To Detect The Expression Of NF-kB In Acute Myeloid Leukemia By Real-time Quantitative Polymerase Chain Reaction

Background and ObjectiveNuclear factor-kappa B is one kind of protein complex which express in almost every cell. It can be combined with enhancer B ofκlight chain of immunoglobulin and can promoteκgene express. NF-κB nearly exist in all cells.The NF-κB family contain NF-κB1 (P50), NF-κB2 (P52), RelA(p65),RelB,c-Rel, and so on. The common is that they posses a highly conservative Rel homeodomain which composed by 300 amino acids. This region is nucleic localization which can combined with DNA, dimmer and interact with IκB.NF-κB which combine with inhibited protein IκB present inactive condition in cytoplasm. It can be activated by many activator, for example TNF, growth factor, oxydizing agent, virus and drug.The activeκB depart from the inhibited protein of NF-κB, move into nucleus, combine with theκB sequence of the target gene and enhance its'expression. NF-κB is functional nuclear regulatory element which participate into immunity, inflammation, tumor, cell cycle regulatiom, apoptosis by regulating target gene.Study demonstrate NF-κB lead to tumor occur because cytokine or its'receptor is activated, which promote the growth or inhibit the apoptosis of unregulated lymphocyte. In addition, NF-κB affect cell cycle or DNA duplication which can lead to tumor. Experiment in vitro indicate that the inhibition of NF-κB can make tumor cell apoptosis. There is constitute or highly active NF-κB in AML and it is related with the subtype of AML. There is no report about the expression of NF-κB mRNA in AML by RT-PCR in China. This study detects the expression level of NF-κB mRNA in common and in AML patients by RT-PCR and explore the possible mechanism of AML.Patients and MethodsThe 60 primary AML patients were from Henan Cancer Hospital and Henan People's Hospital out-patient from 2008.9 to 2010.3 28 cases were male patients,32 cases were female patients, the mean age is 38 years old. Among the patients M1 were 3 cases,M2 was 22 cases,M3 were 18 cases,M4 were 7 cases, M5 were 10 cases.6 healthy volunteers were recruited as control normally.The total RNA was extracted by trizol.The integrity and density of RNA were detected. Reverse transcription Kit is RevertAidTM First Strand cDNA Synthesis Kit purchased from Fermentas company. Reaction conditions were 42℃60min,70℃5min,4℃5min.The RNA was conserved and to be used at-20℃.PCR was carried out in a final volume of 25μL containing 3μl cDNA,0.25μl of each primer,3μl ultrapure water,12.5μl PCR reaction liquid, Cycling conditions were 1 cycle,3minutes at 94℃; 30 cycles,1 minute at 94℃,45 seconds at 58℃, and 1minute at 72℃; and 1 cycle,7 minutes at 72℃. The PCR product was electrophoresis by 2% agarose gel (90V,30min)The quantitive assay of Polymerase chain reaction (PCR) amplification was carried out by ABI3700 RT-PCR Instrument.The primer of NF-κB was designed and synthesized by TAKARA.The forward primer of NF-κB was 5'CTG AAC CAG GGC ATA CCT GT3'.The reverse primer of NF-κB was 5'GAG AAG TCC ATG TCC GCA AT3'.The product of amplifica-tion was 197bp.The forward primer of GAPDH was 5'TCCTCTGA-CTTCAACAGCGACACC3'. The reverse primer of GAPDH was 5'TCTCTC-TTCCTCTTGTGCTCTTGG3'. The product of amplification was 206bp.The PCR reaction kit was SYBR Prime ScriptTM RT-PCR Kit (Perfect Real Time) from TAKARA company.The reaction volume was 50μl. Cycling conditions were 1 cycle,3minutes at 94℃; 40 cycles,30 seconds at 94℃,30 seconds at 57℃, and 45 seconds at 72℃; and 1 cycle,10 minutes at 72℃.The dissociation curve was drawed every 30 seconds.ResultsThe electrophoretogram of agarose gel:The amplification product of NF-κB cDNA and GAPDH cDNA can be seen in every group. The amplification strap of GAPDH located about 206bp.The amplification strap of NF-κB located about197bp. The NF-κB Real-time PCR amplification:The cDNA comparing with 0.01-100ng total RNA was used as model and was ampificated by RT-PCR. Then the standed curve and dissociation curve were made. The dissociation temperature of NF-κB was about 90℃and the dissociation temperature of GAPDH was about 87℃from the picture. This can explain the PCR amplification product was single product.The standed curve and amplification efficiency of NF-κB was the same as GAPDH.The expression of NF-κB mRNA in different subtype of AMLThe CT value of NF-κB and GRAPH were unified. That isΔCt equal Ct NF-κB minus Ct GRAPH.The expression of NF-κB mRNA in different group was expressed by x±s. The expression of NF-κB mRNA in experiment group was higher than control group(P<0.05). Using mean square analysis, the expression of NF-κB mRNA in M4 group(4.346) was higher than M2 group(2.099)(P<0.05). It is higher in M5 group (5.060) than M1(2.428), M2, M3 (2.694) group.There is no difference in other group.Conclusion1. The expression of NF-κB was high in AML. The up-regulation of NF-κB may play important role in the development of AML2. The NF-KBmRNA level was different in different subtype of AML3. It is possible to cure AML at molecule level by inhibiting the activity of NF-κB.