Overexpression Of Oncogene Bmi-1 Promotes The Self-renewal Of Embryonic Stem Cells

Embryonic stem cells(ESCs)depend on a number of signal pathways', synergistic effect to maintain pluripotency,the investigation of the regulation mechanisms could not only help us to understand the molecular mechanism of embryonic development, but also to provide rationale for the clinical application of embryonic stem cells and cancer stem cells.But we still can't explain the complex self-renewal regulation mechanisms, identification of new regulating signaling pathways can lead to better understanding of the development of embryonic stem cells. CSCs exist in the tumor tissuses and have similar characteristics with ESCs.Previous studies have found that the ESCs'stemness factors Nanog and Oct-4 are also expressed in CSCs, and participate in the regulation of self-renew. Thus we hypothese that there are similar regulation signal pathways in the CSCs and ESCs. Oncogene Bmi-1 is overexpressed in many cancers, and plays an important role in the regulation of self-renewal in CSCs, hemopoietic stem cell and neural stem cells. Similar to CSCs, elevated expression of bmi-1 was also found in ESCs, therefore, we predict bmi-1 may also play an important role on the maintenance of ESCs pluripotency .In order to investigate the potential function of bmi-1 in ESC, the bmi-1 Eukaryotic expression vector was constructed and tranfected into mouse embryonic stem cell strain CCE, then the stable transfectant was established and used as a cell model to study the infuences of ESC proliferation, self-renew and differentiation caused by the overexpression of bmi-1.The reseach contents and results include :1. The construction of bmi-1 exression vector: The bmi-1 cds was amplified with mBmiCDSF/mBmiCDSR primers,with EcoRI and SalI restrict enzyme sites added no both ends, The PCR fragment was then cloned into expression vector pCI-GFP to get pCI-GFP-bmi1 eukaryotic expression vector .2. The expression of EGFP-bmi1 protein in CCE: pCI-GFP and pCI-GFP-bmi1 were transiently transfected into CCE, fluorescence microscope was used to observe the expression of fluorescin EGFP. Cells tranfected with pCI-GFP-bmi1 has the green fluorescin expressed in the cell nucleus, and cells transtected with pCI-GFP has the uniformity distribution.3. The screen and identification of stable transfectants: The transfected cells was cultured in the ES culture medium supplemented with G418, 12 days later, the transfectants were identified and named pCGB/CCE and pCG/CCE. Confocal microscopy, semi-quantitive RT-PCR and western-blot were used to detect the expression of EGFP-bmi1, Our results indicate that EGFP-bmi1 protein was expressed and localizates in the nucleus.4. The influence of CCE cell proliferation by bmi-1: We use proliferation curve to display the proliferation capability of cells. Compared with pCG/CCE, pCGB/CCE generates more quickly under the same culture medium. The result indicates that the overexpression of bmi-1 could promote the generation of ESCs.5. The influence of CCE cell self-renewal by bmi-1: To detecte the self-renew of CCE, alkaline phosphatase staining was used in the clone formation essay, compared with pCG/CCE, pCGB/CCE has a higher ratio of AP positive clones. Semi-quantity RT-PCR and western-blot results indicates that the expression of stemness factor such as Nanog and Oct-4 were boh upregulated. We can conclude that bmi-1 can promote the self-renewal of ESCs.6. The influence of CCE cell differentiation by bmi-1: The stemness factors and differentiated factors were detected by RT-PCR under the condition of induced differentiation, the results indicate that the stemness factors in the pCGB/CCE cells have a higher expression level than in pCG/CCE cell, and the differentiation factors such as GATA6 and GATA4 expressed inversely.In conclution, we investigated function of bmi-1 in ESCs. Compared with the control, Bmi-1 overpressed cells exhibit increased potential of proliferation and self-renew and decreased potential of differentiation, which indicates that bmi-1 participates in the regulation of pluripotency and differentiation of ESCs, providing new clues for further research.