Relationship Between ATM Gene And Radiation Damage Repair Of Hepatoma Carcinoma Cell

BackgroundPrimay hepatocellular carcinoma (HCC) is one of the common malignancy in our country,HCC has the incidence rate of 20/hundred thousand and the second mortality rate in the total cancer mortality. For now,the first therapy way is surgical intervention,but the rate of exairesis is low(20%)because the low lever of diagnose and symptom emerging lately.with the development of radiotherapy equipment and technology,3 dimensional conformal radiation therapy(3DCFR) is become one of the therapy for hepatoma. Nevertheless, radical cure dose of hepatoma is more high than tolerated dose of hepatoma because of relatively radiation fastness of hepatocellular carcinoma. advanced stage patient who have hepatopathy confine the radiation therapy dose,the above factor lead to the low effect of radiation therapy for hepatoma. so how to elevate the radiosensitivity is difficult,but it may be a new way for therapy of hepatoma.ionizing radiation induce damage of DNA in cell in first,then lead to apoptosis.if the effect is low,there are two reasons:ionizing radiation can not effect tumor cell because of the cell itself or the microenvironment around tumor cell,the other is the repair of cell after ionizing radiation,the later is the major factor. single-strand break (SSB) and double-strand break (DSB) are major ways of the damage of DNA in cell.if they are not repaired in time,it will lead to cell death,mutation and loss the reproductive activity.so,repair of DSB is one of reasons for radiation fastness of hepatocellular carcinoma. There are two repair mechanisms:homologous recombination repair and nonhomologization end-joining.the later is the major way in mammalian,the newly study manifest that the survival of cell is depend on the repair ability other than DSB number.When IR result in DSB,the first stage is DNA-damage checkpoints.it consist of complex phosphorylation cascades in which DNA-damage ensorscan detect unrepaired DSBs and recruit transducer kinases. These are key players in the DNA damage response because they mediate the hosphorylation of several effector kinases and ultimately activate the appropriate effectors. Among the checkpointplayers, the mammalian transducer kinases ATM and ATR (ataxia telangiectasia and Rad3-related) together with DNA-PKcs (DNA-dependent protein kinase catalytic subunit) play a central role in triggering the checkpoint response through activation of the effector kinases CHK1 and CHK2, which propagate the signal. Likewise, in yeast Saccharomyces cerevisiae, DNA lesions trigger the recruitment of ATM and ATR orthologs Tell and, more important, Mecl, responsible for the phosphorylation of the effector kinases Rad53, Chkl and Dunl. In addition, several mediator proteins modulate the activity of the transducer kinases by interactions with multiple components of the DNA-damage response pathway. Thus, the S.cerevisiae Rad9 protein is a mediator of the checkpointresponse for DNA damage occurring all over the cell cycle. Rad9 orthologs that act as ATM mediatorsin vertebrates include MDC1 and the breast andovarian cancer-specific tumor suppressor BRCA1. The second stage is response for DNA lesion,there are repair factor,cell cycle factor, transcription factor,Checkpoint transducers are also responsible for one of the earliest events that occur after the formation of DSB, that is histone H2AX C-terminal tail phosphorylation,referred to as r-H2AX. In mammals, r-H2AX interacts with the mediator protein MDC1, which, at the same time, interacts with ATM and MR(X)N, thus generating further r-H2AX and amplifying the signal. The spreading of r-H2AX to larger chromatin domains on either side of a DSB is responsible for the stable accumulation of MDC1, MR(X)N, ATM and several other proteins implied in the DSB response. These chromatin modifications occurring around a DSB have been described as recruiting chromatinremodelling complexes that may have a role in DSB repairWhen repair proteinum was recruitmented in end of DNA,the repair is riming.there are two major repair ways:NHEJ and HR.NHEJ is the pathway that repairs DSBs by re-their two ends together. It appears to be thepowerful DSB-repair pathway because itpotential to ligate any kind of DSB ends without requirement for a homologous sequence, as opposed to the alternative HR DSB-repair pathway.there are three factors in NHEJ mechanism:MRX,Ku,DNA ligase,When DSB emrgeing, MRX