Molecular Mechanism Of Circadian Gene Clock In Regulation Of NIH3T3 Cell Cycle

In order to adapt to the changes in the 24-hour light environment on the earth,mammals have evolved their biological clocks to coordinate their physiology and behavior.The molecular mechanism of the biological clock system is a complex negative feedback regulation loop.CLOCK,as the transcriptional activator of this loop,has important position.Current studies have shown that there is a certain diurnal variation in the expression of mammalian cell cycle genes,and overexpression or knockdown of Clock in cells will have a certain impact on the proliferation time of cells,but it is still unclear.The laboratory had knocked out 23 circadian clock genes in the early stage,and after testing,it was found that the proliferation of cells did not differ significantly.In order to study the coupling mechanism between the biological clock and the cell cycle,and based on the work of the previous laboratory,to study the mechanism of coupling between the circadian clock and the cell cycle,we used Clock as the connection point between the two oscillators,knocked out Clock in NIH3T3 B1-B10 cells using the CRISPR/Cas9 gene editing system,and obtained monoclonal cells Clock-F12,to explore Clock's regulation of the cell cycle and its internal mechanism.The Lumicycle reporter experiment found that the biorhythm in Clock-F12 cells was disordered and the amplitude disappeared.By MTT colorimetric method and cell counting method,it is found that the cell proliferation cycle is significantly reduced compared with WT,and the cell scratch and confocal observation single cell experiment results show that the scratch healing rate and migration ability of Clock-F12 are significantly reduced.From the above experimental results,it is speculated that the absence of the Clock gene may block the progress of the cell cycle,resulting in inhibition of cell growth and migration.Flow cytometric analysis of the distribution of cell cycle phases at multiple time points and transferring an exogenous Clock into Clock-F12 have all confirmed that the Clock-F12 cell cycle was blocked in the G1 phase.In order to explore the internal mechanism of G1 phase arrest,we separately characterized the transcription and translation levels of Clock-F12 cell cycle regulatory genes,and found that the transcription level of the regulatory gene Ccnd1(Code Cyclin D1)associated with G1 phase in Clock-F12 cells has a downward trend.The protein level also decreased significantly,and the phosphorylation levels of the two sites of the Rb protein S780 and S807/811(p Rb-S780 and p Rb-S807/811)were also significantly reduced,all of which may cause the cells to be arrested in the G1 phase.In addition,the transcription level of transforming growth factor Tgf-?1 is up-regulated in the G1 phase,and the transcription level of the downstream cell division cycle gene Cdc25 a that it inhibits decreases.The changes in the transcription level of these genes may affect the activity of the important Cyclin D1/CDK4-6 complex in the G1 phase,which causes the cell cycle to be blocked.In the transcriptome sequencing of Clock-F12 cells,the PI3K-Akt signaling pathway and the ECM receptor interaction pathway enriched by the KEGG pathway are involved in cell proliferation and migration,which is in line with the hypothesis that the loss of Clock affects the cell growth state.At the same time,pathways such as the Ras signaling pathway,which is significantly enriched in genes,are also related to malignant tumors.By exploring the internal molecular mechanism of the Clock gene regulating the cell cycle,it is helpful to further understand the coupling between the circadian clock and the cell cycle.Since the change of the circadian clock may affect the results of cancer treatment,this experiment also provides some ideas for delaying the growth of tumors.