The Effect Of H2AX Gene Silence On The Radio Sensitivity Of Esophageal Cancer Cells

The studies in vivo and in vitro have been showed that themodification of H2AX plays a key role in the regulation of a variety ofcell response and DNA double-strand breaks, including DNA repair, cellcycle testing and tumor inhibition. In view of the key role of H2AXionizing radiation, we hypothesized that silent H2AX may affect thecells response and DNA repair in ionizing radiation-induced damage ofcells, and indirectly improve the radiation sensitivity.In this study, we firstly observed the protein expression levels ofH2AX and the nuclear spots formation in different dependent timemanner or dose manner ionizing radiation by the different esophagealcancer cells: both ECA109and TE13are on behalf of various degrees ofdifferentiation of esophageal squamous cell carcinoma. Secondly, wesuccessfully constructed a stable transfection cell lines: the radioactiveresist human esophageal cancer ECA109cell which is low levelexpression H2AX by RNA interference technology. Lastly, we exploredthat the relationship H2AX, MDC1and53BP1protein expression beforeand after transfection in vitro and in vivo, the changes of the nucleusspots formation, and the impact of ionizing radiation on cell cycle. Atthe same time, we also observed that the changes of the tumor tissuessizes and cell cycle after ionizing radiation in nude mice. Therefore, toexplore the mechanism of the silence H2AX improving the esophagealECA109cell radio sensitivity for possible future gene therapy providesmore theoretical support.Part I The expression of γH2AX protein in time-effect anddose-response between different esophageal cancer cell linesObjective: The time-effect and dose-effect in protein expression and nuclear foci of γH2AX were studied after irradiation in differentesophageal cancer cell lines.Methods: We studied dose-response and time-effect relationship inprotein expression and nuclear foci of γH2AX by western blotting andImmunofluorescence staining in different esophageal cancer cell linesafter irradiation which then was compared for differentiation.Result:1. There is a certain amount protein expression of γH2AX inECA109and TE13cell lines when not be irradiated.2. The proteinexpression of γH2AX reached the first highest point of time graduallyadvance with increasing radiation dose in ECA109and TE13cells.3.Two cell lines detected1hour after ionizing radiation were found thepresence of γH2AX protein expression in a dose-dependent, the greaterthe dose, the higher the amount of protein.4. The protein expression ofγH2AX was producing fast, and slowing subsided in ECA109afterirradiation8Gy. After irradiation within24h γH2AX protein expressionwere restored to the level before irradiation.5. The recession time ofγH2AX protein in TE13cells was far greater than in ECA109cells, andit cold not be restored to the level before irradiation within24hours.6.Ionizing radiation-induced the γH2AX nuclear foci in a dose-compliancein cell ECA109, the greater the radiation dose, the greater the number offoci. The foci of γH2AX produced fast, and slowed subsided afteraccepted8Gy irradiation. The γH2AX foci reverted to the pre-irradiationlevel after irradiation24h.7. Received greater dose, generated morenuclear foci of γH2AX in TE13cells.Conclusion: Irradiation induced more protein expression and morefoci of γH2AX and the change also more difficult to restore topre-irradiation levels in TE13, compared with ECA109. There werepositive doses of compliance in both protein levels and the number offoci in the nuclear of γH2AX in two different esophageal cancer cells. Part II Construction shRNA viral vectors of silence the H2AX geneand Establishment of the stable esophageal ECA109cell line of lowH2AX expressionObjective: To build up stable esophageal ECA109cell line ofsilencing H2AX gene by RNA interference. Providing the conditions tostudy the relationship between silence H2AX and radio sensitivity invivo and vitro experiments.Methods: To design multiple silent H2AX gene sequence by RNAinterference, transfect the tool cells after connected with the plasmid,then select the best silencing effect of the target gene by western blottingmethod. The target gene was imported to the lentiviral vector, followedby transfection of esophageal cancer cells. The esophageal cancerECA109cell lines with low expression of H2AX protein were selectedby antibiotic. The silencing effect of primary,5,10and15generationswere detected from the RNA level and protein lever through RT-PCRand western blotting.Results:1.Double-labeled plasmid with silent H2AX gene sequencewas successfully constructed.2. PSC-3gene sequence had the bestsilencing effect through western blotting test.3. The silencing effect ofH2AX in RNA and protein levels was detected