and Ku join the end quickly for degradation,Meanwhile,it can stimulate ligase. MRXis the only protein complex shared by NHEJ and HR DSB-repair pathways in yeast. MR(X)N is composed of Rad50/RAD50, Mre11/MRE11 and Xrs2/NBS1 proteins. Rad50 shares the structural assembly of SMC (structural maintenance of chromosomes) proteins. Rad50 has a high-affinity DNA binding domain and seems to be able to bridge DNA molecules together. This property could help maintain DSB ends close to each other to facilitate their paring and ligation in the NHEJ reaction [8]. Mrel 1 shows in vitro DNA nuclease activities. It cleaves DNA structures that can be found at DSB ends such as hairpin structures and 3'single-strand overhangs at the single-/double-stranded transition. It also carries a 3'to 5'exonuclease activity.. It is phosphorylated in response to DSB formation as part of the signalling pathway triggering cell cycle arrest. In vertebrates, the MRN complex is analogous to yeast MRX, although there is no definitive evidence that MRN is involved in NHEJ. Yeast MRX has been shown to be one of the first complexes to bind DSB ends after their occurrence..the function of MRX is tethering DNA and recruit ligase. In vertebrates,Ku is part of a larger complex called DNA-dependent protein kinase (DNA-PK) whose catalytic subunit DNA-PKcs is also required for NHEJ [45]. DNA-PK functions as a DNAend-bridging factor in NHEJ the Ku complex seems to participate in NHEJ by stabilizing DSB ends and by preventing 5'-end resection. Ligase compose of Lig4/Ligaseâ…£, Lif1/XRCC4,Nej1/XLF,together with Ku, ligase can join the end of DNA which can not pair or affinity, there are potential superiority in repairing any DSB.DSB repair by HR corresponds to an exchange or a transfer of identical or quasi-identical sequences between the DNA molecule carrying the DSB and another intact DNA molecule. HR is a faithful mechanism if the DNA template used for repair is identical to the original DNA sequence present at the DSB. Otherwise, HR repair of DSBs can lead to local mutations or even more deleterious genome rearrangements. HR mostly involves proteins encoded by genes of the RAD52 epistasis group composed of,RAD51, RAD50,RAD52, RAD54, RAD55, RAD57, RADACHTUNGTRENUNG59, RDH54, MRE11 and XRS2 Classical HR is mainly characterized by three successive steps:1) resection of the 5'-ended DNA strand at break ends, followed by 2) strand invasion into a Homologous DNAduplex and strand exchange, and 3) resolution of recombination intermediates.The central steps of HR mechanisms:D-loop formation and strand exchange These reactions are mostly performed by a nucleoprotein filament composed of the 3'single-stranded DSB end coated with the Rad51/RAD51 recombinase protein.The Rad51-filament assembly requires the binding of RPA to the 3'single-stranded DSB ends. RPAis a heterotrimeric complex that binds ssDNA with high affinity and is believed to remove its secondary structures. RPA binds DSB ends shortly after resection and directly interacts with Rad52. Rad52 is the factor needed for almost all recombination mechanisms.ATM(ataxia-telangiectasia mutation) gene is the mutation gene of AT(ataxia-telangiectasia),it located into 11q22-23, total length of the gene is 150kb, exon is 12kb,and 66 extrons, encoding a ATM proteinum which have 3056 residues of amino acids,it is one member of phosphatidylinositol 3 kinase(PI3K)ATM gene is important for repair the DNA lesion induced by ionization radiation(IR),when IR result in DSB,the sign of lesion will lead to activation and autophosphorylation of ATM proteinum, dimeride of ATM is divided.together with MRN(X),MDCl,it can magnify sign of lesion and recruit reparative factor. The expression of ATM is up-regulation after phosphorylation,and make lots of target molecules which located downstream phosphorylation,such as p53,CHK2,BRCAl,meanwhile,sign of DNA lesion is transmised to DNA-PK, ku proteinum, form a complex, influencing repair of DNA lesion.And p53 is a key proteinum for DNA lesion checkpoint. Through the machine of specificity post-transcriptional regulation,p53 can interact with p21 or GADD45, and activate JNK or p38MAPK signal pathway to paticipate into apoptosis,and regulate p21 to make G1 blockaged.MDM2 can degrdn p53 by proteasomes which is depended by ubiquitin.ATM activating CHK2 make p53 further phosphorylation and stability, the deactivation of CHK2 can lead to cell cycle G2 stoppage,there are lots of proteinums to