GSDMD介导的细胞焦亡通过上皮-间质转化促进肝肿瘤不完全消融后残癌侵袭转移的研究

Radiofrequency ablation for hepatocellular carcinoma can be affected by a variety of factors. Rapid progression and metastasis of residual tumors can often been observed after insufficient ablation. However, there are few studies on the biological characteristics of residual tumors such as invasion and metastasis after incomplete ablation, and the mechanism involved in this process has not been clearly understood. We found that there was pyroptosis phenomenon in the marginal tumor tissues affected by sublethal hyperthermia. In addition, the proportion of pyroptotic cells was significantly higher than that in unablated tumor tissues. Based on the results, we propose the hypothesis that sublethal hyperthermia activates GSDMD-mediated pyroptosis pathway, increases the release of IL-1β, changes the inflammatory microenvironment of tumor, and induces epithelial-mesenchymal transformation during radiofrequency ablation. It can promote the invasion and metastasis of residual cancer cells by the mechanism. Furthermore, it can effectively reduce the progress of residual cancer after incomplete ablation by inhibiting the expression of GSDMD. The study will investigate the expression level of important molecules related to pyroptosis and epithelial-mesenchymal transformation and the correlation between them by using the incomplete ablation hepatoma model and hepatoma cell lines with shRNA silencing GSDMD expression based on the experiments of molecule, cells and animal. This study will provide a new theoretical basis for revealing the new mechanism of invasion and metastasis of residual cancer after incomplete ablation and improving the clinical efficacy of hepatocellular carcinoma.

射频消融治疗肝癌时会受到各种因素影响，由于消融不足可导致残余肿瘤快速进展、转移。但是，有关不完全消融后残癌侵袭转移的研究极少，该过程所涉及的机制尚不清楚。我们研究发现在消融边缘亚致死性高温区肿瘤组织中存在焦亡现象，焦亡细胞比例明显高于未消融的瘤组织。据此我们提出假说：射频消融治疗肝癌时亚致死性高温激活了GSDMD介导的细胞焦亡通路，引起IL-1β释放增加，肿瘤炎性微环境发生改变，诱发了上皮-间质转化，进而促进残癌细胞的侵袭转移，通过抑制GSDMD表达可以有效降低不完全消融后残癌的进展。本研究拟从动物、细胞和分子水平，通过小鼠肝癌不完全消融模型、体外模拟消融所致的亚高温、建立shRNA沉默GSDMD表达的肝癌细胞系，检测残癌组织和体外肝癌细胞中焦亡关键分子及上皮-间质转化相关分子的表达情况及两者之间表达的相关性。本研究将为揭示消融后残癌侵袭转移的新机制以及提高肝癌临床疗效提供新的理论依据。

本研究通过用亚致死性高温（43℃）水浴加热肝癌细胞的方式，模拟热消融时残余肿瘤受到的热损伤。实验分为4组：37℃水浴组、GSDMD—37℃水浴组、43℃水浴组、GSDMD—43℃水浴组。Transwell实验和划痕实验检测细胞侵袭迁移能力。RT-PCR和Western blot检测细胞中焦亡关键分子（GSDMD、Caspase-1、IL-1β）及EMT相关分子（E-cadherin、N-cadherin、Vimentin）表达情况，分析两者之间表达的相关性与细胞侵袭迁移能力的关系。在体观察小鼠亚致死性高温处理的人肝癌细胞接种后肿瘤长径变化。结果发现亚致死性高温能够抑制人肝癌细胞的增殖、迁移及侵袭能力，并不诱导其发生EMT样改变；沉默GSDMD介导的细胞焦亡能够抑制EMT，从而抑制人肝癌细胞的增殖、迁移及侵袭能力；受亚致死性高温影响的残癌组织生长速度与正常肝癌组织生长速度无显著差异。因此，介于GSDMD在细胞焦亡中的作用，可针对肿瘤侵袭转移作用，为肿瘤治疗提供新的靶点，为相关临床药物的研发提供新的思路。