The Expressional Circadian Rhythm Of Clock Genes Bmal1and Per2in Raji、Hut-78、LY-8and HL-60Cell Lines

ObjectiveExplore the transpriptional levels of Bmal1and Per2in Haematological tumorcell lines, measure the circadian rhythms of them and fit the circadian rhythms incosine function. Using the consine function to calculate the peak and trough times inthe transpriptional levels of clock genes and provide theoretical basis for the furtherstudy of the relationship between transpriptional levels of clock genes and tumorgrowth.Object and Methods1. Object1.1Human Burkett’s lymphoma cell line Raji.1.2Human cutaneous T cell lymphoma cell line Hut-78.1.3Diffuse large B cell lymphoma cell line OCI-LY8.1.4Myeloid leukemia cell line HL-60.2. Methods2.1Cell culture. Four kinds of cell lines were cultured in vitro. The condition ofculture includes culture medium of RIPA1640containing10%fetal bovine serummedium,5%CO2and37℃environment. Keep the culture medium changed every other day.2.2The synchronous induction of rhythms of the clock gene expression. Replace theculture medium with0.5%fetal bovine serum and culture for2days for starvationand then change the culture medium with50%fetal bovine serum in RIPA1640for2hours for the inducement of synchronous rhythm, then change the culturemedium with no serum RIPA1640medium. Mark this moment as ZeitgeberTtime0(ZT0),4hours later mark the time as ZT4, and so on.2.3Measure the content of mRNA. Extract total RNA by Trizol at differenttimes(ZT0,4.8,12,20,24) in different cell lines, test the purity and integrity ofthem and synthesis cDNA by using reverse transcription Kit and measure thecontent of the expression of Per2and Bmal1by Real-time PCR.2.4Statistic analysis. Conduct homogeneity of variance test and test of normarlity for△Ct value. Conduct single factor analysis of variance for data which meet theconditions.Data expressed by Mean+SD,consider P<0.05as significant.Setect thesignificant data for cosine fitting.Use the consine analysis software and CronoLabsoftware to get the rhythm parameters.The funtiong for fitting is: F（t）=M+Acos(ω t+φ),among which M stands for mesor median,A stands foramplitude, φ for peak phase and ω for conversion time.Results1. The expression of clock gene Bmal1induced by the serum shock in Raji cell lineshave no statistic significance at different times across the day (P>0.05), thusindicates that the serum shock can not induce the rhythm expression of Bmal1inRaji cell line.2. Circadian rhythms could be found in the transcriptional levels of clock genesBmal1and per2in serum shocked OCI-LY8、Hut-78、HL-60cell lines, and it wasalso found in the transcriptional level of Per2in serum shocked Raji cell line. Thetranscriptional circadian rhythms in clock genes can be described with cosinefunction3. The transcription levels of Bmal1gene in OCI-LY8cells peaks at ZT0and troughs at ZT12, that of Per2peaks at ZT12and troughs at ZT4; The transcriptionlevels of Bmal1gene in Hut-78cells peaks at ZT4and troughs at ZT8, that ofPer2peaks at ZT12and troughs at ZT0; The transcription levels of Bmal1gene inHL-60cells peaks at ZT0and troughs at ZT8, that of Per2peaks at ZT8andtroughs at ZT20; The transcription levels of Per2gene in Raji cells peaks at ZT12and troughs at ZT0.4. Fit the variation of transcription levels of different genes in different cells intocosine function, with which t (ZT times) for variable: The relative transcriptionallevel of Bmal1in OCI-LY8could be fit into F（t）=13.9+5.4cos（15t）,that of per2could be fit into F（t）=9.6+4.1cos（22.5t+90）；The relative transcriptional levelof Bmal1in Hut-78could be fit into F（t）=10.6+6.0cos（45t+180）,that of per2could be fit into F（t）=7.5+5.0cos（15t+180）；The relative transcriptional levelof Bmal1in HL-60could be fit into F（t）=18.9+3.5cos（22.5t+180）,that of per2could be fit into F（t）=7.0+3.6cos（15t+240）；The relative transcriptional levelof Per2in Raji could be fit into F（t）=9.6+3.6cos（15t+180）.Conclusion1. The circadian rhythms of the transpritional levels of Bmal1and Per2inOCI-LY8、Hut-78and HL-60cell lines can be induced by serum shock while thatof Bmal1cannot be induced in Raji cell lines.2. The circadian rhythms of the transpritional levels vary according to the types ofcell lines.The circadian rhythms of the transpritional levels of Bmal1and Per2inOCI-LY8、Hut-78and HL-60cell lines can be described through consine function.