肝星状细胞通过线粒体代谢转变调控乳酸穿梭影响肝癌干细胞干性的机制研究

Hepatic stellate cells (HSCs), as important interstitial cells in liver cancer microenvironment, play an important role in the malignant process of liver cancer. However, the role of HSCs in hepatocellular carcinoma (HCC) stem cell pool and its regulatory mechanism is unclear. Previous studies by the applicant found that the metabolic shifts of mitochondria play an important role in the malignant transformation of tumors and the self-renewal of stem cells, suggesting that the mitochondrial metabolism mode might affect the cell fates in the tumor microenvironment. Furthermore, we found that after co-culture with the HCC cells, the HSCs were activated with the metabolic pattern change mediated by the AMPK/PGC-1α signaling pathway, meanwhile promoting the stemness of HCC by enhancing the lactate uptake by tumor cells. This study is intended to systematically detect the metabolic phenotype of HSCs in the liver cancer microenvironment on the basis of previous studies. With the use of inhibitors of aerobic glycolysis, we are going to explore the relationship between HSCs metabolic patterns and activation state, as well as the role of metabolites, lactic acid, in promoting stemness of HCC stem cells. Finally, we explored the mechanisms of AMPK / PGC-1α signaling pathway in HSCs mitochondrial metabolic shifts and elucidated the molecular mechanism by which HSC affects the stemness of HCC through lactic-acid shuttle. Taken together, these studies will not only enhance our understandings of the metabolic coupling in the cancer microenvironment, but also provide a theoretical basis for the developing a promising target to reduce the degree of malignancy of liver cancer.

肝星状细胞（HSC）作为肝癌微环境中重要的间质细胞，在肝癌恶性进程中扮演重要角色。然而，HSC对于肝癌干细胞池的作用及调控机制尚不明确。申请人前期研究发现线粒体代谢转变在肿瘤的恶性转化及干细胞的自我更新中扮演重要角色，提示线粒体代谢模式可能影响肿瘤微环境中的细胞命运。进一步，申请人发现与肿瘤共培养后，HSC发生激活，伴随AMPK/PGC-1α信号通路介导的代谢模式转变，并通过增强肿瘤细胞的乳酸摄取，促进肝癌干性。本研究拟在前期基础上，系统检测肝癌微环境中HSC的代谢表型。利用有氧糖酵解抑制剂，研究HSC代谢模式与激活状态的关系，并明确代谢产物乳酸对肝癌干细胞的影响。最后通过激活AMPK/PGC-1α信号，探究在HSC线粒体代谢模式转变中的作用，阐明HSC通过乳酸穿梭影响肝癌干性的分子机制。通过以上研究，不仅增强对肿瘤微环境中代谢偶联的认识，也为研发新靶点降低肝癌恶性程度提供理论依据。

本研究利用肝星状细胞（HSCs）与肝癌细胞体外共培养及体内成瘤模型，深入探究了肝癌纤维化微环境中HSCs与肝癌的相关作用关系，并阐明代谢中间产物乳酸在微环境中的偶联作用。. 本研究发现HSCs在肝癌共培养后发生AMPK/PGC-1α反向调控的糖酵解代谢转变，通过释放代谢中间产物乳酸，促进肝癌的干性、侵袭与转移。进一步研究发现，乳酸通过抑制多囊泡内体的降解，促进与细胞膜融合，促进了肿瘤外泌体的释放。机制研究发现，乳酸通过发挥乳酸化的作用影响外泌体降解释放过程。通过靶向抑制乳酸摄取，能有效抑制肝癌恶性进展。揭示了代谢偶联及乳酸化翻译后修饰在肝癌发生发展中的重要作用。. 鉴于肝癌多发生在肝纤维化背景下，深入探究HSCs的激活机制对于肝癌的发生具有重要意义。利用Nestin-GFP转基因小鼠模型，发现HSCs在肝纤维化中高表达巢蛋白Nestin，机制研究发现Nestin通过抑制TGFβ受体I的泛素化，促进了Smad2/3的磷酸化及HSCs的激活。通过AAV6靶向HSCs中Nestin的表达可有效逆转肝纤维的进展。由此推测肝癌纤维化微环境中激活的HSCs亦表达Nestin，通过抑制HSCs中Nestin的表达可抑制纤维化微环境，进而有效遏制肿瘤恶性进展。. 此外，肿瘤本身warburg效应是肿瘤恶性表型的特征之一。本研究通过分析发现，环状RNA——circFNDC3B通过编码具有功能的多肽circFNDC3B-218aa，影响肿瘤糖酵解转变，进而影响肿瘤的生长。提示除了微环境对肿瘤的相互作用，肿瘤可通过自身机制调控糖代谢变化。. 最后，肝脏缺血再灌注损伤影响了肝癌切除术后肿瘤复发。通过对缺血再灌注损伤模型的研究，发现CD4+T细胞通过表达CD154，促进缺血再灌注损伤。而通过间充质干细胞胞外囊泡（MSC-EVs）的输注，可通过稳定中性粒功能、减轻蛋白质紊乱折叠及影响钙离子信号通路，下调CD154的表达，有效保护了肝缺血再灌注损伤。