Study On Variation Of Radiosensitivity Of Esophageal Carcinoma Cells By Lentiviral Vector-mediated Down-regulation Of Gene MDC1 And 53BP1

Esophageal carcinoma is one of the most common malignant tumor in our country and more than 50 persent of patients who suffered from esophageal carcinoma aroud the world exist in China. Radiotherapy(RT) is one of the most important methods to therapied esophageal carcinoma, however, esophageal carcinoma has lower 5 years survival rate but higher failed control rate and recurrent rate after RT. It is still a challenged question how to enhance radiosensitivity of esophageal carcinoma cells, to decrease local recurrent rate, and to increase local control rate and long-time survival.There has a close relationship between cell cycle and radiosensitivity. Checkpoint kinase control the cell cycle course, DNA is beleved the main target of irradiation, when irradiation induced DNA damage, system of DNA damage repair and checkpoint of cell cycle is actived immediately, arrest of cell cycle were induced to enhance repair for DNA lesions. Mediator of DNA damage checkpoint 1 and p53-binding proteins1 (MDC1 and 53BP1) are very important protein kinase for repair damaged DNA and arrest of cell cycle, retarded cell cycle course. MDC1 and 53BP1 has no effect to growth of somatical cells, but to transfer and enlarge signal to down-stream proteins. In recent years, some studies report that MDC1 and 53BP1 express in many nomal and tumor cells, inhibited protein expression of MDC1 and 53BP1, it could release retarded cell cycle and increase sensitivity to killing tumor cells after irradiation. To date, it is still not report about protein expression of MDC1 and 53BP1 in esophageal carcinoma cells, and how to regulated cell cycle of esophageal carcinoma cells after DNA damage.In this report, firstly, expression of MDC1 and 53BP1 were detected in different type of esophageal carcinoma cells, change of cell cycle and radiosensitivity were detected in esophageal carcinoma cells after radiation alone in vitro, we also observed MDC1 and 53BP1 forms nuclear foci in response to DNA Damage. Secondly, we designed three expression sequences of shRNA target to MDC1 and 53BP1 respectively, and cloned the sequences to lentivirus work vector pSIH1-H1-copGFP. The recombined vectors were identified by restriction enzyme analysis and sequencing. Lentivirus was produced after pSIH1-H1-copGFP and three package plasmids co-transfected to 293T cells with the help of lipofectamine 2000. Esophageal carcinoma cells were infected by recombined lentivirus, positive cell clones were selected to amplification culture, these were stabilised transfected esophageal carcinoma cells which was down-regulate the gene MDC1 and 53BP1. Finally, MDC1 and 53BP1 nuclear foci formation, protein expression of CHK1 and CHK2, change of cell cycle and radiosensitivity were detected in esophageal carcinoma cells after radiation alone or combined with transfection. We also observed the change of the tumor volume to evaluation the the effect of low expression of gene MDC1 and 53BP1 on radiation sensitivity esophageal cancer in vivo.Part I Effect on cell cycle and expression of MDC1 and 53BP1 after irradiation in esophageal cancer cellsObjective:To observe expression of MDC1 and 53BP1 in different type of esophageal carcinoma cells, observe effect on radiosensitivity, cell cycle and expression of MDC1 and 53BP1 protein, MDC1 and 53BP1 nuclear foci formation in response to DNA Damage after irradiation in esophageal cancer cells in vitro.Methods: In three human esophageal carcinoma cell lines of TE-1,TE-13 and ECA109, expression of MDC1 and 53BP1 mRNA were detected with reverse transcriptase-polymerase chain reaction(RT-PCR), expression of MDC1 and 53BP1 protein were measured with immunohistochemistry assay, western blotting and indirect immunofluorescence assay respectively. Radiosensitivity were analysized with clonegentic assay and MTT methods, change of cell cycle were measured with flow cytometry(FCM) and expression of MDC1 and 53BP1 protein were detected with western blotting after irradiation in TE13 and ECA109 esophageal caicinoma cells in vitro, Immunofluorescence images were captured using a Zeiss fluorescence microscope.Results:①Expression of MDC1 and 53BP1 in TE-1,TE-13 and ECA109 cells were observed in level of mRNA and protein in this report.