USP44启动子高甲基化通过调控p53蛋白稳定性促进阴茎癌恶性进展的作用及机制研究

The inguinal lymph node metastasis of penile cancer is the most important factor for treatment and survival. However, the molecular mechanism of lymph node metastasis has not been reported yet. Five different biological characteristics of cell lines which provide experimental basis were previously successfully established. mRNA-seq, Western-Blot and qRT-PCR showed that the decreased expression of USP44 was regulated by promoter methylation and involved in lymph node metastasis. Our primary investigations also showed that decreased expression of USP44 promotes the degradation of p53 ubiquitin, which further affects the biological characteristics of penile cancer cells, such as EMT, dryness and chemosensitivity. In this proposal, we planned to study the function and mechanisms of USP44 and its promoter methylation on the progression of penile carcinoma through regulating p53 protein stability by multidisciplinary, multidimensional and multimodel technology. This study is expected to elucidate the mechanism of metastasis of penile cancer at the molecular level, and provide theoretical basis for predicting markers and afford a new approach therapy for lymph node metastasis of penile cancer.

阴茎癌淋巴结转移是决定治疗及预后的关键因素。目前，阴茎癌淋巴结转移的分子机制研究尚处于空白阶段。我们将前期成功构建5株不同生物特性的细胞株作为实验基础。通过配对的阴茎癌原发灶组织与癌旁组织RNA-seq，Western-Blot及qRT-PCR检测发现USP44在阴茎癌中低表达，受启动子甲基化调控且参与淋巴结转移。初步实验提示USP44低表达促进p53蛋白泛素降解，进而影响阴茎癌细胞EMT、肿瘤细胞干性、化疗敏感性等生物学特性。本研究将运用多学科、多层面、多模型的技术全面阐明USP44启动子甲基化调控p53蛋白稳定性而发挥抑癌作用的具体分子机制。有望从分子水平阐释阴茎癌淋巴结转移机制，为隐匿性/早期转移性阴茎癌患者提供可能的预测标志物，为后续治疗方案的选择提供参考证据，为寻找潜在的治疗靶点提供理论依据。

项目背景：阴茎癌淋巴结转移是决定治疗及预后的关键因素。目前，阴茎癌淋巴结转移的分子机制研究尚处于空白阶段。.研究内容：体内、外实验明确USP44在阴茎癌中表达情况及USP44 对阴茎癌细胞生物学功能的调控；mRNA-seq, Western-Blot等实验明确阴茎癌细胞中USP44表达的甲基化调控机制及USP44调控阴茎癌发生发展的具体分子机制。.重要结果：USP44可能是阴茎癌的潜在抑癌基因。初步体内、外实验提示USP44低表达与阴茎癌的生长及侵袭转移有关。随后研究发现阴茎癌中USP44表达下调可能与其启动子区高甲基化有关。.项目研究证实：USP44mRNA和蛋白表达水平在阴茎癌细胞中显著低于正常细胞，且与其启动子区甲基化水平呈显著负相关，采用去甲基化药物5-Aza处理，可降低USP44启动子区甲基化水平和促进其表达。过表达USP44可显著抑制阴茎癌细胞增殖、迁移、侵袭能力，而敲低USP44则呈相反的作用。Co-IP/MS分析发现USP44可与鸟嘌呤核苷酸从头合成的关键酶IMPDH2相互结合，且抑制IMPDH2表达，采用蛋白酶体抑制剂MG132可逆转USP44对IMPDH2表达的影响。质谱检测发现，USP44显著减少阴茎癌细胞ATP和GTP含量。以上结果提示，USP44可能通过稳定E3连接酶对IMPDH2的降解，从而抑制鸟嘌呤核苷酸从头合成，抑制阴茎癌的进展。.关键数据及其科学意义：本研究将运用多学科、多层面、多模型的技术全面阐明USP44启动子甲基化调控的具体方式而发挥抑癌作用的具体分子机制。有望从分子水平阐释阴茎癌淋巴结转移机制，为隐匿性/早期转移性阴茎癌患者提供可能的预测标志物，为后续治疗方案的选择提供参考证据，为寻找潜在的治疗靶点提供理论依据。