The Function And Mechanism Study Of ING3and DFO On Stem Cell Genomic Stability

Cells sustain genomic stability by cell cycle checkpoint, cell cycle regulation, DNA replication regulation, DNA repair and programming apoptosis or necrosis. Any endogenous or exogenous factor affecting these processes may lead genomic instability thus resulting in bad consequence such as cancer. Self-renew and pluripotent properties of stem cell make it more sensitive to these genomic instability factors. Thus it is necessary to reach a better and more comprehensive understanding of stem cell genomic stability in order to make the best and greatest use of its potentials in basic study and clinical application.Our studies focus on potential influences of ING3, ROS and DFO on genomic stability of stem cells with hope to find small molecules or proteins playing important roles in this process. In order to screen small molecules facilitating long term culture of stem cells in vitro, we treat stem cells with DFO, a hypoxia mimicking agent and might a genomic stability protector like DMOG. We find DFO suppresses stem cell proliferation or self-renew, and increase apoptotic cells with gradually raised DFO concentration. AP staining result suggests DFO treat will not lead stem cell differentiate. At the same time, we also find that in appropriate concentration (50μM), DFO treat will obviously increase pluripotent genes expression whereas high concentration (100(μM) DFO treat will compromise this promoting property. In cell cycle analysis, we find, consistent with our previous observations, after DFO treat, cells in G2/M phase gradually decrease whereas more and more cells are arrested in G1phase or be programmed to die. At the same time, our lab observes a basal γH2AX accumulation in stem cells without any treat, but there is no obvious difference on γH2AX foci amount between MEFs and stem cells after IR. We then detect the ROS level, the main endogenous DNA lesions factor, suspecting that this phenomenon may result from different ROS level in these cell lines. However, it ends with same or lower ROS level in stem cells than in MEFs. And Western blot detection of upstream proteins of γH2AX in DNA damage repair pathway displays no consistent high basal accumulation of these proteins in stem cells with γH2AX. These results suggest γH2AX may not exist as a marker of DNA damage, but play other roles in stem cells. But its activation and functional mechanism still remain unclear. Besides, we also discover a high level basal expression of ING3, an Ⅱ type tumor suppressor gene, in stem cells, and ING3 expression decreases upon differentiation of stem cells, suggesting it may play an important role in stem cells. A further study finds that ING3level decrease after IR, but the mechanism is still unclear. In addition, there is no obvious change in ING3expression level in stem cells after50J/m2UVC treat. To further explore the potential function of ING3in stem cells, ING3knockdown stable cell line is being constructed with commercial lentiviral shRNA plasmids.All these above results suggest stem cells may have more complex mechanisms to sustain genomic stability and our work may contribute to a better understanding of stem cell genomic stability and its future application in clinical therapies.