去泛素化酶OTUD6A通过抑制HDAC6泛素化降解促进结肠癌侵袭和转移的机制研究

Colon cancer has a high rate of metastasis, patients with metastasis have poor prognosis. Epithelial mesenchymal transition (EMT) is the key process for cancer cell invasion and metastasis. Histone deacetylase 6 (HDAC6) plays a vital role in EMT, and posttranslational modifications of HDAC6 may regulate EMT via affecting the expression level of HDAC6 or its deacetylase activity, thus further contributing to cancer cell invasion and metastasis. Ubiquitination plays an important role in maintaining HDAC6 stability, and our previous study have found that SPOP is an E3 ligase of HDAC6. However, the deubiquitinase which reverses the ubiquitinational degradation of HDAC6 has not been reported. Our preliminary experimental results showed that: the expression level of deubiquitinase OTUD6A and HDAC6 in colon cancer were positively correlated. Cellular experiments results showed that OTUD6A could specifically bind to HDAC6, knockdown of OTUD6A could decrease the expression level of HDAC6, as well as suppress EMT. Thus we propose the following hypothesis: OTUD6A deubiquitinates HDAC6, subsequently regulates EMT, and therefore promote colon cancer invasion and metastasis. And we try to certificate this hypothesis via clinical samples, cellular and animal experiments. The present program will help us to better understanding the role and mechanism of OTUD6A in colon cancer invasion and metastasis, which will provide theoretic foundation for diagnosis and therapy of colon cancer.

结肠癌转移率高，出现转移者预后差。上皮间质转化（EMT）是癌细胞获得侵袭和转移能力的关键过程，组蛋白去乙酰化酶6（HDAC6）在EMT中起重要作用，翻译后修饰可通过影响HDAC6表达水平和/或其去乙酰化酶活性调控EMT，参与肿瘤侵袭和转移。泛素化在HDAC6稳态平衡中发挥关键作用，我们已报道SPOP是HDAC6重要的E3泛素连接酶之一，然而逆转其泛素化降解的去泛素化酶尚未见报道。预实验发现结肠癌组织中去泛素化酶OTUD6A和HDAC6表达水平呈正相关。细胞实验结果显示OTUD6A可特异性结合HDAC6，敲降OTUD6A可下调HDAC6表达水平，并抑制EMT。据此提出假说：OTUD6A抑制HDAC6泛素化降解，调控EMT，进而促进结肠癌侵袭和转移，并拟从临床组织、细胞和动物水平验证上述假说。本研究有望阐明OTUD6A在结肠癌侵袭和转移中的作用及分子机制，为结肠癌的诊断和治疗提供新的理论依据。

结直肠癌转移率高，预后差，蛋白质翻译后修饰及铁死亡在结肠癌的侵袭和转移过程中发挥重要作用。我们的前期研究发现组蛋白去乙酰化酶HDAC6在结肠癌中呈高表达，并与结肠癌患者预后差相关。同时前期实验证实结肠癌组织中去泛素化酶OTUD6A和HDAC6表达水平呈正相关，OTUD6A可特异性结合HDAC6，敲降OTUD6A可下调HDAC6表达水平，提示OTUD6A为HDAC6的去泛素化酶。本项目以人结肠癌组织标本、人结肠癌细胞株为研究对象，使用Western blot、Real time PCR、免疫共沉淀、免疫组化等方法，探索OTUD6A去泛素化修饰HDAC6对结肠癌细胞铁死亡的影响及其在结肠癌侵袭和转移中的作用，同时探索OTUD6A上游调控miRNA。研究发现：1）OTUD6A 在结肠癌组织中呈高表达，OTUD6A可促进结肠癌细胞的生长增殖，OTUD6A高表达与患者结肠癌肿瘤大小、浸润深度有关；2）OTUD6A 可抑制 HDAC6泛素化降解；3）OTUD6A可通过上调HDAC6表达水平，进而上调GPX4表达水平，从而抑制铁死亡，促进结肠癌细胞发生发展；4）反向预测OTUD6A的上游miRNA，证实miR-424-5P可调控OTUD6A表达水平；5）初步研究发现胃腺癌组织中OTUD6A蛋白呈高表达，且与肿瘤浸润程度、分化程度及TNM相关。本研究证实了OTUD6A去泛素化修饰HDAC6调控铁死亡促进结肠癌发生发展、侵袭和转移的具体机制，并探索出其上游miRNA，为结肠癌诊断和预后提供了潜在有效预测分子及miRNA，为结肠癌的分子机制的研究提供了新思路，同时也为结肠癌的小分子药物的开发提供了依据。