A Research On The Function Of Notch2 In The Determination Of Cell Fate After Mitotic Arrest

Currently, Cancer has become one of the most threatening deseases to human’s health, and it has become a globally important medical problem to prevent the development of Cancer and have a better treatment for Cancer. As tumor cells have the potential to proliferate unlimitedly, drugs that targeting mitosis such as Taxol and vinblastine have been used in the clinical treatment of Cancer for a long time. Drugs targeting mitosis such as Taxol will interfere with the function of microtubules and impair the formation of a proper bipolar spindle, which would cause a delay in mitosis and result in cells’death. It’s just according to this principle that drugs targeting mitosis are used to kill cancer cells for the treatment of Cancer. However, in recent years some research showed that with the treatment of anti-mitotic drugs, in addition to death in mitosis, the majority of cells have two other fates and they would either enter abnormal mitosis or just exit the mitosis and slip into the next interphase. How to enhance the proportion of apoptosis cells and reduce the proportion of non-apoptosis cells after mitotic arrest is a problem to be solved.As to research about mitosis, most of the research focuses on the modification and degradation of protein during mitosis while new protein which is synthetized during mitosis draws little attention. In this study we used the Ribosome profiling technology which is based on high-throughput sequencing and is in gradual perfection to sequence genome samples from both normal mitotic cells and mitotic arrest cells in the translational level. After the completion of sequencing, we analysed the data from sequencing and acquired some genes which had difference in the translational level between normal mitosis and mitotic arrest. Then we screened and identified some important genes from those genes which had translational differences we acquired. Notch2 is one of the genes which have translational differences between normal mitosis and mitotic arrest. Notch2 belongs to the Notch family and was reported to have a close relationship with cell proliferation, apoptosis and individual’s development. This study focuses on how the Notch2 functions in the determination of cell fate after mitotic arrest and which Notch ligand functions upon mitotic arrest.The result showed that the protein level of Notch2 increased in mitotic arrest cells compared with normal mitotic cells and the protein synthesis of Notch2 increased after mitotic arrest. This result further verified the correctness and reliability of the results of sequencing in our study. Time-lapse results and Immunofluorescence results showed that the knockdown of Notch2 didn’t affect the progress of mitosis. After mitotic arrest, the knockdown of Notch2 would increase the proportion of cells which died in the sub-G1 phase following abnormal mitosis. This phenomenon indicated that Notch2 may play a role in the survival of cells which were in the sub-G1 phase. The detection result of the protein level of Notch2 in different phases of the cell cycle showed that, during mitosis and mitotic arrest cleaved-Notch2 was modified and degraded. Moreover, during mitosis and mitotic arrest the mRNA level of Hesl which is a classical downstream molecule of Notch2 declined obviously in contrast to G1、S phase and the transcriptional activity of Notch2 was low. After mitotic arrest, Notch2 mainly functioned in sub-G1 phase rather than in the period of mitotic arrest. The knockdown of Notch2 would decrease the mRNA level of cyclinD1, thus after mitotic arrest Notch2 knockdown may cause G1 arrest and promote cell death in sub-G1. The knockdown of Notch2 would suppress the increase of the mRNA level of p27Kipl induced by mitotic arrest, thus may affect the probability of G1 arrest. As to anti-apoptotic molecules Mcl-1 and Bcl2, the knockdown of Notch2 would suppress the increase of the mRNA level of Mcl-1 and Bcl2 induced by mitotic arrest, which may result in the increase of the proportion of cell death in sub-G1. At last, we studied the Notch ligands which activated Notch2 upon mitotic arrest. We found that with Taxol treatment, the possible ligand activated Notch2 was JAG1 and we screened JAG1 shRNA for future JAG1’s functional experiment.