The Effect Of DLK1 On Cell Growth Of Human Hepatocellular Carcinoma And Its Mechanisms

Human Delta-like 1 homolog (Drosophila) (DLK1), also named as pre-adipocyte factor 1 (Pref-1), fetal antigen-1 (FA1) or pG2, is a maternally silenced and paternally expressed imprinted gene at 14q32. DLK1 is widely expressed in a variety of murine and human embryonic tissues, but it is only present in a limited number of adult tissues, such as the adrenal gland, the pituitary, the placenta. DLK1 protein possesses 383 amino acids, is a type I transmembrane protein, including a intracellular tailer, a transmenbrane domain and an extracellular motif, which contains six cysteine-rich EGF like repeats similar to those found in the Delta/Notch/Serrate family of signaling molecules. Therefore, DLK1 belongs to the epidermal growth factor-like homeotic family. DLK1 has pleiotropic function: (1) inhibition of adipocyte differentiation, (2) involved in wound healing, (3) modulating the differentiation of hematopoietic stem cells and hepatic stem cells, such as: liver oval cells, hematopoietic stem cells (HSC), bone marrow derived stem cell (BMSC) et al. DLK1 is an important factor of the early liver fibrogenesis and cirrhosis. Although DLK1 has been proved to be imprinted in the most of adult tissues, the recent studies showed that DLK1 was highly expressed in lots of cancers including small cell lung carcinomas, neuroendocrine tumors and liver cancers, implying that it might contribute to tumorigenesis.DLK1 was found to be significantly up-regulated in hepatocellular carcinoma (HCC) specimens, whereas the transcript of the gene was weakly expressed in adjacent non-tumor livers using cDNA microarray. Based on this previous study, (1) immunohistochemical (IHC) was performed to detect the expression of DLK1 using tissue microarray containing 57 HCC specimens and the matched non-cancerous liver tissues. We found that DLK1 was up-regulated in 71.9% (41/57) HCC specimens, whereas the protein of DLK1 was weakly expressed in adjacent non-cancerous livers, implying that there is an unknown correlation between DLK1 and hepatocarcinogenesis. (2) To characterize the biological functions of DLK1 on HCC cells, we established SMMC-7721-DLK1-GFP, SMMC-7721-GFP and Huh7-DLK1-GFP, Huh7-GFP stable cell lines and confirmed the overexpression of DLK1 using RT-PCR and western blot analysis and further visualized the subcellular localization of DLK1 by immunofluorescence assay. By using the BrdU incorporation method, colony formation assay and tumorigenicity in nude mice, we found that the exogenous DLK1 expression can significantly promote the proliferation, colony formation and tumorigenicity in SMMC-7721 and Huh7 HCC cells. (3) In order to explore the underlying mechanisms of DLK1 contributes to the hepatocarcinogenesis, we have analyzed the differential gene expression profiles between DLK1-overexpressing SMMC-7721 cells and its empty vector control by using Affymetrix GeneChip Human Genome U133 Plus 2.0 microarrays, where 283 genes were up-regulated, and 180 genes were down-regulated. Real-time PCR was performed to verify the microarray data by randomly choosing four up-regulated genes: DLK1 , mitogen-activated protein kinase 1 (MAPK1), tumor suppressor candidate 3 (TUSC3), interferon-inducible protein 16 (IFI16) and three down-regulated genes: Janus kinase 1 (JAK1), matrix metallopeptidase 16 (MMP16) , Caspase-1. (4) Based on previous research that some DLK1 up-regulating liver tumors also express high levels of IFI16, we chose IFI16 for further investigation. The up-regulation of IFI16 induced by DLK1 was further verified using a semi-quantitative RT-PCR and western blot assay. Further, DLK1 can augmen the transcriptional activity of IFI16 promoter assessed by Luciferase reporter assay, suggesting that IFI16 is a down-stream target gene of DLK1 signaling pathway. Western blot analysis showed that IFI16 can decrease the expression of p53 and p21. Consistently, DLK1 overexpressing cells had an approximately 10% increase in the number of cells in S phase cycle progression analyzed using flow cytometry. However, knock-down of IFI16 using siRNA distinctly abrogated the DLK1-induced proliferation in those cells. Furthermore, DLK1 overexpression up-regulated the expression of IFI16 at mRNA level in human primary HCC. These findings suggest that IFI16 might be an essential downstream effector of DLK1 in HCC cells, and required for DLK1-induced cell growth acceleration.In conclusion, the overexpression of DLK1 could significantly accelerate HCC cell growth in vitro and in vivo, and we could presume the signal transduction pathways regulated by DLK1 in cell proliferation by comparing the differential expression profiles. The mechanisms research of DLK1 promoting HCC cell growth is very helpful for us to understand the mechanisms of DLK1 involved in HCC genesis. These findings provide a clue to investigate the further mechanisms of DLK1 in cell growth and hepatocarcinogenesis.