Function And Regulation Of Novel Gene, HESRG, In Human Embryonic Stem Cells And Its Expression In Human Intracranial Tumors

Stem cells are capable of self-renewal and pluripotency. According to their derivation, stem cells can be divided into embryonic stem cells and adult stem cells. Embryonic stem cells was derived from inner cell mass of early blastocyst or predorminal germ cells, which was proven to be pluripotent. Under suitable conditions, ES cells can differentiated into tissue and cells from all the three germ layers. The pluripotency of ES cells makes it significant in the future utility for cell differentiation, embryonal development research, gene function research, trans-gene animal preparation, establishment of human disease model, drug discovery, cell or tissue transplantation, tissue engineering and even regenerative medicine. Human embryonic stem cell line, hES cell line, was firstly established by Thomoson et.tal in1998. It was selected as top science research development in1999. After that, numerous researches aimed at human embryonic stem cells. Today, researches on embryonic stem cells and adult stem cells are still the peer field of biology and medicine research.Self-renewal and pluripotency are the fundamental characteristics of human embryonic stem cells. Mechanism of self-renewal and pluripotency are the key-point for stem cell biology research and the basic for cell alternation and tissue engineering. Recently, as the discovery of induced pluripotent cells, studies of self-renewal on ES cells provided a way for understanding cell reprogramming mechanism and establishment of individual regenerative medicine. Self-renewal of ES cells involves several elements including cell signaling pathways, core transcriptional factors, cell cycle factor, DNA methylation and stability of chromatin, among which studies on core transcriptional factors as Oct4, Nanog and Sox2and signaling pathways as canonical WNT pathway, FGF/PI3K pathway and TGF(3pathway have reached a relatively high level. However, temporary data can hardly explain maintaining of relf-renewal and pluripotency of ES cells entirely. It is widely thought that there must be many undetected fields for new genes and new mechanism involved in this.In the field of neurobiology, treatments for recovery of neurotraumatic disorder and degenerative disease as Parkinson's disease, Huntington's disease and Alzheimer's disease remain unsettled. Recently, discovery of neural stem cells and researches about pluripotency of neural progenitors broke the myth that neural tissue is lack of ability for regeneration. Despite of numerous obstruct to be settled, transplantation of stem cells has been thought to be a possible resolve for refractory disease of nerve system. According to differentiation of ES cells and iPS cells in vitro, various types of neural cells could be derived such as neural stem cells, functional neuron and glial. So, differentiation of ES cells in vitro can not only provide a fantastic cell model for researches on early development of embryo and cell differentiation but also provide abundant donor resource for cell transplantation in future. As a result, researches on neural differentiation of human embryonic stem cells are significant.In our previous work, according to micro-array screening, e-cloning and RACE, our research group (complied by researchers from molecular pathology lab, Cancer research institution and department of neurosurgery, Xiangya Hospital) cloned a novel human gene named as HESRG, human embryonic stem cells related gene, which has been recruited by GenBank with ID number DQ445779. It locates at3p14.3in human chromosome.Based on cloning full-length of HESRG, our previous work found that:1. Sequence blast indicated that partial homologous sequences were found only in primates such as Pan troglodytes and Macac mulatta;2.By fusion gene expression strategy, Western blot and immunohistochemistry, it have been confirmed that the ORF of HESRG comprises669bp and encodes a24kilo-dalton protein composed of222amino acids. Bioinformatical analysis has shown that HESRG has a nuclear localization signal and contain a leucine zipper domain, suggesting it may act as a transcription factor in hES cells;3.Preliminary study has indicated that overexpression of HESRG prompted hES cells to differentiate into mesoderm, while downregulation of HESRG expression by RNAi induced hES cells to differentiate into ectoderm, suggesting that HESRG may play a key role in self-renewal of hES cells;4.Further research confined regulation of Oct4in its expression. Except our group, there is no report about HESRG all over the world. Based on works mentioned above, our research was aimed at further function of HESRG and its signaling regulation. Besides, although our preliminary researches found no expression of HESRG in common tumor cell lines such as HT-29, Caski, K562, CNE-2, HEK293and THP-1, expression pattern of HESRG in human tumors remains unknown. In this piece of work, we are going to test its expression in human central nervous system. For we found one of those ESTs constructed HESRG, BF223023, origins from germ cell tumors, we predict HESRG probably expresses in germ cell tumors. As a result, we paid close attention to its expression in intracranial germ cell tumors.This work is constructed by the following parts:[Cultivation and identification of human embryonic stem cells and expression of HESRG in hES cells]Human embryonic stem cell lines, H1and H9, were utilized for our research. According to RT-PCR, Real-time PCR and immunocytofluorescence, we detected expression of pluripotent markers, Oct4and Nanog, and species specific markers, TRA-60-1and SSEA-3in human embryonic stem cell lines, H1and H9. Synthesis with preliminary works, we confirmed our cultivation strategy was suitable for proliferation of pluripotent hES cells, which could be used for following research. RT-PCR, Real-time PCR and immunocytofluorescence showed high expression of HESRG in both H1and H9cell lines at both mRNA and protein level. Immunocytofluorescence detected HESRG protein located in nuclear of hES cells, which indicates that HESRG may function as transcriptional factors. We analyzed expression