after transfection byRT-PCR and western blotting method. H2AX gene low expression inesophageal cancer cell lines at the level of RNA and protein decreasedby52%and70%, respectively.4. The silencing effect of H2AX in RNAand protein levels are stable at5、10and15generations.Conclusion: We successfully designed the RNA sequences to reduceH2AX gene expression and constructed the stable esophageal ECA109cell lines with low expression of H2AX.Part III The impact to radio sensitivity through silencing H2AX inECA109in vitro experimentsObjective: To observe protein (γH2AX, MDC1and53BP1)expression, interaction changes, nuclear foci and changes in cell cycle. Methods:1. The protein expression change of γH2AX, MDC1and53BP1after silencing H2AX in ECA109、ECA109-NC and silenceH2AX groups triggered by8Gy irradiation was observed throughwestern blotting.2. The change of relationship between γH2AX, MDC1and53BP1after silencing H2AX was tested byCo-immunoprecipitation(COIP).3. The change of γH2AX, MDC1and53BP1foci after silencing H2AX was observed by Immunofluorescence.4. The changes in cell cycle were detected by flow cytometry afterirradiation8Gy24hours.5. We detected cell proliferation and radiosensitivity change after silencing H2AX by MTT assay and colonyformation assay..Results:1. The expression of γH2AX increased in E+R (irradiationECA109) and NC+R (negative vector irradiation) group, the increaselevel of γH2AX was reduced in H+R (silent H2AX irradiation) group.There was no impact to the expression of MDC1and53BP1aftersilencing H2AX.2. The relationship between γH2AX, MDC1and53BP1became closely after irradiation. The close relationship trigger byirradiation was reduced between three proteins after silence H2AX.3.1It was found nuclear foci of γH2AX、MDC1and53BP1changed overtime, the most at1h, then the number gradually reduced, and close to thelevel before irradiation at12h, the rule of three kind of foci consistentwith each other.3.2The foci of γH2AX, MDC1and53BP1reducedafter silence of H2AX.4.1No significant difference was found inapoptosis rate between different groups.4.2G0/G1phase proportiondeclined after irradiation24h; silence of H2AX cold reduce the decline;The G2/M phase was increased after irradiation; silence of H2AX coldreduce the increase. S phase after irradiation showed no significantdifference between groups.5. There was no significant impact onproliferation after silence H2AX gene.6. Colony formation assayconfirmed silence of H2AX could increase radio sensitivity.Conclusion: ECA109-H group had higher radio sensitivity than ECA109and NC group, indicating silence of H2AX could increase radiosensitivity. The mechanisms include defective cell cycle checkpoints andabolishment of foci formation for several important mediator andeffectors proteins in the DNA damage response to IR.Part IV The vivo studies of radio sensitization in esophageal cancerECA109after silence of H2AXObjective: Included cell cycle、protein expression and tumor size innude mice xenografts after silence H2AX were studied. From vivoexperiments proved silence H2AX cold increase radio radio sensitivity.Methods:1.60nude mice were divided into Control、Control+R、NC、NC+R、H and H+R groups.2. The corresponding cells wereinoculated, the seeded cells of2×106/50μl/mice, preparation of nudeesophageal model.2. Observed the tumor growth between differentgroups.3. To give6MV-X line single15Gy irradiation after inoculation22days, and each group were killed5nude mice after irradiated24h,tumor specimens were packing required, protein levels were detected byWestern blotting, RT-PCR and flow cytometry analysis RNA levels andchanges in cell cycle.Results:1. All nude mice survived and tumor formation. The tumorvolume in silence H2AX group was smaller than the other two groups.2.H2AX was low expressed in RNA and protein level in the resection oftumor tissue.3. The relationship between γH2AX, MDC1and53BP1became closely after irradiation in vivo. The close relationship trigger byirradiation was reduced between three proteins after silence H2AX.4.The tumor growth were fastest in control group, silence H2AX slowedthe xenografts growth. The growth slowed down after irradiation, whilesilence H2AX cold increases the decline.5. The apoptosis rate wasdifferent in Control, NC and H group, H group was higher than the othertwo groups.6. The proportion of G0/G1after irradiation; The proportionof G2/M increased after irradiation, silence H2AX cold reduce theincrease. The S phase was reduced after irradiation, while the S phase was increased after silence H2AX.Conclusion: Silence H2AX cold increased radio sensitivity. Themechanisms include defective cell cycle checkpoints、enhance apoptosisrate and abolishment of foci formation for several important mediatorand effectors proteins in the DNA damage response to IR.