基于肝特异性敲除小鼠研究LASS2在肝细胞癌发生发展中的作用及机制

The LASS2 gene was cloned in 2001, which is highly expressed in liver. It remains unclear whether LASS2 plays an important role in the occurrence and development of hepatocellular carcinoma (HCC). Our preliminary experiments have shown that the liver-specific LASS2 knockout mice could spontaneously develop HCC. We also found that the expression of LASS2 gene in the HCC specimens was significantly lower than that in adjacent tissues. The above results suggested that LASS2 was likely to play an important role in the occurrence and development of HCC. It is known that LASS2 can inhibit the activity of proton pump V-ATPase, and the level of V-ATPase activity is an important determinant of activation of Wnt/β-catenin pathway. Analysis of gene expression profiles indicated that deletion of LASS2 led to the abnormal activation of the Wnt/β-catenin pathway. Therefore, we hypothesize that LASS2 deletion will lead to increased V-ATPase activity, activation of Wnt/β-catenin pathway and spontaneous HCC formation. In this research, we will verify this possibility at levels of whole animal, cell and molecular models. The implementation of this project will provide new ideas for elucidating HCC pathogenesis and finding new targets for HCC diagnosis and therapy.

LASS2是2001年克隆到的基因。在人体组织器官中，LASS2在肝脏中的表达量是最高的。LASS2在肝细胞癌（HCC）发生发展中是否起重要作用还未明确。我们前期研究发现，LASS2肝特异性敲除小鼠自发形成HCC，并且LASS2基因在人HCC组织中的表达量明显低于癌旁组织。以上提示LASS2在HCC的发生发展中很可能起重要作用。目前已知LASS2抑制质子泵V-ATPase活性，而V-ATPase活性水平是Wnt/β-catenin 通路能否激活的决定因素之一。基因表达谱芯片分析表明，LASS2敲除小鼠肝脏中Wnt/β-catenin 通路异常激活。我们推测：LASS2 缺失引起V-ATPase活性提高，进而Wnt/β-catenin通路激活，最终HCC自发形成。本项目拟从整体动物、细胞和分子不同水平验证这一可能性。本项目的实施，为阐明HCC发病机理、发现新的HCC诊断和治疗靶标提供新思路。

人体组织和器官中，LASS2在肝脏中的表达最高。LASS2与肝脏多种疾病的发生发展都密切相关。本研究是基于LASS2肝特异性敲除（LASS2-/-）小鼠进行的。我们发现，LASS2-/-小鼠自发形成肝细胞癌（HCC），并且LASS2缺失使Wnt/β-catenin信号通路异常激活。我们利用人HCC细胞系进一步研究，结果发现，LASS2表达下降，引起质子泵V-ATPase活性增加，Wnt/β-catenin信号通路被异常激活，最终促进人HCC细胞发展。而LASS2表达升高，引起质子泵V-ATPase活性降低，Wnt/β-catenin信号通路被抑制，最终抑制人HCC细胞发展。本课题明确了LASS2在HCC发生发展中所起的重要作用，并且阐明了其分子机制，为HCC的预防和治疗提供新的研究思路和靶点。在完成上述实验内容的基础上，我们还探讨了LASS2在肝脏脂肪变性发病机制中的作用。用正常饲料或高脂饲料（HFD）饲喂LASS2-/-和野生型小鼠，结果表明，LASS2-/-小鼠对HFD诱导的肝脏脂肪变性具有缓解作用。在小鼠肝细胞系AML12中降低LASS2的表达，可以显著提高V-ATPase活性，细胞的H+分泌显著增加，细胞内H+浓度显著降低。我们还观察到，降低LASS2的表达，可以显著降低FFA处理的AML12细胞内ROS水平。相对应的，在AML12中升高LASS2的表达，可以显著降低V-ATPase活性，细胞的H+分泌显著降低，细胞内H+浓度显著升高，细胞内ROS水平显著升高。