Study Of Effect In Proliferation And Apoptosis And P16Promoter Hypermethylation Of U266Cell Lines Treated By Decitabine

Objective:Multiple myeloma is a genetically complex disease,which ischaracterized by an abnormal plasma cell stem derived from B lymphoma cellclonal proliferation in the bone marrow.And cant be cured at present.But inrecent years it has made great progress in MM treatment,especially withpeople gradually realized the particular interest of Epigenetics in tumormorbidity mechanism.Targeted drugs treatment become more and morecommonly nowadays, including demethylation treatment.The domestic and foreign research has confirmed that MM patients’celllines exist in abnormal hypermethylation,which mostly happens in the CpGabound island of tumor suppressor genes promoter area,such as P15INK4Bgene, P16gene, P53gene and so on,then lead to the genesilence.Decitabine(DAC),belongs to demethylation drugs, is a nucleosideanalogue.Phosphorylated decitabine integrates into DNA in the process ofDNA replication,to participate in DNA synthesis.Phosphorylated decitabineinsteading of cytosine covalent bond to DNA methyltransferases(DNMTs),bycovalent bonding the DNMTs sulfydryl group of cysteine residues lead tothe enzyme inactivation.The FDA (U.S. Food and Drug Administration)hasapproved decitabine to be used in the treatment of myelodysplastic syndromes(MDS) formally. The basic research shows that the deletion and mutation ofP16gene exists in human malignant tumors in general. And it has reportedthat the mutation rate of P16gene range30%to80%in the detection ofvarious of cell lines of malignant tumor. P16gene methylation is also commonin multiple myeloma.In order to further confirm the mechanism of proliferation and apoptosisinduced by DAC in MM cells. This experiment choose human multiplemyeloma cell U266cell lines,to observe proliferation suppression and apoptosis-oriented function by DAC, to detect the expression changes of theDNA methyltransferases DNMT3AmRNA and tumor suppressor geneP16mRNA,to test the changes of methylation status of P16gene after DACfunction. Thus to provide theoretical basis for the new treatment of Multiplemyeloma.Methods:1The conventional method to cultivate U266cell lines，which is a humanMultiple myeloma cell. The cells were half passed every48--72hours.Logarithmic growth cells were used in the experiment. Each experimentwas repeated three times.2CCK-8method to detect different concentrations DAC influence theproliferation of U266cell line: the final concentration of the DAC respectively0.2、0.5、1.2mmol/L.Observing cells inhibition rate after single drug groupswere roled for24h,48h,72h.Computational formula: Cell Proliferationinhibition rate(%)=(1-OD value of experimental group/OD value of controlgroup)×100%.3AnnexinV/PI double staining to detect DAC of different concentrationsimpact in U266cell line apoptosis, the drug concentration was the same asaforementioned groups.Observing the apoptosis rate after each single-agentwas roled for24h,48h,72h;4PI staining, FCM (flow cytometer) to detect the cell cycle distribution afterdifferent concentrations DAC roled for72h.Firstly70%ethanol was used tobreak the cell membrane.After cells under RNase incubation for1hour,Propidium idodide staining for30min,then FCM to detect.The drugconcentration was the same as CCK-8experiment.5Real-time Quantitative PCR method to test the expression ofDNMT3AmRNA and P16mRNA after DAC function in U266cells for48h.Collect the U266cells roled by different concentration DAC for48h,extract total RNA,reverse transcript cDNA,real-time Q-PCR method testthe expression of P16gene mRNA and DNMT3AmRNA.Record the objectivegene and reference gene CT value,applicate formula F=2-△△CT，△△CT value =(objective gene CT value of Text group-reference gene CT value of Textgroup)-(objective gene CT value of Control group-reference gene CT value ofControl group),calculate the relative expression amount.The primer sequencesas following: DNMT3A upstream sequences:5’-TATTGATGAGCGCACAAGAGAGC-3’; downstream sequences:5’-GGGTGTTCCAGGGTAACATTGAC-3’. Fragment length:113bp.P16upstream sequences:5’-CGGAAGGTCCCTCAGACATC-3’; downstreamsequences:5’-TCATGAAGTCGACAGCTTCCG-3’.Fragment length:385bp.β-actin upstream sequences:5’-CTAGAAGCATTTGCGGTGGAC-3’.Fragment length:510bp.6Methylation specific PCR method (MSP method) to test changes ofmethylation status of P16gene by DAC function for48h. The drugconcentration was0.2,0.5,1.2mmol/L respectively, and also set controlgroup.MSP method procedure:Firstly hydrosulphite modify DNA,cytosinewithout methylation in DNA fragment convert to uracil,while methylatedcytosines unchanged.Then design the primer for methylated andnon-methylated sites respectively.PCR method amplify DNA fragment.If theprimer for methylated sites could amplify DNA fragment,it prove that existmethylation in detected sites;If the primer for non-methylated sites canamplify DNA fragment,it prove that exist non-methylation sites.MSP is aqualitative analysis way.7Statistical analysis:The software of SPSS13.0was used.Firstly make theTest of Normality. Mean±standard expresses deviation grouped data.Carryout the Homogeneity of variance test,if P>0.1,think the Variance homogeneity,then the