Mutations In The Loop Region Of The Bmal1 Gene Affect The Circadian Clock And Cell Cycle Of NIH3T3 Cells

In nature,many organisms have biological rhythms,which are formed by the evolution of these organisms as they gradually adapt to the earth's 24-hour day and night environment.There is an internal "biological clock" in every organism.In mammals,the core of this internal "biological clock" is mainly located in the suprachiasmatic nucleus(SCN)of the brain,and its main molecular mechanism is the transcription translation feedback loop(TTFL).CLOCK and BMAL1 are transcribed and translated by Clock and Bmal1 genes,respectively,assembled in the cytoplasm to form a heterodimer,which binds to the E-box near the promoter to drive the transcription of other circadian clock genes,such as Period(Per1,2,3)and Cryptochrome(Cry1,2),Rev-erb,Ror,Dbp,Tef,Hlf,E4bp4 and clock control genes(CCGs).The expressed PER and CRY proteins gradually accumulate in the cytoplasm and undergo dimerization.During CK1?/?-mediated phosphorylation,one part is transferred to the nucleus,and the other part is degraded by the 26 S proteasome.The PER-CRY complex that enters the nucleus binds to CLOCK:BMAL1 to inhibit the transcriptional activity of CLOCK:BMAL1,and indirectly inhibits their own expression level.The gene-edited NIH3T3 B1-B10 G5 cell line with mutations in the Loop region of the Bmal1 gene constructed by our laboratory was edited from the CCACAACCAGCGA sequence to the TCGC sequence at positions 156 to 168 of the 13 th exon of the Bmal1 gene.Trigger changes in the cell clock and cell cycle.Firstly,compare and analyze the nature of the amino acids before and after the mutation of BMAL1 protein.Secondly,the changes in cell phenotype are detected by the following methods,mainly including: 1.Using cell counting method to determine the cell proliferation ability;2.Using cell streaks respectively Method and tracking the movement of single cells to detect cell migration;2.Use LumiCycle cell rhythm meter to detect the oscillation of cell biological rhythm genes;3.Use flow cytometry to analyze the cell cycle;4.Use neutral red staining to analyze the presence of lysosomes in cells Changes in the internal properties;5.Detect the transcription level of the core circadian clock genes of the cell;6.Determine the ability of BMAL1 and CLOCK mutations in the Loop region to form heterodimers through the dual luciferase reporter gene experiment.Through the above experiments,we have a preliminary understanding of the effects of mutations in the Loop region of the Bmal1 gene on the biological clock and cell cycle.Our results show that the mutations in the Loop region of the Bmal1 gene can change the cell phenotype,including: 1.Change the morphology of the cell,making the cell morphology larger by 36.09 ± 17.14 ?m2;2.Significantly reduce the cell proliferation rate;3.Change The properties of the lysosome in the cell,including the location of the lysosome,the concentration of the distribution,the number,or the p H of the lysosome,are the result of the combined effect;4.It reduces the cell migration activity,but does not completely inhibit the cell's activity.Migration;5.Changed the cell cycle distribution,making cells more concentrated in the G2/M phase;6.Changed the intracellular rhythmic gene expression,including reducing the amplitude of rhythmic gene expression,shortening the cell rhythmic gene expression cycle,etc.;7.Changed the oscillating trend and oscillating level of the transcription level of cell biological rhythm genes;8.It was verified that the loop region of the Bmal1 gene is not a key factor affecting the binding ability of the BMAL1 protein and the CLOCK protein.Through the above experimental results,we have a preliminary understanding of the effect of the Loop region mutation on the function of the BMAL1 protein and its initiation in the cell,and further deepen the understanding of the function of the flexible Loop region between the bHLH and PAS domains of the BMAL1 protein.,Suggesting that the Loop region may play an important role in the circadian clock function of BMAL1 and its participation in other cell physiological activities.In the future,we will conduct research on how the Loop area affects the biological clock and cell cycle.