pattern of HESRG in mES cell line, C57/BL6, and hES cell line and found that HESRG specifically expressed in human embryonic stem cells but not in mES cells, which is consistent with bioinformatics analysis and implies HESRG is species specific and whether differences of HESRG between mES cells and hES cells is the reason for differences of biological behavior between them worth for further study. Further more, we tested expression level of HESRG in both undifferentiated hES cells and spontaneous differentiated hES cells and found down-regulation of HESRG in differentiated hES cells at both mRNA and protein level. According to results mentioned above, we suggest that HESRG is a novel gene associated with undifferentiated status of hES cells[Study for function of HESRG in hES cells through RNA interference]By previously established strategy for siRNA interference through Lipofectamine RNAiMAX, we silenced HESRG in hES cells. We found that hES cells appeared canonical differentiated phenotype after interference of HESRG. Acquired cells turned to be classic phenotype of neural progenitor cells at day9after interference. According to Real-time PCR, we found that pluripotent marker gene such as Oct4, Nanog, Sox2down-regulated dramatically, which implied the loss of pluripotency of hES cells. By detecting markers of neural progenitor cells, Nestin, Soxl, Musashi-1and PAX6, and markers of terminal differentiated neural cells, βⅢ-tubulin and GFAP, we found that acquired cells after interference of HESRG showed charaterisitcs of neural progenitor cells. These cells would express marker of neuron (3111-tubulin after consistent cultivation for2weeks. As a result, we suggest that HESRG could participate in the maintaince for self-renewal and pluripotency of hES cells through repressing neural differentiation.[WNT signaling pathway and HESRG]By bioinformatic analysis, we found there several potential binding sites showing (A/T)(A/T)CAA(A/T)GG sequence for LEF/TCF family in WNT signaling pathway on sequences around promoter of HESRG, so we predicted that HESRG could be directly regulated by WNT pathway. In hES cells, when WNT signaling pathway was activated by specific inhibitor of GSK-3, BIO, expression of pluripotent marker gene Oct4, Nanog, Sox2could be maintained at relatively high level with undifferentiated state of hES cells. At the same time, expression of HESRG could be also maintained at a high level. We constructed luciferase report plasmid, pGL3-basic-HESRG upstream2061bp, which contained transcriptional binding site of LEF/TCF protein family around promtor of HESRG. When transfected with plasmid pGL3-basic-HESRG upstream2061bp and activated by WNT pathway through BIO, hES cells would show relatively high activity of luciferase. Such result showed that WNT pathway activated HESRG through activation of HESRG promoter. We tested expression abundance of subtypes of LEF/TCF family and found that TCF-3expressed most in hES cells. We specifically silenced TCF-3by siRNA interference and found that expression level of HESRG could be maintained at a relatively high level even under conditions without external WNT signal after interference of TCF-3. We co-transfected plasmid pGL3-basic-HESRG upstream2061bp and si-RNA-TCF-3into hES cells and found that hES cells transfected with luciferase report plasmid would show high activity of luciferase without external WNT signaling stimulation, which indicated that TCF-3repressed activity of HESRG promoter. According to results of bioinformatics analysis, we designed PCR primers containing binding site of LEF/TCF family around HESRG promoter and we found that TCF-3directly combined with DNA sequences at upstream2000bp of HESRG. According to results mentioned above, we suggest that WNT pathway could activate HESRG and maintain its expression in hES cells by direct combination of TCF-3.[Expressino of HESRG in intracranial germ cell tumors]We used RT-PCR to systematically investigate the expression of HESRG in various types of intracranial tumors and found that HESRG was expressed only in germinoma and embryonal carcinoma, but hardly in other types of brain tumors. Real-time PCR results further confirmed this expression pattern. Subsequently, we tested intracranial non-germ cell tumors and intracranial germ cell tumors by immunohistochemistry. Our results showed HESRG was expressed strongly and diffusively in the nuclei of tumor cells in intracranial germinoma and embryonal carcinoma as well as human embryonic stem cells. No positive staining signal was observed in any other type of intracranial tumors. In germinomas,25of31showed intensive (3+) expression,4cases showed moderate (2+) immunostaining and the remaining2cases showed weak (1+) immunostaining. In embryonal carcinoma,6of9showed intensive (3+) immunostaining and3of9showed moderate (2+) immunostaining. These results suggest HESRG is a novel, sensitive and specific biomarker for intracranial germinoma and embryonal carcinoma.In conclusion, based on our researches mentioned above, we suggest that:1. Expression of HESRG can be detected in human embryonic stem cells but not in mouse embryonic stem cells. HESRG protein locates in nuclear of hES cells. Expression of HESRG will be down-regulated according to spontaneous differentiation of hES cells. 2. HESRG participates into the maintaining of undifferentiated status and self-renewal of hES cells. Repressing its expression through RNAi interference will result neural differentiation of hES cells.3. Canonical WNT pathway regulates expression of HESRG. By selective inhibitor of GSK-3, BIO, activated WNT pathway is capable of maintaining undifferentiated status of hES cells and activate HESRG expression. hES cells under non-conditioned medium will spontaneously differentiate without BIO and expression of HESRG will decrease. WNT pathway directly regulated HESRG through TCF-3binding to the promoter and maintaining its expression in hES cells.4. Intracranial germinoma and embryonal carcinoma express HESRG at both mRNA and protein level, while no expression can be detected in other intracranial tumors and normal brain tissues. HESRG is a sensitive and specific biomarker for intracranial germinoma and embryonal carcinoma.