TGF-β介导AGO1/Smad2复合体调控上皮间质转化促进肝癌细胞侵袭转移的分子机制研究

Hepatocellular carcinoma (HCC) is one of the most common cancers in China, and its recurrence and metastasis in the treatment process is the most important factor affecting the prognosis of patients. Thus, exploring an appropriate therapeutic target is the key to solve the problem. In our previous studies, high expression of AGO1, which is an important component of the RNA-induced silencing complex, could promote the proliferation and metastasis in cell lines. Its high expression in HCC specimens could predict the poor prognosis. Preliminary mechanism studies demonstrated that AGO1 interacted with Smad2 to activate the phosphorylation of Smad2 and Smad4, thereby promoting Snail transcription and mediating the occurrence of epithelial mesenchymal transformation (EMT).The results indicated the close correlation between AGO1 and TGF-β pathway, AGO1 high expression predicted the poor prognosis. Based on these evidences, we proposed a hypothesis that TGF-β may promote the binding of AGO1 and Smad2 and activate phosphorylation, and up-regulate the transcription of N-cadherin and Snail, leading to the invasion and metastasis of HCC cells by EMT. Through this study, we expect to prove that TGF-β can mediate the binding of AGO1 and Smad2 to form a complex, and then explore the molecular mechanism of this complex involved in the regulation of EMT of HCC cells, and eventually clarify the possibility of AGO1/Smad2 complex as a new target for treating the invasion and metastasis of HCC, providing a new therapeutic idea for the clinical treatment of HCC metastasis.

肝癌是我国常见癌种之一，其在治疗过程中复发转移是影响患者预后的最主要因素。找到合适的治疗靶点是解决问题的关键。申请人前期研究发现RNA诱导沉默复合体的重要组分AGO1在肝癌细胞中高表达促进其增殖转移，在临床标本中高表达提示患者预后较差；初步机制研究发现AGO1与Smad2相互作用，激活Smad2和Smad4磷酸化，促进Snail转录，介导上皮间质转化（EMT）发生。前期研究提示AGO1紧密关联TGF-β通路，影响患者预后。基于此申请人提出假说：TGF-β可能促进AGO1和Smad2结合并激活磷酸化，上调N-cadherin和Snail的转录，介导肝癌细胞EMT，进而发生侵袭转移。通过本研究期望证实TGF-β介导AGO1与Smad2结合为复合体，明确该复合体参与调控肝癌细胞EMT的分子机制，探索AGO1/Smad2复合体作为肝癌治疗新靶点的可能性，为肝癌复发转移临床治疗提供新思路。

背景与目的:肿瘤相关中性粒细胞(tumor -associated neutrtrophils, TAN)在促进抑制性肿瘤免疫微环境和免疫逃避方面发挥关键作用，限制了免疫检查点抑制剂在不同癌症(包括肝细胞癌)中的治疗效果。Argonaute 1 (AGO1)是RNA沉默的关键。然而，AGO1在肿瘤微环境中的作用尚不清楚。.方法:我们对小鼠皮下HCC模型进行了Cy-TOF流式细胞术分析，探讨AGO1在肿瘤微环境中的作用。我们通过细胞因子抗体阵列来鉴定Ago1释放的趋化因子。我们通过肿瘤/中性粒细胞共培养、多重免疫荧光和肿瘤小鼠模型，研究了AGO1与趋化因子(C-C motif)配体25 (CCL25)的关系。我们使用RNA-seq, ChIP，荧光素酶报告器和CO-IP等方法以深入了解AGO1/SMAD2/SMAD4/ creb结合蛋白(CBP)复合体的潜在机制。我们使用CCL25中和单抗治疗免疫功能正常的小鼠。.结果:我们发现AGO1促进了肿瘤微环境中N2中性粒细胞的浸润。AGO1通过分泌CCL25招募N2中性粒细胞。AGO1通过骨形态发生蛋白受体、IA型/Smad2信号通路促进CCL25的表达。高表达的AGO1和CCL25在HCC样品中呈正相关，可以招募更多的N2中性粒细胞。此外，CCL25中和抗体可以抑制AGO1高表达的肿瘤。AGO1与Smad2相互作用促进其磷酸化。AGO1、Smad2和CBP形成一个复合物，诱导Smad4乙酰化并刺激CCL25转录。.结论:AGO1通过招募Smad2和CBP乙酰化Smad4，并通过增强CCL25表达招募N2 TAN促进肿瘤发生。CCL25是HCC进展的潜在生物标志物和HCC的治疗靶点。