TLR4介导的PKM2转录及活性调控对人肝癌细胞能量代谢的影响及机制研究

Hepatocellular carcinoma (HCC) is one of the most commonly occurring malignant tumors in clinical. In the process of the evolution from hepatitis to HCC, there was the change of the patterns of cellular energy metabolism. The research for mechanisms of cellular energy metabolism changes is an important subject to the treatment of HCC. The material in the tumor microenvironment can promote the proliferation of liver cancer, in which the transmembrane transduction is mainly completed through TLR4. TLR4-mediated hepatocellular carcinoma cell proliferation process is accompanied by the changes of energy metabolism. PKM2 is the key enzyme in the process of aerobic glycolysis of tumor cells. TLR4 regulates both the nuclear transcription and the activity of PKM2, but its regulatory mechanism is still unclear. On the basis of our previous project and work, the subject intends to reveal the influence of TLR4-mediated transcriptional regulation and activity regulation of PKM2 for energy metabolism of human liver cancer cells and its mechanism from the level of clinical tissue specimens, the nude mouse model of hepatocellular carcinoma and the liver cancer cell lines, using the common technique of gene and protein research, such as luciferase reporter gene, EMSA, ChIP, real-time RT-PCR, IMAC technology, Co-IP, western blot, etc. The completion of our subject will provide reliable theoretical basis for improving molecular biological mechanism, and open up a new way for developing the appropriate treatment strategies and greatly improving the effect of the treatment in the occurrence and development of liver cancer.

肝癌是临床上最常见的恶性肿瘤之一，从肝炎到肝癌的演变过程中，伴随有细胞能量代谢方式的改变，研究细胞能量代谢改变的机制是肝癌治疗的重要课题。肿瘤微环境中的物质能够促进肝癌的增殖，其跨膜转导主要是通过TLR4来完成。TLR4介导肝癌细胞增殖过程伴随其能量代谢的改变；PKM2是肿瘤细胞有氧糖酵解过程中的关键酶；TLR4对PKM2的核转录及活性均有调节作用，但其调控机制尚不清楚。在我们的前期课题及工作基础之上，本项目拟从临床肝癌组织标本、肝癌裸鼠模型和肝癌细胞系三个水平，应用荧光素酶报告基因、EMSA、ChIP、real-time RT-PCR以及IMAC、Co-IP、western blot等基因研究和蛋白质研究常用技术，揭示TLR4介导的PKM2转录及活性调控对人肝癌细胞能量代谢的影响及机制。本课题的完成为完善肝癌发生发展的分子生物学机理提供可靠的理论基础，为开发相应的治疗策略并大幅提高肝

肝癌是临床上最常见的恶性肿瘤之一，从肝炎到肝癌的演变过程中，伴随有细胞能量代谢方式的改变，研究细胞能量代谢改变的机制是肝癌治疗的重要课题。肿瘤微环境中的物质能够促进肝癌的增殖，其跨膜转导主要是通过TLR4来完成。TLR4介导肝癌细胞增殖过程伴随其能量代谢的改变；丙酮酸激酶M2型(Pyruvate Kinase M2, PKM2)是肿瘤细胞有氧糖酵解过程中的关键酶；TLR4对PKM2的核转录及活性均有调节作用，但其调控机制尚不清楚。.我们首先通过检测了细胞内ATP 含量、细胞培养液中乳酸含量及葡萄糖浓度的变化，证实在TLR4介导肝癌细胞增殖过程中伴随有能量代谢的改变；有氧糖酵解是肿瘤细胞能量代谢的最主要特征，而导致肿瘤细胞糖酵解增强的重要原因之一是关键酶合成增加、活性增强。在肿瘤细胞的糖酵解过程中PKM2 发挥重要作用。我们首先对比了在肿瘤微环境物质的作用下高表达TLR4 肝癌细胞株和低表达TLR4 肝癌细胞株PKM2 表达含量的差异，证实了TLR4 对PKM2 的核转录调节。应用常用基因信息学分析数据库（Genome Browser、TFSEARCH）对PKM2 的基因序列进行分析。利用PCR及重叠PCR 技术构建野生型及突变型PKM2基因的启动子区，利用DUAL Reporter Assaysy stem处理并测定荧光强度, 结合体外细胞实验，发现与肿瘤密切相关的转录因子GATA2能结合PKM2启动子上的AGATTTTTATCTCA序列，证实GATA2 参与了肝癌细胞TLR4 对PKM2 的转录调控；此外，通过免疫共沉淀、Western blot和免疫荧光技术证明TLR4介导的PKM2活性调节是通过TLR4/MyD88蛋白与PKM2直接结合、调节 PI3K通路TLR4介导的信号转导是通过ERK通路磷酸化PKM2三种方式进而影响其活性及能量代谢。进一步，我们利用慢病毒整合的低表达TLR4的肝癌细胞株，构建裸鼠皮下成瘤模型。证实低表达TLR4的肝癌细胞的致瘤能力大大减弱，且PKM2的表达显著减低。.本项目从临床肝癌组织标本、肝癌裸鼠模型和肝癌细胞系三个水平，揭示TLR4介导的PKM2转录及活性调控对人肝癌细胞能量代谢的影响及机制，为肝癌发生发展分子机制提供理论依据并为肝癌的治疗效果开辟一条崭新的途径。