repair DSB,such as BLM,BRCA1,H2AX,Mre11,NBS1,XRCC4,Rads etc.ATM gene is a "hot gene for study radiation fastness of hepatocellular carcinoma in recently years.some studies confirm there are relationship between ATM gene and breast cancer,and Li verified that ATM gene which is depression can elevate the radiosensitivity in uterine cervix cance.but rare study in hepatoma.we verified ATM gene is overexpression in hepatoma patient in prophase experimentation.so we assume that the radiosensivity will be elevated if we inhibit the ATM gene in hepatoma cell.wo design the siRNA sequence which can inhibit the ATM gene in hepatoma cell in first,detect the level of depression,then observe the repair of DNA lesion.Objective:1. To design the siRNA sequence which can inhibit the ATM gene in hepatoma cell,and detect the depression rate in level of mRNA and proteinum.2. To observe Influence of siRNA targeting against ATM on DNA damage repair for hepatoma cells after X irradiationMethods:1. siRNA sequence was designed and synthesised which can was inhibit ATM gene high performance in hepatoma cell.According to the genetic information, three siRNA sequences of ATM was designed, And desigened the quantitate PCR amplification primer for ATM gene according the software of primer(Primer Premier 5.0),upstream: 5-ACTGGCCTTAGCAAATGC-3'downstream:5-TTGCAGCCTCTGTTCGAT-3'2. the transfection effect and depression level after siRNA transfect into hepatoma cell.First,we chosed the better hepatoma cell HepG2,then the three siRNA sequences were transfected into the hepatoma cell by liposome, detected the transfection effect with fluorescence microscope after 6 hours.And chosed the effective concentration and time. The expression level of ATM mRNA and protein was detected by qRT-PCR and Western blotting respectively. 3. influence of cell biology in hepatoma cell after ATM gene depression,we chosed the siRNA which can inhibit ATM gene high-performance,and transfect-ed into hepatoma cell.The cell proliferation was assessed by MTT assay.we irradiate the hepatoma cell after 24 hours when transfection. the changes in cell cycle and apoptosis were evaluated by flow cytometry.the level of DNA lesion was detected by neutrality comet assayRESULTS:1. the valid siRNA sequence was boltingedThe valid siRNA sequence which can depress ATM gene was boltinged fluorescence microscope manifest that the level of transfect in three siRNA sequence are 90%,every siRNA sequence was transfected into HepG2,wo extract totalRNA after 12 hours,and we get the the value of OD260/OD280 is 1.96,it comfirm that RNA is pure; RNA electrophoretogram manifest that the RNA is integrity. qRT-PCR demonstrated that the best inhibitory effect on ATM mRNA expression in HepG2 is 72% in the third siRNA sequence when 75nmol/mL. western blot demonstrated the inhibition ratio on protein expression is 78% in 48h after transfection. And the depression of proteinum isgradually upgrade after 72 hours2. influence of cell biology in hepatoma cell after ATM gene depression,The high-performance siRNA sequecnce was transfected into hepatoma cell after 24 hours,we collect the cell.The proliferation among three groups(HepG2ATM,HepG2NC and lipofectamine2000) cells was not marked change by MTT. After transfection,we irradiate the hepatoma, FCM display that the number of cells at G1,G2,S and Mstage have not distinguish before radiation,but G2/M stage in HepG2ATM group was obviously increased, and the number of cells at S stage was manifestly decreased after X irradiation,from 31.8% to 11.1%. FCM showed that it have not distinguish before radiation,yet the period of apoptosis ratio in HepG2ATM group is 2 folds than that of the negative control group. The neutrality comet assay demonstrate that the level of DNA lesion in HepG2 ATM group is more than that in negative control group.CONCLUSION:1. we bolting the siRNA sequence which can high-performance inhibit the ATM gene in hepatoma cell,and get the optimal concentration and the optimal time.2. the hepatoma cell proliferation have not change after ATM gene was depressed.3. the cell of HepG2 ATM group was irradiated,the FCM show that G2/M stage in HepG2ATM group was obviously increased, and the number of cells at S stage was manifestly decreased.meanwhile The earlier period apoptosis ratio was increased, The neutrality comet assay demonstrate the level of DNA lesion is manifest. The above show that ATM gene can influence the repair of DNA lesion in hepatoma cell.