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

Abstract

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.