The Functional Characterization Of Two Hepatocellular Carcinoma-related Genes: KIAA0101 And DLK1

Hepatocellular carcinoma (HCC) is one of the most prevalent and lethal cancer in Asia and Africa. In China, it ranks in the second death rate among malignancies. The development of HCC is with multi-factors in etiology, multi-step and multi-gene involvement in carcinogenesis and progression. A broad spectrum of genes have been involved in HCC development related to their genetic or epigenetic alteration, including p53, p16, p21, p27, beta-catenin, PTEN and Rb etc. Recently, advanced methods such as cDNA array were introduced into studies on functional genomics of HCC and revealed a number of genes with novel sequences and unclarified function, were involved in HCC development or progression. Our laboratory has found a spectrum of genes with abnormal expression pattern in HCC. Among them, we selected KIAA0101 and DLK1 for further investigation.KIAA0101, also named OEATC-1, consists of 888 bp and encodes a protein with 111 amino acids. It has been recently reported as a novel gene related to thyroid, non-small cell lung carcinoma and colon cancer. But there is no report about its expression in liver cancer. Based on our previous cDNA results, the increased mRNA expression of KIAA0101 was further validated in 8/15 HCC tissues as compared with their counterpart non-cancerous liver tissues using the semi-quantitative RT-PCR method. After preparing its specific rabbit poly-clonal antibody, we found that KIAA0101 was surprisingly down-regulated expression at protein level in 25/30 HCC tissues (83%). Utilizing tissue array and immunohistochemical survey, it was also demonstrated that no (-) or very weak (+) signals were detected in 107 HCC tissues (T) out of 161 paired HCC (T) and non-cancerous liver(NT) tissues (66.4%) while such signals were only detected in 24 non-cancerous tissues (14.9%)( P<0.05). Thesedata demonstrated that the results of KIAA0101 mRNA were inconsistent with those from protein expression in HCC tissues. Dual immuno-fluorescence and enriched mitochondrial protein Western blot were performed and indicated that the KIAA0101 gene product was predominantly localized in mitochondria of human HCC cells, and the minor portion was found in nuclei. Furthermore, when KIAA0101 plasmid was transfected into HCC lines, the KIAA0101 overexpression could inhibit the cell growth significantly (P<0.05) and this effect may be mediated through the blockage of the cell cycle transition from G1 to S phase. Our results implied that the KIAA0101 was possibly involved in the development of HCC.DLK1 (Pref-1) is also an up-regulated gene revealed by our cDNA array experiment. It is a transmembrane protein containing six epidermal growth factor (EGF)-like repeats, a single transmembrane domain and a short intracellular tail. DLK1 is an imprinted gene present in animals from birds to mammals. This protein is described to participate in embryonic growth, hematopoiesis, wound healing and adipocyte differentiation. Also, DLK1 has been investigated and found to be high expressed in several cancers including neuroblastoma, small cell lung carcinoma, myelodysplastic syndrome (MDS), brain and pituitary tumors, but its involvement in human hepatic cancer has not yet been documented. In mouse fetal liver tissues, DLK1 was found to be highly expressed; while there is no expression in adult liver tissue. It has been reported that the hepatoblast cells was isolated based on the expression of DLK1 expression from the fetal liver tissue and the DLK1⁺ cells showed highly proliferative potential than the DLK1^- cells.In our reports, the DLK1 was found up-regulated in about 30% of cancer tissues from HCC patients at both mRNA and protein level. The multiple tissue Northern blot showed that DLK1 was expressed at highest level in placenta, moderate in testis, ovary and heart, weak in skeletal and pancreases, and non-detectable in other tissues including normal liver tissue. By in vitro cell growth experiments, DLK1 could both promote the HCC SMMC7721 cell growth significantly in both stable and transient over-expression conditions. Also, the same results were obtained from the in vivo nude mouse xenograft tumorigenecity assay. For better understanding its molecular mechanism, we compared the synchronized cell cycle progression after nocodazol traeatment between the DLK1 over-expressed cells with the control using the FACS analysis. The results showed that the DLK1 could accelerate G₂/M checkpoint transition and enter the S phrase earlier. At about 16 hours after release, the S phrase in thepopulation from DLK1 over-expressed cells reached its peak; while in the control cells the time to reach the peak of S phrase was about 21 hours after release. We further investigated cyclin D,E, B and p34cdc2,p21 etc related with cell cycle and found that the p34cdc2 protein has decreased its phosphrylation level at Tyr-15, though its total protein level had not alteration. Furthermore, by examing the in vitro p34cdc2 kinase activity, the enzyme activity was elevated in DLK1 over-expressed cells. In addition, we found that the DLK1 and AFP could co-express in the same tumor cells in primary HCC.Taken together, we demonstrated that DLK1 was over-expressed in liver cancer tissues from a subset of HCC patients and that the DLK1 over-expression could promote the HCC cell line growth in vitro and in vivo. It is speculated that DLK1 is involved in HCC development and progression.