Epigenetic regulation of SLIT-ROBO pathway in hepatocellular carcinoma

Abstract

Hepatocellular carcinoma is an aggressive, chemotherapy-resistant cancer and the prognosis of affected patients is very poor. Therefore, unraveling molecular components of hepatocarcinogenesis not only leads to the improvement of diagnostic and prognostic tools but also may reveal novel therapeutic targets. We previously defined the differential expression of SLIT-ROBO genes in HCC. To explore the mechanisms of the inactivation of these genes, we analyzed the hypermetylation of SLIT1, SLIT2, ROBO2 and ROBO3 genes in a group of HCC cell lines consisting of four high-AFP expressing well-differentiated (HUH7, Hep3B, PLC/PRF5 and HepG2) and low-AFP expressing poorly-differentiated cell lines (Focus, SKHep1, Snu387 and Snu423). We first demonstrated that the transcription of all studied genes can be rescued upon treatment of cell lines with 5-Aza-2‟-deoxycytidine (5- Aza-dC). Next, we analyzed the methylation of SLIT-ROBO genes by methylation specific PCR. All genes were found to be at least partially methylated, except ROBO3, which displayed a heavily methylated pattern. in silico analyses of the 5‟ upstream sequence of ROBO2 gene revealed a putative promoter region, an enhancer in the first intron and a CpG island. Methylation –specific PCR amplification of ROBO2 by using primers selected from this CpG island supports the potential of this region as a gene regulatory site. Therefore, it is worthy to extend the methylation analyses of SLIT-ROBO pathway in HCC patients. Furthermore, mechanistic studies on ROBO3, which was shown to counteract the overexpressed ROBO1 effects may shed light into the role of ROBO3/ROBO1 axis and the potential of ROBO3 as a tumor suppressor gene in HCC.