comparisons between two groups use t test,One-way analysis ofvariance was used for comparing means in groups more than two;IfP≤0.1,think the variance heterogeneity,use the Nonparametric test.P<0.05wasindicated statistical significance.Results:1Effect of DAC on the proliferation of U266cells:Different concentration(0.2mmol/L,0.5mmol/L,1.2mmol/L) DAC acted on U266cells for24h,48h,72h respectively.One-way analysis ofvariance was used to analyze datas,show that the DAC inhibited U266cellproliferation, and with the time, dose dependent. The proliferation inhibitionrate increased obviously. After24h:5.30±1.08(%)；7.50±0.44(%)；22.33±2.18(%).After48h：11.17±4.75(%)；20.17±5.16(%)；30.40±2.26(%).After72h：25.80±6.52(%)；43.70±6.71(%)；59.33±2.30(%).Satistical analysis was performed between each treatmentgroup,in addition to comparison between24h and48h group in the0.2mmol/LDAC concentration(p=0.101)，the comparison among groups was statisticallysignificant.(P<0.05).2Effect of DAC on the apoptosis of U266cells:After AnnexinV/PI double staining,the results was analyzed by flowcytometer.At the same time,under different concentrations,the cell apoptosisrate increased with drug concentration increased,suggest with the dosedependent;And under the same concentrations,with the timeextension,apoptosis rate increased,suggest with the time dependent.After24h,the apoptosis rate of control group,0.2mmol/L group,0.5mmol/L group,1.2mmol/Lgroup:8.23±0.70(%),13.30±0.75(%),17.38±0.63(%),27.58±1.32(%).After48h:11.33±1.02(%),16.75±1.84(%),25.78±6.27(%),37.53±1.36(%)；72h：19.25±1.20(%),27.70±1.85(%),35.23±3.74(%),49.18±5.70(%). Satisticalanalysis between the treatment groups, and bettween the treatment groups andcontrol group,the difference was statistically significant (P <0.05).U266cellapoptosis rate was promoting with the time dose dependent.The experimentalresults also showed that DAC mainly induced early apoptosis of U266cell.The early apoptosis rate is:After24h:5.90±0.93(%),7.43±0.42(%),11.93±4.02(%),16.68±4.51(%).48h:6.93±0.53(%),11.15±1.72(%),19.98±7.11(%),30.40±2.36(%).72h:16.70±0.85(%),19.68±2.84(%),30.95±5.29(%),42.75±6.19(%).With the time and dose dependent.3Influence of DAC on U266cell cycle distribution:The results was analyzed by flow cytometer. After DAC of differentconcentration acted on U266cells for72h,it shows that with the dose increased,G0/G1phase cells gradually increased;S phase and G2/M phasecells both decreased.In control group,0.2mmol/L group,0.5mmol/Lgroup,1.2mmol/L group,the cell rate of G0/G1phaserespectively:82.01±1.68(%),89.21±1.22(%),92.86±1.13(%),96.75±0.48(%)；Satistical analysis between the treatment groups, and bettween the treatmentgroups and control group,the difference was statistically significant (P <0.05).The cell rate of S phaserespectively:2.32±0.57(%),1.19±0.28(%),0.92±0.37(%),1.08±0.71(%); theNonparametric test was used to analysis datas,it was found that it was notsignificantly different among each group.(P=0.075).The cell rate of G2/Mphase respectively:13.87±2.28(%),9.12±1.52(%),5.80±0.47(%),2.02±0.84(%).Satistical analysis between the treatment groups, and bettween the treatmentgroups and control group,the difference was statistically significant (P <0.05).Suggst decitabine arrests U266cells at G0/G1phase,and with the dosedependent.4Influence of DAC on the DNMT3A mRNA and P16mRNA expression:Real time Q-PCR results showed that0.2,0.5,1.2mmol/L three differentconcentration DAC in U266cells for48h,compared with the control groupDNMT3A mRNA expression level gradually reduced with drug concentrationincreasing.Set up the control group was1,the relative expression amount ofthe objective gene is0.77±0.13,0.42±0.11,0.24±0.18respectively.it was nosignificantly different between0.5mmol/L and1.2mmol/L DACgroup(P=0.204).The comparison between other groups， was statisticallysignificant (P <0.05).Compared with control group, P16mRNA expressionlevel gradually elevated with the drug concentration increasing, expressquantity is1.67±0.34，2.48±0.37，3.23±0.30respectively.Each group wasstatistically significant (P <0.05).5Test P16gene methylation level before and after DAC effect:MSP method was used in0.2,0.5,1.2mmol/L three differentconcentrations DAC groups for48h,compared with control group.It detectedthat CpG island of P16gene promoter exist hypermethylation in U266cell lines.After decitabine treatment, non-methylation band appear in gel, it presentpartial methylation status.And with the increasing of drugconcentration,methylation bands to reduce the brightness,non-methylationbands brightness increased.MSP method results showed that DAC couldreverse P16gene methylation of U266cell line partially.Conclusions:1DAC inhibits U266cell proliferation, and promotes cell apoptosis,especiallypromotes cell early apoptosis, in time and dose dependent.2DAC could influence the cell cycle of U266cells,cells were arrested inG0/G1phase,furthermore inhibits cell proliferation,,in dose dependent.3DAC can reduce the amount of DNMT3AmRNA expression, increase theamount of P16mRNA expression,with the dose dependent.4U266cells presents hypermethylation status in promoter region of P16gene,DAC can reverse methylation status of P16gene partially,and increasethe expression of it.