荜茇酰胺通过靶向TrxR1来上调ROS发挥抗肝癌的作用和机制研究

Piperlongumine (PL) is a natural product isolated from medicinal plants, was recently identified as selectively toxic to cancer cells mainly through generation of ROS. However, PL has not yet been tested in hepatocellular carcinoma (HCC), and the exact target of PL remains unclear. Our previous research showed that TrxR1, the critical enzyme in regulating ROS levels, was overexpressed in human HCC. Meanwhile, we found that PL could induced significant apoptosis, and increases the level of ROS, along with inhibition of TrxR1 activity in HCC cells. Therefore, we hypothesized that PL selectively kills HCC cells by targeting the TrxR1 to increase ROS generation. .This project plans to identify TrxR1 is the direct target of PL at the molecular level, and clarify the signaling pathways of apoptosis through activating ROS and targeting TrxR1 protein in HCC cells. Furthermore, we will verify the anti-tumor activity of PL against HCC and the impact of TrxR1 on the development of HCC in vivo. Ultimately, we propose to analyze correlation between the development of HCC and TrxR1 by associating with clinical tissue samples. This research project will make a valuable contribution to the evidence base about TrxR1 as a direct target of PL, approach to clarify the molecular anti-hepatoma mechanisms of PL, and provide a novel target for the treatment of HCC.

荜茇酰胺是重要的中药单体化合物，对多种肿瘤细胞具有抑制活性，主要通过激活ROS来诱导肿瘤细胞凋亡。但它的直接作用靶点尚不明确，它对肝癌的作用尚无报道。TrxR1是调节ROS的重要蛋白酶，我们前期研究发现其在人肝癌组织中高表达；同时，前期研究还发现荜茇酰胺在肝癌细胞中能够明显诱导凋亡、上调ROS水平，并伴随着对TrxR1活性的抑制。我们推测，荜茇酰胺可能是通过靶向TrxR1来上调ROS水平、发挥抗肝癌作用。本项目拟在分子层面验证TrxR1为荜茇酰胺的直接作用靶点；在细胞层面上深入阐明TrxR1和ROS介导荜茇酰胺抗肝癌活性的分子机制和信号通路；在动物体内验证荜茇酰胺的抗肝癌活性和TrxR1对肝癌发生发展的影响；最后结合临床组织样本分析TrxR1与肝癌发生发展的相关性。本课题将确证TrxR1为荜茇酰胺的直接作用靶点，阐明荜茇酰胺抗肝癌的分子机制，同时为TrxR1作为肝癌治疗的靶点提供新的依据。

荜茇酰胺是重要的中药单体化合物，对多种肿瘤细胞表现出较强的选择性活性抑制，在抗肿瘤方面极具应用潜力，但其对肝癌的作用尚不清楚，且潜在的作用靶点及分子调控机制仍未知。基于项目组前期研究结果我们推测TrxR1和ROS在荜茇酰胺的抗肝癌调控中可能起关键作用。为进一步确认荜茇酰胺的抗肝癌分子作用机制，围绕荜茇酰胺和肝癌主要开展了以下研究内容：（1）验证ROS在荜茇酰胺抗肝癌活性中的作用；（2）细胞和分子层面上确证TrxR1是荜茇酰胺的直接作用靶点；（3）阐明荜茇酰胺通过抑制TrxR1诱导肝癌细胞凋亡的下游分子作用机制；（4）动物层面验证荜茇酰胺的抗肝癌效果及其机制；（5）临床肝癌组织中TrxR1的表达和活性与肝癌疾病进程的相关性。研究结果明确了荜茇酰胺可靶向抑制TrxR1酶的活性并增加肿瘤细胞内ROS水平，从而激活内质网应激途径最终诱导肝癌细胞凋亡，其可作为潜在的抗肝癌药物，也进一步确证了TrxR1是肝癌的重要药靶。同时，对荜茇酰胺在转录层面对肝癌疾病进程的影响也进行了探索，发现荜茇酰胺能够诱导LINC01391上调来抑制HCC细胞增殖，进一步深入研究发现LINC01391是通过ICAT抑制Wnt/β-catenin途径并发挥抗肝癌作用。不仅如此，依托本项目还研究确认microRNA-590-5p/YAP1轴、CPEB1以及NEAT1等多个不同的作用靶点在肝癌疾病进程中扮演了重要角色。本项目的研究成果为肝癌的诊疗提供了新的思路以及多个新的药物干预靶点。