Department of Anatomy and Cell Biology, Osaka Medical College, Takatsuki, Osaka, Japan.
Int J Oncol. 2013 Mar;42(3):817-22. doi: 10.3892/ijo.2013.1778. Epub 2013 Jan 17.
The genetic and epigenetic events of hepato-carcinogenesis are relatively poorly understood. By analyzing genes from human hepatocellular carcinoma (HCC) with restriction landmark genomic scanning, several aberrantly methylated genes, including Delta-like 3 (DLL3), have been isolated. In this study, we investigated the function of DLL3 in hepatocarcinogenesis. Methylation of the DLL3 gene in HCC cell lines was investigated with methylation-specific PCR and expression of DLL3 mRNA in HCC cells was examined by RT-PCR. Reactivation of DLL3 expression by treatment with a demethylating agent was examined in methylation-silenced HuH2 cells. Human DLL3 cDNA was cloned and DLL3 function was examined by restoring DLL3 expression in HuH2 cells. The effects of DLL3 on cell growth were evaluated by colony formation assay. Induction of cell death by overexpression of DLL3 was examined by flow cytometric assay using Annexin V and PI. Apoptotic cells were detected by TUNEL staining and the amount of single-stranded DNA was measured by ELISA. As a result, the promoter region of the DLL3 gene was methylated in four of ten HCC cell lines. This aberrant methyl-ation correlated well with the suppression of RNA expression and a demethylating agent reactivated DLL3 expression in methylation-silenced HCC cells. Interestingly, the restoration of DLL3 in the methylation-silenced HuH2 cells led to growth suppression on colony formation assay. Flow cytometric assay with Annexin V and PI showed that this growth suppression by DLL3 expression is associated with the induction of apoptosis. Furthermore, these apoptotic effects were confirmed by TUNEL staining and measurement of single-stranded DNA. These results suggest that DLL3 was silenced by methylation in human HCC and that it negatively regulates the growth of HCC cells.
肝癌发生的遗传和表观遗传事件相对了解较少。通过对人肝癌（HCC）的基因进行限制性标志基因组扫描分析，分离出了几个异常甲基化的基因，包括 Delta-like 3（DLL3）。在这项研究中，我们研究了 DLL3 在肝癌发生中的作用。用甲基化特异性 PCR 分析 HCC 细胞系中 DLL3 基因的甲基化，用 RT-PCR 检测 HCC 细胞中 DLL3 mRNA 的表达。用去甲基化剂处理来检测沉默甲基化的 HuH2 细胞中 DLL3 表达的重新激活。克隆人 DLL3 cDNA，并在 HuH2 细胞中恢复 DLL3 表达来检测 DLL3 功能。通过集落形成试验评估 DLL3 对细胞生长的影响。用 Annexin V 和 PI 通过流式细胞术检测过表达 DLL3 诱导的细胞死亡。用 TUNEL 染色检测凋亡细胞，用 ELISA 测量单链 DNA 的量。结果，在十个 HCC 细胞系中的四个中，DLL3 基因的启动子区域发生了甲基化。这种异常甲基化与 RNA 表达的抑制以及去甲基化剂在沉默甲基化的 HCC 细胞中重新激活 DLL3 表达密切相关。有趣的是，在沉默甲基化的 HuH2 细胞中恢复 DLL3 导致集落形成试验中的生长抑制。用 Annexin V 和 PI 的流式细胞术显示，DLL3 表达引起的这种生长抑制与细胞凋亡的诱导有关。此外，TUNEL 染色和单链 DNA 的测量证实了这些凋亡作用。这些结果表明，DLL3 在人 HCC 中被甲基化沉默，并且它负调控 HCC 细胞的生长。
