Zuo Wentao, Ma Haoyang, Bi Jianghui, Li Tiaolan, Mo Yifeng, Yu Shiyu, Wang Jia, Li Beiqing, Huang Jinfeng, Li Yongwen, Li Li
College of Basic Medical, Guilin Medical University, Guilin, Guangxi, P. R. China.
College of Pharmacy, Guilin Medical University, Guilin, Guangxi, P. R. China.
Gastroenterol Rep (Oxf). 2024 Nov 4;12:goae094. doi: 10.1093/gastro/goae094. eCollection 2024.
Intrahepatic and extrahepatic metastases contribute to the high recurrence rate and mortality of hepatocellular carcinoma (HCC). Constitutive activation of nuclear factor-κB (NF-κB) is a crucial feature of HCC. NF-κB p65 (p50-p65) is the most common dimeric form. Ser536 acts as an essential phosphorylation site of RelA/p65. However, the effect of RelA/p65 Ser536 phosphorylation on progression and metastases during intermediate and advanced HCC has not been reported.
Phosphorylation of RelA/p65 (p-p65 Ser536) and NF-κB p65 were detected by using immunohistochemical staining in HCC tissue samples. The biological effects of RelA/p65 Ser536 phosphorylation were evaluated by using xenograft and metastasis models. NF-κB p65 nuclear translocation was detected by using Western blotting. The binding of NF-κB p65 to the , , and promoters was detected by using chromatin immunoprecipitation. The biological effects on proliferation, migration, invasion, and epithelial-mesenchymal transition were assessed by using tetrazolium-based colorimetry, colony formation, EdU incorporation, flow cytometry, cell wound healing, and transwell assay.
NF-κB p65 is highly expressed, while p-p65 Ser536 is not well expressed in intermediate and advanced HCC tissues. experiments demonstrated that a phosphorylation-mimetic mutant of RelA/p65 Ser536 (p65/S536D) prevents tumor progression and metastasis. experiments showed that p65/S536D inhibits proliferation, migration, and invasion. Mechanistically, RelA/p65 Ser536 phosphorylation inhibits NF-κB p65 nuclear translocation and reduces NF-κB p65 binding to the , , and promoters.
RelA/p65 Ser536 phosphorylation was detrimental to NF-κB p65 entry into the nucleus and inhibited HCC progression and metastasis by reducing , , and . The phosphorylation site of RelA/p65 Ser536 has excellent potential to be a promising target for NF-κB-targeted therapy in HCC.
肝内和肝外转移导致肝细胞癌（HCC）的高复发率和死亡率。核因子κB（NF-κB）的组成性激活是HCC的一个关键特征。NF-κB p65（p50-p65）是最常见的二聚体形式。Ser536是RelA/p65的一个重要磷酸化位点。然而，RelA/p65 Ser536磷酸化在中晚期HCC进展和转移中的作用尚未见报道。
采用免疫组织化学染色检测HCC组织样本中RelA/p65（p-p65 Ser536）和NF-κB p65的磷酸化情况。通过异种移植和转移模型评估RelA/p65 Ser536磷酸化的生物学效应。采用蛋白质印迹法检测NF-κB p65核转位。采用染色质免疫沉淀法检测NF-κB p65与、和启动子的结合。采用基于四氮唑的比色法、集落形成、EdU掺入、流式细胞术、细胞划痕愈合和Transwell实验评估对增殖、迁移、侵袭和上皮-间质转化的生物学效应。
NF-κB p65在中晚期HCC组织中高表达，而p-p65 Ser536表达不佳。实验表明，RelA/p65 Ser536的磷酸化模拟突变体（p65/S536D）可阻止肿瘤进展和转移。实验表明，p65/S536D抑制增殖、迁移和侵袭。机制上，RelA/p65 Ser536磷酸化抑制NF-κB p65核转位，并减少NF-κB p65与、和启动子的结合。
RelA/p65 Ser536磷酸化不利于NF-κB p65进入细胞核，并通过减少、和抑制HCC进展和转移。RelA/p65 Ser536的磷酸化位点具有成为HCC中NF-κB靶向治疗的有前景靶点的巨大潜力。
