular mechanisms are scantily understood. Further work demonstrated that CCAT2 could bind and regulate Block of Proliferation 1 (BOP1), and interestingly, the overexpression of BOP1 also promoted CIN in CRC. Based on these findings, we proposed the following hypotheses: firstly, CCAT2 binds directly to BOP1 to modulate its protein stability and subcellular localization, in addition, CCAT2 transcriptionally up-regulates BOP1 by Myc; and secondly, BOP1 promotes the mis-segregation of chromosomes, thus mediates the inductive effect on CIN and CRC progression of CCAT2. A series in vivo and in vitro experiments were then designed to elucidate the comprehensive regulation effect of CCAT2 on BOP1. Furthermore, the potential interacting region between CCAT2 and BOP1 and the beneath molecular mechanisms would also be investigated. This study also aimed to elucidate the mediatory role of BOP1 on CCAT2-induced CIN and CRC progression and its potential mechanism. This project will lead to a better understanding of CIN and the development of therapeutic strategies targeting CIN in CRC.

染色体不稳定性（CIN）在结直肠癌发生、发展中发挥着关键性作用，目前以CIN为切入点的肿瘤治疗策略进展十分缓慢。本课题组前期研究发现长链非编码RNA-CCAT2可诱导结直肠癌发生CIN，但分子机制不明。深入研究发现CCAT2可结合并调控BOP1蛋白，且这一蛋白可促进染色体分离异常导致CIN。因而我们推测：1）CCAT2可通过直接结合BOP1改变其蛋白稳定性及亚细胞定位，并在转录水平上通过Myc正向调控BOP1，最终上调BOP1；2）BOP1继而诱导染色体分离异常促进CIN及结直肠癌发生发展。本课题拟从分子、细胞、动物和人体组织水平分别研究CCAT2对BOP1的多层次调控作用及二者的作用位点和机制，揭示BOP1在CCAT2促进细胞染色体分离异常导致CIN及结直肠癌发生发展中的作用及其潜在的分子机制。本课题的开展有助于深入了解结直肠癌CIN的发生机制，为靶向CIN的临床干预途径提供理论依据。

染色体不稳定性可促进肿瘤转移和耐药，其调控具体机制厄待进一步研究。 既往研究证实长链非编码RNA(lncRNA)CCAT2与染色体不稳定性相关，但对于CCAT2如何调控这一过程以促进肿瘤发生发展的机制尚不明确。我们通过一系列的体内外研究证实CCAT2的高表达可诱导结直肠癌细胞系中发生染色体不稳定性。微阵列分析和质谱分析表明，CCAT2的表达与核糖体合成的基因的密切相关。通过后续体内外研究，我们发现CCAT2可直接与核糖体合成蛋白(BOP1)相互结合并使其降解延缓，最终提高其蛋白水平，在转录后水平上上调BOP1。此外，CCAT2还可通过调控MYC继而激活了BOP1的表达在转录水平上调BOP1。在结直肠癌细胞系中，我们也证实过表达BOP1可促进染色体结构及数目异常，此外BOP1可促进肿瘤的增殖，反之敲低BOP1后肿瘤增殖能力降低。上述结果证实了BOP1在染色体不稳定性形成及肿瘤发生发展中的作用。通过进一步探究发现BOP1通过促进Aurora B激酶的活性形式来调控染色体异常，这可能是其调控的具体机制之一。在CCAT2转基因小鼠中也可观察到相较于正常足足，BOP1在腺瘤组织中表达增高。临床标本分析也发现与非肿瘤组织相比，CCAT2及BOP1在微卫星稳定肿瘤中均表达增多，在具有微卫星不稳定性的肿瘤中却没有观察到这一现象。 此外CCAT2及BOP1水平表达呈正相关，且高表达CCAT2及BOP1患者的预后较差。通过该研究，我们证实了CCAT2可调控BOP1的表达来促进染色体不稳定性和肿瘤发生发展。该研究为开发针对该通路的策略来治疗微卫星稳定性的结直肠肿瘤患者提供了新思路。该项目最终发表SCI论文2篇。