②The percentage of G0/G1, G2/M and S phase of TE-13 cells was changed dependence on doses increasing of irradiation. At 12h after irradiation with 1Gy and 2Gy, arrest of G2/M stage was gradually increased and reached the peak at 24h after irradiation with 5,10,15Gy (P<0.05). Apoptotic index of TE-13 cell was obviously elevated at 12h,.24h and 48h after irradiation with 15Gy (P<0.01). however, Expression of MDC1 and 53BP1 proteins in TE-13 cells were no significant changes at 48h after irradiation with 5,10,15Gy (P>0.05), compared with the control group③MDC1-containing foci were clearly present within 5 min after IR. The average number of MDC1-positive foci per cell increased with time and peaked 30 min after irradiation. Gradually, the number of MDC1-positive foci decreased to background levels within 4 h. We have the similary result about 53BP1 foci in esophageal caicinoma cells.Conclusion: Expression of MDC1 and 53BP1 protein were universely exsited in different type of esophageal carcinoma cells, G2/M arrest and apoptosis of TE-13 cells were remarkable increasing after different doses irradiation. Meanwhile, No changes of expression of MDC1 and 53BP1 proteins in TE-13 and ECA109 cells were investigated after irradiation. Irradiation can affect the number of gene MDC1 and 53BP1 nuclear foci but not expression of MDC1 and 53BP1 protein in esophageal cancer cells.Part II Construction and identification of recombined MDC1 and 53BP1-shRNA lentivirus and establishment of stabilized tranefected esophageal carcinoma cell linesObjective: To obtain stabilized transfected esophageal carcinoma cell lines which targeting MDC1 and 53BP1 gene, observe the change of expression of MDC1 and 53BP1 mRNA and protein, It offered a good tool for us to research on the radiosensitivity of ECA109 esophageal caicinoma cells by RNAi targeting MDC1 and 53BP1 gene.Methods: Three specific target sequences were selected according to MDC1 and 53BP1 mRNA sequence respectively, and a non-specific control was designed. The complementary DNA which contained both sense and antisense oligonucleotides were synthesized. After phosphorylation and annealing, these double strands DNA were cloned to EcoRI and BamHI sites of pSIH1-H1-copGFP. Then the product pSIH1-MDC1-shRNA1,pSIH1-MDC1-shRNA2,pSIH1-MDC1-shRNA3 and pSIH1-53BP1- shRNA1,pSIH1-53BP1-shRNA2,pSIH1-53BP1-shRNA3 and pSIH1-negative was confirmed by electrophoresis and sequencing. Compare the RNAi effect of three specific plasmids harboring different shRNA expressing cassette by Real-time RT-PCR. pSIH1-MDC1-shRNA and pSIH1-53BP1-shRNA were cotransfected along with pPACKH1- Lentivector Packaging system into 293T to package lentivirus particles.48h after transfection with specific or control lentiviral vectors, selected for stable integrants by using copGFP reporter gene, the effect of RNAi were identificated by Real-time RT-PCR and western blotting.Result:①It was confirmed by digestion and sequencing that MDC1 and 53BP1 shRNA expression structure is correctly cloned to pSIH1-H1-copGFP.②After transfected with MDC1 and 53BP1 shRNA, mRNA expression of MDC1 and 53BP1 genes were inhibited obviously in ECA109 cells .The inhibition of pSIH1-MDC1-shRNA1,pSIH1-MDC1-shRNA2,pSIH1-MDC1-shRNA3 were 68%,78%,61%respectively while pSIH1-53BP1- shRNA1,pSIH1-53BP1-shRNA2,pSIH1-53BP1-shRNA3 were 81%,74%,65%.③Successfully established the MDC1 and 53BP1 knocking down cell lines: ECA109/MDC1and ECA109/53BP1.④The mRNA and protein expression of MDC1 and 53BP1 genes were inhibited obviously in ECA109/MDC1and ECA109/53BP1, The inhibition of mRNA and protein expression of ECA109/MDC1 were 66%and 58%,while ECA109/53BP1 were 65%and 52%.Conclusion:①After transfected with MDC1 and 53BP1 shRNA, mRNA expression of MDC1 and 53BP1 genes were inhibited obviously in ECA109 cells.②Successfully established the MDC1 and 53BP1 knocking down cell lines: ECA109/MDC1and ECA109/53BP1.Part III Effect on expression of CHK1 and CHK2 and distribution fo cell cycle after irradiation in esophageal carcinoma cells with RNA interference.Objective: To observed effect on expression of CHK1 and CHK2, formation of MDC1 and 53BP1 nuclear foci and change of cell cycle after radiation in esophageal carcinoma cells with RNA interference.Methods:①Observed the effect of knockdown of MDC1 and 53BP1 on proliferation ability of ECA109 cell by MTT assay, radiosensitivity of ECA109 cell by clone conformal assary after radiation.