人肝癌细胞系Slit／Robo启动子甲基化状态及基因表达的研究

Analysis of the expression of Slit/Robo genes and the methylation status of their promoters in the hetpatocellular carcinoma cell lines

目的研究Slit／Robo信号通路相关基因Slit1、Slit2、Slit3和Robo1、Robo3在多种人肝癌细胞系中的表达及甲基化状态，探讨与肝癌发生和发展的关系。方法提取9种人肝细胞癌细胞株（Hep3B、HepG2、PLC／PRF／5／PRF／5、SMMC7721、BEL-7402、MHCC97-H、MHCC97-L，LM3、LM6）及对照细胞株L02的基因组DNA和总RNA，采用半定量逆转录聚合酶链反应技术和甲基化特异性聚合酶链反应技术检测Slit1、Slit2、Slit3和Rob01、Rob03基因的基因表达水平与启动子甲基化状态。实验数据应用Pairedf检验。结果Slit1、Slit2、Slit3基因除个别细胞株外，在不同转移潜能细胞株中均发生了DNA甲基化，同时Slit1和Slit3在mRNA水平几乎均不表达，Slit2基因表达程度在不同转移潜能的细胞株之间存在差异，随着转移潜能的增加表达大致呈下降趋势。作为Slit2受体的Robol基因在10株肝癌细胞株中均发生甲基化修饰，但除在SMMC7721、BEL-7402、L02不表达外，其余7种细胞株均有表达。Rob03基因相关CpG岛在9种肝癌细胞株中均未发生甲基化，同时其在mRNA水平均无表达。结论Slit／Robo可能在肝癌发生和发展中发挥作用。而Robo3则在肝癌中不发生表达而且其表达沉默可能不受甲基化方式调控。

Objective To analyze the expression of genes in the Slit/Robo signaling pathway, and the methylation status of their promoters in hetpatocellular carcinoma （HCC） cell lines. Methods Genomic DNA and total RNA were isolated from 9 HCC cell lines of different metastatic ability （Hep3B, HepG2, PLC/PRF/5, SMMC-7721, BEL-7402, MHCC97-H, MHCC97-L, LM3, LM6） and a control cell line L-02. The expression profiles of Slitl, Slit2, Slit3, Robol, and Robo3 were analyzed by reverse transcription polymerase chain reaction（RT-PCR）. The methylation status of the promoters was detected by methylation specific polymerase chain reaction （MSP）. Results The promoters of Slitl, Slit2 and Slit3 genes were almost methylated in all the HCC cell lines. The Slitl and Slit3 RNAs were not detected in most of the cell lines. Furthermore, the mRNA Slit2 was decreased gradually as the metastatic potential of the cell lines increased. As the candidate ligand of the Slit2 gene, Robol was frequently methylated in HCC cell lines whereas its mRNA was detected in all of these cells except SMMC-7721, BEL-7402 and L-02. Robo3 was unmethylated in HCC cell lines while its mRNA was not detected in these HCC cell lines. Conclusion The hypermethylation status of Slit/Robo signaling pathway related genes is a universal event in the HCC.The hypermethylation status of Slitl, Slit2, Slit3 genes associated with the loss of expression or reduced expression. Those data suggest that Slit/Robo pathway may play a significant role in the progress or metastasis of HCC.