类泛素蛋白FAT10调控Capn4表达在结肠癌侵袭转移中的作用及机制研究

Our team found that ubiquitin-like protein FAT10 can promote the invasion and metastasis of malignant tumors via stabilizing its substrates (cancer research), but the expression and role of FAT10 in colorectal cancer (CRC) are unknown. Recently, we demonstrate that Capn 4 play an important role in the progression of CRC. Further, our preliminary studies suggest that FAT10 was highly expressed in CRC tissues and positively correlated with Capn4 expression. Furthermore, downregulation of FAT10 could inhibit the expression of Capn4 in CRC cells and reduce the invasion and migration ability of CRC cells. Moreover, direct binding between endogenous FAT10 and Capn4 was identified in CRC cells by Co-IP and immunofluorescence co-localization assay. Besides, our findings indicate that Capn4 is degraded by the Ubiquitin-proteasome pathway (UPS), but blocking UPS could relieve the regulation of FAT10 on Capn4. Consequently, we hypothesize that FAT10 stabilizes Capn4 expression through inhibiting the ubiquitination and degradation of Capn4, leading to promote the invasion and metastasis of CRC. To test this hypothesis, we will construct stable transfected cell lines and nude mice transplantation tumor model, to investigate the effect of FAT10 on the invasion and metastasis of CRC by regulating Capn4. We further demonstrate the molecular mechanism through which FAT10 regulating Capn4 expression by in vitro ubiquitination experiments. This study will provide new clues for molecular diagnosis and targeted therapy of CRC.

本团队研究发现类泛素蛋白FAT10稳定底物表达促进恶性肿瘤的侵袭转移（Cancer Research），而结肠癌中的表达及作用不详。最近我们研究证实Capn4在结肠癌进展中发挥重要作用。预实验发现：FAT10在结肠癌组织中高表达，与Capn4表达呈正相关；下调FAT10抑制结肠癌细胞中Capn4表达，并降低细胞的侵袭迁移能力；免疫共沉淀和免疫荧光共定位发现FAT10与Capn4直接结合；泛素-蛋白酶体途径（UPS）介导Capn4的降解，阻断UPS途径可解除FAT10对Capn4的调控作用。据此，我们推测：FAT10通过抑制Capn4泛素化降解从而稳其表达，促进结肠癌侵袭转移。本项目通过构建稳定转染细胞株及裸鼠原位移植瘤模型，明确FAT10通过调节Capn4促进结肠癌侵袭转移；利用体外泛素化实验等技术阐明FAT10调控Capn4表达的分子机制。本研究将为结肠癌分子诊断、靶向治疗提供新的线索。

结直肠癌 (colorectal cancer CRC) 是严重危害人类健康的恶性肿瘤之一，其发病率位居恶性肿瘤第三位，死亡率居世界第四位。文献报道类泛素蛋白FAT10通过稳定底物表达促进恶性肿瘤的侵袭转移，而在结肠癌中的表达及作用不详。近期我们研究证实Capn4在结肠癌进展中发挥重要作用，但Capn4在结肠癌中如何被调控尚不明确。我们通过体外构建稳转细胞株及体内动物实验，明确FAT10通过调节Capn4促进结肠癌侵袭转移；利用体外泛素化实验等技术阐明FAT10调控Capn4表达的分子机制。研究发现：1、FAT10在结肠癌中高表达，且与患者临床分期差以及不良预后相关。体内体外实验证实干扰FAT10的表达后能抑制结肠癌的侵袭迁移能力，而过表达FAT10后能促进结肠癌的侵袭迁移能力。2、在结肠癌细胞中过表达FAT10可上调Capn4的蛋白表达水平而不影响其mRNA水平，并且可促进结肠癌细胞的侵袭迁移能力，在之基础上调Capn4后能逆转因下调FAT10后导致结肠癌细胞侵袭转移能力的下降，这说明Capn4是FAT10调控结肠癌侵袭转移的关键分子。随后检测结肠癌组织中FAT10和Capn4的蛋白表达水平，发现两者表达正相关。3、机制上明确了泛素-蛋白酶体途径（UPS）介导Capn4的降解，阻断UPS途径可解除FAT10对Capn4，证实了FAT10与Capn4直接结合且FAT10与泛素竞争结合Capn4导致其泛素化水平下降。本项目是对FAT10、Capn4基因研究的继续和深入，研究结果将有助于阐明结肠癌中FAT10调控Capn4的分子机制，并为FAT10、Capn4涉及的调控通路和结肠癌的治疗等提供新的理论依据。