| BackgroundAs known to all, Ionizing radiation, chemotherapy drugs and exogenous and endogenous products of cell metabolism can cause different forms of DNA damage. DNA double-strand break, (DSB) is the most serious damage. After DNA damage, cells will start the appropriate repair pathway to repair it.In higher eukaryotes, repair of DSB is dependent on Homologous recombination repair (HR) and Non-homologous end joining(NHEJ) [1,2].Missing the function of some key protein or the repair process is blocked by exogenous factors, which can make the DNA damage can not be repaired or incomplete repair. And ultimately it lead to cell mutation, Canceration and apoptosis because of Genomic instability.So the research of repair pathway has important significance to the treatment of tumors.Homologous recombination repair is a very accurate way of repair which exchange and recombine the sister chroatid by using homologous fragments as template. HR repair is usually in the cell cycle S phase, G2 phase and M phases. Because homologous recombination repair of semi-repair time takes several hours, with the characteristics of slow repair efficiency [3]. Homologous recombination repair can be divided into the following steps:(1) Identification of DNA damage sites;(2) Processing of DNA damage Sites;(3) Synthesis of invasive and repair of chain;(4) formation of the holliday-junction and dissociation of the holliday-junction. As follow:after ionizing radiation, DSBs were produced. ATM binds to DNA damage sites by detecting them, then make H2AX phosphorylate. Phosphorylation of H2AX make NBS1 bind to DNA, then NBS1,MRE11 and RAD50 form the complex of MRE11/RAD50/NBS1 (MRN).MREl 1 of the MRN, can process at the end of the 5'â†'3' direction of DNA break site, then exposed 3'single-stranded DNA ends, which bind to several replication protein A (RPA). AS a result, DNA single strand (dss) is stable and Preventing to the formation of secondary structure. RAD51 take the place of RPA which binding 3'end of the single-stranded DNA and nucleoprotein filament is formed by covering exposed DSS. Nucleoprotein filament identify the homologous DNA template and make DNA chain process to Holliday-junction by the help of RAD51. Holliday-junction was divided into two Double-stranded DNA molecules by the function of nuclease and ligase[5].Many genes and proteins take part in HR repair. Such as:ATM; BRCA1; BRCA2; RAD51; XRCC2; XRCC3 etc. ATM gene plays a very important role in HR repair. ATM gene was found in a patient who suffered ataxia telangiectasia which is induced by mutations of ATM. Because of ATM deficiency,cells were became unstable; Sensitive to ionizing radiation and cell cycle was arrested. The functions of ATM gene in cell signal pathways were as follow:1) AS a cell cycle checkpoints which is activated in phase G1/S; phase S and phase G2/M. ATM can be phosphorylated including p53; c-Ab1; RPA ect which activate signal pathways. The 15th serine of p53 is actived by ATM phosphorylation make the check point in phase Gl/S work [6].The complex was form between Nbsl/Nibrin and Merll/Rad50 by ATM making Nibrin activate. Phase G2/M checkpoint is relate to phosphorylation of the 68th threonine at Chk2 N terminal [6]. (2) Repair of DNA damage, DNA damage can activate ATM by phosphorylation of BRCA1; phosphorylation of the 988th serine; phosphorylation of CtBP-interacting protein (CtIP).ATM control the repair pathway by making BRCA1 and RAD51 active. (3) The regulation of apoptosis, some study comfirm that rats which has defect in ATM gene can make apoptosis faster than the normal ones [7].The purpose of the treatment of cancer is to kill as many cancer cells and minimal impact on normal tissue cells. According to unlimited proliferation of tumor cells, some drugs were produced which can make Cell cycle arrest or inhibit tumor cells proliferation. But all of them have Side effects which led the damage of normal tissue. So a mount of study were taken to DNA damage repair pathways, in order to produce some drug can inhibit tumor cell proliferation but less normal tissue damage. The homologous recombination (HR) repair is an important role in DNA repair. Therefore, inhibition of HR or utilize the defect of HR may be another important therapy to cancer. There are more and more researches between express of ATM gene and radiation sensitivity were taken in Radiotherapy. The functions of ATM gene in cell signal pathways were:1) Active the cell cycle checkpoints; 2) Regulate DNA repair and apoptosis. These functions are significance to DNA repair[14]. By regulating the phosphorylation of protein kinase ATM, controlling phosphorylation of several downstream protein to regulate the DNA repair after Ionizing radiation. DNA repair defect in ATM is the reason for the increase of radio sensitivity [15]. ATM kinase blockers and Small molecule inhibitors which inhibit activity of ATM interrelated enzymes can lead to the lack of cell cycle checkpoint. As a result, the radio sensitivity of tumor cells are increased.Objective:1. Construct HepG2 cell which has ATM gene silenced (Anti-ATM HepG2), detect interference effect of ATM gene. 2. According to dose, time and cell type, compare the level of DNA damage and repair by yH2AX analysis.Methods:1. Construction of interference vectorAccording to design of target genes, synthesize 4 pair miRNA oligo sequence. Each pair oligo construct four miRNA vectors and transformed into competent DH5 by annealing, recombinant clones and inserting to the miRNA expression vector. Clones were picked respectively for colony PCR screening. Finally, Positive clones were sequenced to verify inserting sequences are consistent with the design of oligo sequences.2. Detect interference effect of Interference vector in HepG2.The four interference vectors were transiently transfected into HepG2 cells. Calculate target gene and reference gene expression by qPCR.3. According to dose, time and cell type, compare the level of DNA damage and repair byγH2AX analysis.According to experimental requirements, group the cell and process them (Ionizing radiation,γH2AX analysis). Finally, analyze the level of DNA damage and repair by Number of focus.4. Statistical analysisAll data was expressed as the form of x±s, all analysis were carried out using the SPSS 13.0 software package. (test:α=0.05).Expression of ATM gene by qPCR was analyzed with one-way ANOVA and LSD method for multiple comparison.Dunnett's T3 method was employed for heterogeneinty of variance,γH2AX analysis of DNA damage by immunofluorescence assay was analyzed with factorial analyze.Results: 1. Successfully to construct four interfering vectors:pcDNA6.2TM-GW/mir7-ATM-1, pcDNATM6.2-GW/mir7-ATM-2, pcDNATM6.2-GW/mir7-ATM-3, pcDNATM6.2-GW/mir7-ATM-4. all of them are consistent with the design of oligo sequences.2. According to target gene and reference gene expression by qPCR, HepG2+ mir7-ATM-4, the ATM gene expression level (2-ΔΔCT) is 0.47,0.53 efficiency of ATM silenced, on the top of 4 Interference vector.3. By calculating theγH2AX of each group and statistical analyze the average focus. The number of yH2AX focus in each dose group or time group is obviously different from each others (each p<0.05); According to the number of yH2AX focus in dose group and at different time points, we can estimate the appropriate level of DSB repair.Conclusion:1. Successfully to construct four interfering vectors.2. HepG2+mir7-ATM-4, the efficiency of ATM silenced, on the top of 4 Interference vectors.3. By inhibiting expression of ATM, DNA repair via homologous recombination is reduced.Namely, by inhibiting the expression of ATM to inhibit DNA repair pathway of homologous recombination double-strand breaks, affecting the efficiency of DNA damage repair... |
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