②Distribution of cell cycle and apoptosis index were measured with folw cytomitry 1h,2h,12h,24h and 48h after irradiated by 5Gy.③After transfected, expression of CHK1 and CHK2 protein and mRNA were detected with western blotting after radiated by 5Gy, formation of MDC1 and 53BP1 nuclear foci were detected with Zeiss fluorescence microscope.Result:①After transfected, proliferation ability of ECA109 cell were not influenced, p > 0.05.②Percent of G0/G1 stage were significantly decreased at 12h after irradiated by 5Gy, obvious decreased percent of G0/G1 stage were detected in, while retardance of G2/M stage were observed at 12h after irradiated by 5Gy,the degree of retardance of G2/M stage was more serious in ECA109-M and ECA109-B cells than in ECA109,ECA109-N cells . Percent of S stage and apoptosis were not influenced in this report. We have obtained similarity results at 24h,48h after irradiated by 5Gy.③D0 value and SF2 value were 3.06Gy and 0.91 in ECA109 cells, D0 value were 2.90,1.88,2.07Gy and SF2 value were 0.89,0.84,0.79 respectively with clonegenetic assay in ECA109-N,ECA109-M,ECA109-B cells.④After knocking down expression of MDC1 and 53BP1 , expression of CHK1 and CHK2 on level protein were not influenced, level of CHK2-T68 phosphated expression were decreased 1-24h after irradiation,there was not significant on level of CHK2-T68 phosphated expression in ECA109,ECA109-N, ECA109-M, ECA109-B cells 48h after irradiation. The number of MDC1 and 53BP1 nuclear foci were significant decrease in ECA109-M,ECA109-B cells.Conclusion: Radiosensitivity is higher in ECA109-M,ECA109-B cells than ECA109-N,ECA109 cells, arrest of G2/M stage were also released 12h after irradiated by 5 Gy.After transfected, expression of CHK1 or CHK2 gene at the level of mRNA and protein were not inhibited, but the gene CHK2T68 were markedly inhibited after irradiated by 5 Gy.The formation of MDC1 and 53BP1 nuclear foci were also inhibited after irradiated by 5 Gy.PartⅣThe effect of RNAi targeting MDC1 and 53BP1 gene on radiation sensitivity of esophageal cancer in vivoObjective: To investigate the effect of RNAi targeting MDC1 and 53BP1 Gene on radiation sensitivity of esophageal cancer in vivo. It offers a powerful new strategy for cancer gene therapy combined with radiotherapy.Methods: 64 male BLBA/C/nu nude mice were randomly divided into eight groups and eight kinds of prepared cells were subcutaneously injected on the paw pat of mice(2×106/100μl/mice):①The control group(ECA109 group);②The negative group(ECA109-N group);③MDC1 knocking down cell group(ECA109-M group);④53BP1 knocking down cell group(ECA109-B group). Every group was divided into two groups A:irradiation group;B: unirradiation group. Tumor growth were monitored every other day since the sixth day after injection. Tumor volume were measured with calipers. The expression level of CHK1 and CHK2 protein were examined in different groups by western blotting . Apoptotic cell and cell cycle distribution were detected in transfected cells by FCM assay.Result:①Visible tumors were detectable by day 7 after implantation,the tumor volumes were no significant among all the groups.②After irradiation by 15 Gy, ECA109-M group and ECA109-B group'volume are similar ,but these two groups'tumor volume were smaller than the other groups; the growth inhibition rate increased, but the relative growth rate was decreased significantly(p<0.05). The q value which reflect the radiosensitizing effect in ECA109-M+R and ECA109-B+R groups was more than 1 which were 1.36, 1.45 respectively.③Expression of CHK1 and CHK2 on level protein were not influenced, but level of CHK2-T68 phosphated expression were decreased significantly after irradiation by 15Gy. Percent of cell cycle and apoptosis were not significant in our study.Conclusion: RNA interfered expression of gene MDC1 and 53BP1 could inhibit the proliferation of esophageal cancer cell in the nude mice after irradiation.Combining RNA interfered expression of gene MDC1 and 53BP1 and radiotherapy could get better control for esophageal cancer,but this aspect need to explore farther.