HECA通过Wnt通路抑制肝癌细胞增殖、迁移和侵袭

HECA inhibited proliferation,migration and invasion of hepatocellular carcinoma through the Wnt pathway

目的:拟在通过影响肝细胞癌HepG2细胞株中HECA基因表达变化,观察与HECA基因相关联的Wnt通路上重要调节分子的变化,讨论此通路影响肝癌细胞增殖能力状况及其相关作用机理。方法:HECA慢病毒感染肝癌细胞株HepG2,细胞株中稳定上调HECA,用HECA siRNA转染HepG2细胞株下调HECA,在HECA表达上调的细胞株中用qPCR检测Wnt通路中β-catenin、TCF4、CDK2、CDK9、Cyclin A和Cyclin K的mRNA表达水平的变化,用Western blot检测qPCR结果中差异明显的β-catenin、TCF4蛋白表达水平的变化,应用FH535抑制剂处理HECA过表达的HepG2细胞,进一步验证HECA基因与Wnt通路的关系,利用Western blot检测HECA的表达水平,用MTT、划痕、克隆形成以及Transwell实验检测对肝癌细胞侵袭、迁移、增殖能力的影响。结果:qPCR法以及Western blot法对其相关基因表达水平进行测定,结果显示在HepG2细胞株内HECA过表达后,与HECA相关联的Wnt通路重要调节分子中β-catenin、TCF4的表达水平显著降低;FH535抑制剂处理HECA过表达的HepG2细胞,Western blot实验结果显示FH535处理过的过表达组HECA水平更高,细胞功能实验也表明FH535处理过的过表达HECA组,其细胞增殖及侵袭能力下降最明显。结论:HECA基因抑制肝细胞癌增殖及侵袭的分子机制可能为:HECA通过Wnt/β-catenin这一经典通路内β-catenin与TCF4的结合活性降低而发挥作用。

Objective:To discuss the effect of HECA pathway on HCC proliferation capacity and its related mechanism by influencing HECA expression changes in HepG2 cell lines and observing the changes of important regulatory molecules on HECA gene-associated Wnt pathway.Methods:By lentivirus infection in hepatocellular carcinoma cell line HepG2,HECA raised,HECA was down-regulated in HepG2 cell line with HECA siRNA.In HECA orer-expressed cell line,qPCR detected the mRNA expression ofβ-catenin,TCF4,CDK2,CDK9 and Cyclin A and Cyclin K.Western blot testedβ-catenin,TCF4 protein expression level.HECA overexpressed HepG2 cells were treated with FH535 inhibitor to further verify the relationship between HECA gene and Wnt pathway,and HECA expression level was detected by Western blot.Thus,MTT,scratch,clone formation and Transwell test were used to detect the influence on invasion,migration and proliferation ability of liver cancer cells.Results:qPCR and Western blot were used to test the expression levels of related genes.The results showed that after HECA overexpression in HepG2 cell lines,the expression levels ofβ-catenin and TCF4 in HECA-associated important regulatory molecules of Wnt pathway were significantly reduced.Western blot results showed that HECA level in HECA-overexpressed HepG2 cells treated with FH535 inhibitor was higher,and the cell function experiment also showed that the cell proliferation and invasion ability of the HECA-overexpressed HECA group treated with FH535 was most significantly decreased.Conclusion:The molecular mechanism of HECA gene in inhibiting proliferation and invasion of hepatocellular carcinoma may be that HECA plays a role through reduced binding activity ofβ-catenin and TCF4 in the classical pathway of Wnt/β-catenin.