PADI1瓜氨酸化p65促进肺腺癌发生发展的机制研究

The citrullination modification of proteins has been demonstrated playing an important role in the pathological processes of malignant tumor, such as proliferation and metastasis. This process is mediated by peptidylarginine deiminases (PADIs). Interestingly, the second highest number of citrullinated peptides was identified in lung tissue. However, the role of PADIs in the development of lung cancer remains unclear.Our preliminary studies first found that PADI1 was significantly upregulated in lung adenocarcinoma tumor tissues and patients with high PADI1 expression had shorter overall survival. Knockdown of PADI1 suppressed cell growth and inhibited migration in lung adenocarcinoma cell lines. Microarray results following GSEA enrichment and KEGG pathway analysis indicated PADI1 was positively correlated with NF-κB signaling. Interestingly, citrullination of protein have been demonstrated to modulate its cellular localization. We further found NF-κB luciferase reporter activity was significantly decreased in the PADI1-silenced lung adenocarcinoma cell lines. Moreover, western blot revealed that the expression of nuclear p65 was also significantly decreased in PADI1-silenced lung adenocarcinoma cell lines. However, we found knockdown of PADI1 had no impact on the expression of p65 total mRNA and protein and IκB protein. Based on the citrullination function of PADI1, we hypothesized that PADI1 may promote p65 citrullination, which further promotes its nuclear localization, and therefore modulated the expression of cytokines in lung adenocarcinoma. This project sets out to investigate the function of PADI1 in lung adenocarcinoma on levels of clinical samples, cellular and animal study, and further explore the possibility that PADI1 contributes lung adenocarcinoma proliferation and metastasis on p65 through ‘rescue’ experiments. The detailed mechanisms will be investigated by CoIP, protein mass spectrometry and other methods.

蛋白瓜氨酸化修饰促进肿瘤增殖转移等恶性过程。精氨酸脱亚胺酶（PADIs）促进蛋白瓜氨酸化。研究发现肺组织存在丰富的瓜氨酸化，但PADIs是否参与肺癌的发生发展尚不清楚。我们首次发现PADI1在肺腺癌中表达升高并与预后负相关；低表达PADI1抑制肺腺癌细胞增殖及转移。基因芯片结合生信分析提示PADI1通过NF-κB通路发挥作用。研究显示瓜氨酸化修饰影响蛋白的细胞定位。我们发现低表达PADI1抑制NF-κB转录活性，减少核内p65蛋白含量，而不影响p65总mRNA、p65总蛋白及IκB蛋白表达。鉴于PADI1的瓜氨酸化作用，我们推测PADI1可能通过瓜氨酸化p65促其入核，进而调节炎症因子促进肺腺癌的发生发展。本项目将从临床样本、细胞实验和动物实验三个层面，通过质粒构建、免疫沉淀、蛋白质谱等手段，研究PADI1通过瓜氨酸化p65促进肺腺癌增殖转移的分子机制，为肺腺癌的发生发展提供新的理论依据。

近年来研究发现，蛋白瓜氨酸化修饰不仅调节机体的炎症及免疫反应，也参与调节肿瘤细胞的增殖、转移、上皮间质转化、肿瘤细胞耐药等恶性生物学行为。精氨酸脱亚胺酶（PADIs）促进蛋白瓜氨酸化。研究发现肺组织存在丰富的瓜氨酸化，但PADIs是否参与肺癌的发生发展尚不清楚。本项目从临床样本、细胞实验、动物实验、分子机制四个层面，研究PADI1在肺腺癌中的临床意义、分子生物学功能及其作用机制。研究发现：在TCGA肺腺癌数据库中，肿瘤组织中PADI1表达水平显著高于正常组织，且PADI1高表达提示预后不佳，是患者预后不佳的独立危险因素；GEO数据库和我们自己的标本也证实了上述结果。在肿瘤生物学功能方面，分析发现PADI1可能与糖酵解、上皮间质转化、缺氧、炎症反应、KRAS信号激活、有丝分裂激活等肿瘤特征相关；细胞及动物实验证实，低表达PADI1抑制肺腺癌的增殖能力，且细胞实验还发现低表达PADI1抑制肺腺癌转移能力；通过RNA芯片及TMT蛋白组学发现，PADI1可能通过Cell cycle、DNA Replication、TNF signaling pathway、NF-kappa B signaling pathway、p53、apoptosis等信号通路发挥作用。进一步，我们发现，低表达PADI1后，可抑制p53信号通路中的CDC45L、MCM2、p21及MCM4的蛋白表达；与此同时，低表达 PADI1可显著抑制NF-κB的转录活性，CCL5、IL1α、IL1β、IL6、CXCL8等多个与促进肿瘤发生发展的炎症因子表达水平显著下降。项目的实施，发现PADI1可作为肺腺癌预后不佳的独立危险因素，促进了肺腺癌的增殖及转移，初步揭示了PADI1的分子作用机制，从而为肺腺癌的防治提供新的思路和靶点。