UBA2下调CDKN1A表达介导结直肠癌SUMO化促进肿瘤发生发展的作用机制研究

Our team found that SUMOylation was closely associated with colorectal cancer. UBA2, as one of the heterodimer of SUMO activating enzyme subunit 1, played a key role of SUMOylation. UBA2 might be an independent prognostic factor for CRC. The UBA2 expression from the normal intestinal mucosa to neoplasia to adenocarcinoma showed a trend of gradual enhancement. Moreover, CDKN1A was probably the target gene of UBA2 by RNA-seq. UBA2 knockdown could significantly inhibit the malignant biological behavior of cancer cells, promote cancer cell G1 phase arrest and late apoptosis by repressing CDKN1A. Therefore, UBA2, as a key molecule of SUMOylation, down-regulated CDKN1A expression to mediate SUMOylation and to promote tumorigenesis and progression in colorectal cancer. The project will intend to add clinical samples (paired of normal intestinal mucosa to neoplasia to adenocarcinoma) to identify UBA2 gradual expression from normal intestinal mucosa to neoplasia to adenocarcinoma. Through positive and negative intervention of CHEK1 expression and ChIP-Seq, CoIP technology, we aim to confirm the regulation relationship among UBA2, CHEK1 and CDKN1A. Utilizing ChIP-Seq technology to make clear the mechanism how UBA2 down-regulated the CDKN1A pathway. This project may help us to further clarify the mechanism of tumorigenesis and progression in colorectal cancer.

课题组前期发现SUMO化修饰与结直肠癌密切相关；UBA2是SUMO化过程的亚基，对SUMO化起决定作用，并且UBA2是结直肠癌独立预后因素，在结直肠正常黏膜-腺瘤-腺癌过程中表达渐进增强，其表达下调可抑制肿瘤恶性生物学行为；CDKN1A是UBA2作用靶基因，其表达降低可诱导肿瘤细胞发生G1期阻滞和晚期凋亡。据此分析，UBA2作为结直肠癌SUMO化修饰的关键基因，通过下调CDKN1A来增强细胞增殖，减少凋亡，介导结直肠癌SUMO化，促进肿瘤发生发展。本课题拟增加正常粘膜、腺瘤、腺癌配对的结直肠癌样本，检测UBA2在正常粘膜-腺瘤-腺癌病变过程中的差异表达；正负向调控CHEK1和应用ChIP、CoIP技术明确UBA2、CHEK1、CDKN1A上下游调控关系，探讨介导SUMO化修饰的机制；通过ChIP-seq明确UBA2调控CDKN1A下游通路。旨在进一步阐明结直肠癌发生发展的分子机制。

结直肠癌是由正常粘膜-腺瘤-腺癌多因素、多步骤逐渐发展的结果，而这多阶段过程与蛋白质翻译后修饰SUMO化密切相关，SUMO蛋白表达上调能显著增强结直肠癌肿瘤细胞恶性增殖、侵袭和转移能力。为了探索SUMO化在结直肠正常粘膜-腺瘤-腺癌进展的机制，课题组通过免疫组化和二代测序检测了同一配对正常-腺瘤-腺癌样本的同一部位SUMO化三个核心分子SAE1、UBA2 和UBC9的表达，结果显示UBA2在结肠正常、腺瘤、腺癌癌变过程中表达呈渐进性增高，在结直肠癌发生、发展过程中起极其重要作用。通过单因素、多因素和KM生存曲线分析，UBA2是结直肠癌独立预后因素。随后，在细胞功能实验和裸鼠成瘤体内实验中，进一步证实UBA2 表达下调可显著促进肿瘤细胞发生晚期凋亡和G1期阻滞。为了进一步研究UBA2促进结直肠癌发展的具体机制，课题组通过IPA软件和RNA-seq二代测序技术，发现UBA2可通过与CHEK1基因相互作用抑制CDKN1A表达，导致cyclin D1和caspase3/9等周期和凋亡基因的改变，从而增强细胞增殖和减弱凋亡, 促进结直肠癌的发生、发展。随后我们通过正负向调控UBA2和CHEK1，明确CHEK1上下游调控关系和信号通路。为UBA2成为结直肠癌诊断标志物提供理论依据。