The Transcription Factor Nanog Expression Lead To Hek-293 Cells In Malignant Transformation And The Mechanism

Nanog, a new found gene, plays a critical role in maintaining stem cell self-renewal and pluripotency. At first, Nanog was described as a gene specifically expressed in pluripotenct cells including ES, EG and EC cells. However, recent researches demonstrating Nanog expressed in several tumors including seminoma, breast carcinoma, retinoblastoma and prostate cancer, may indicate the general expression of Nanog in tumors. A new research demonstrated that forced expression of Nanog in hematopoietic stem cells lead to malignancy, indicated Nanog is a candidate oncogene and contributing to transformed phenotype.By using RT-PCR and Western-blot we found the high expression of Nanog in many types of tumor. In order to confirm Nanog is an oncogene, a series of experiments were performed. Firstly, we cloned Nanog gene from PA-1, a human teratoma cell line, through RT-PCR. Then, eukaryotic expression plasmid of pcDNA3.1(+)-Nanog was constructed and transfected into HEK-293 cells. Nanog expressed stably in the HEK-293 cell lines which selected by G418 and blank transfected group served as controls. A positive cell clone and a control cell clone were selected randomly, named 293-N1 and 293-C1 respectively. Nanog expression was identified by RT-PCR and Western-blot. Second, through MTT colorimetry assay, soft agar colony formation and nude mice tumorigenesis assay, we found that HEK-293 cells aquired increased growth rate, ability for growing and forming cell clonies on soft agar, and the tumorigenic ability in nude mice. Tumor tissues analysed by paraffin section and H.E. staining show the low degree of differentiation and invasion of tumor, suggest the high degree of malignancy and invasion and metastasis.We analyzed the proteomics of 293-N1 and 293-C1 cells to reveal the tumorigenic mechanism induced by Nanog expression. We confirmed the protein distribution of HEK-293 cells by software at first, and decided to use pH4-7 IPG strip in experiment. Through many experiments we obtained four groups of 2-D gels in good consistency and identified 33 protein spots expressed differentially by software, 29 of which were assayed by MALDI-TOF-MS and 25 proteins were identified at last. Two up-regulated expression proteins FAK and Ezrin, which related to tumor, are identified by semi-quantitative RT-PCR and Western-blot. As many studies showed FAK and Ezrin contributed to proliferation, survival and metastasis of tumor cells, we concluded that a reason of malignant transformation of HEK-293 cells is due to up-regulated expression of FAK and Ezrin.Furthermore, transcription factor Nac1 was up-regulated after expression of Nanog in HEK-293 cells. Subsequently, we proved that there is the interaction between Nanog and Nac1 both in vitro and in vivo by co-immunoprecipitation and GST pull-down. Since both of Nac1 and Nanog are important transcription factors in ES cells, it provided clues to reveal the mechanism of malignant transformation of HEK-293 cells.To sum up, we discovered the malignant transformation of normal cells induced by Nanog, and identified Nanog belongs to oncogene. Analysing the mechanism of malignant transformation by proteomics, we found two up-regulated proteins, which closely related to tumors, may directly attribute to carcinogenesis. Nac1 up-regulated and interacting with Nanog may contribute to this process.