Zheng Dan, Liu Bin-Bin, Liu Yin-Kun, Kang Xiao-Nan, Sun Lu, Guo Kun, Sun Rui-Xia, Chen Jie, Zhao Yan
Liver Cancer Institute, Zhongshan Hospital, Fudan University, Shanghai 200032, China.
Zhonghua Gan Zang Bing Za Zhi. 2009 Mar;17(3):198-202.
To analyze the expression of genes in the Slit/Robo signaling pathway, and the methylation status of their promoters in hepatocellular carcinoma (HCC) cell lines.
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 Slit1, Slit2, Slit3, Robo1, 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).
The promoters of Slit1, Slit2 and Slit3 genes were almost methylated in all the HCC cell lines. The Slit1 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, Robo1 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.
The hypermethylation status of Slit/Robo signaling pathway related genes is a universal event in the HCC. The hypermethylation status of Slit1, 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.
分析Slit/Robo信号通路中基因在肝癌（HCC）细胞系中的表达及其启动子的甲基化状态。
从9种具有不同转移能力的肝癌细胞系（Hep3B、HepG2、PLC/PRF/5、SMMC-7721、BEL-7402、MHCC97-H、MHCC97-L、LM3、LM6）及对照细胞系L-02中提取基因组DNA和总RNA。通过逆转录聚合酶链反应（RT-PCR）分析Slit1、Slit2、Slit3、Robo1和Robo3的表达谱。用甲基化特异性聚合酶链反应（MSP）检测启动子的甲基化状态。
Slit1、Slit2和Slit3基因的启动子在所有肝癌细胞系中几乎均呈甲基化状态。大多数细胞系中未检测到Slit1和Slit3 RNA。此外，随着细胞系转移潜能的增加，mRNA Slit2逐渐降低。作为Slit2基因的候选配体，Robo1在肝癌细胞系中频繁发生甲基化，而其mRNA在除SMMC-7721、BEL-7402和L-02外的所有这些细胞中均能检测到。Robo3在肝癌细胞系中未甲基化，但其mRNA在这些肝癌细胞系中未检测到。
Slit/Robo信号通路相关基因的高甲基化状态在肝癌中是普遍现象。Slit1、Slit2、Slit3基因的高甲基化状态与表达缺失或表达降低相关。这些数据表明Slit/Robo通路可能在肝癌的进展或转移中起重要作用。
