| Objective: radiotherapy occupies a decisive position among the therapeutic methods aimed at liver cancer,radiotherapy can kill the cancer cells,but as the frequency of radiotherapy increases,the tumor focus becomes insensitive to it gradually,which is the so-called radioresistance,which is one of the main reasons for a poor anticancer effect on the patients with liver cancer in the late stage of radiotherapy,thus it is partic?larly important to more deeply study the underlying mechanism of tolerance of liver cancer cell to radiotherapy.In this study,we have ascertained that 1)radioactive ray can promote the expression of early growth response factor(Egr-1)and autophagy-related proteins.2)the molec?lar mechanism related to inducement of radioresistance by liver cancer cell through autophagy.3)Egr-1 can bind to the autophagy-related gene 4B(Atg4B)and activate the subregion binding and in turn control the process of autophagy.In general,the molec?lar mechanism of inducing radioresistance by the promotion autophagy of liver cancer cell by Egr-1 on a condition of exposure to radiotherapy was asercertained.This research res?lt can provide a related scientific theoretical basis for the clinical application in the future and the final conquest of radioresistance.Method: 1.western blotting was used to detect the effect of different ionized radiations on the expression of early recording factor Egr-1 in early stage and the genes related to autophagy including Atg-4b,p62 and LC3 protein.2.after the Egr-1 was competitively inhibited by the adenovirus DN-Egr-1,CCK8 method,clone formation test and Trancewell test were used to detect the change in survival ability,clone formation ability and migration ability of liver cancer cell after being exposed to radiotherapy.3.After the Egr-1 was competitively inhibited by the adenovirus DN-Egr-1,Western blotting method was used to detect the expression of apoptosis-related protein.Luciferase test and CHIP(chromatin immunoprecipitation)test have confirmed that Egr-1 bound to Atg-4b in the liver cancer cell and reg?lated the expression of Atg-4b.5.the autophagy inhibitor 3MA and chloroquine were used to inhibit the autophagy,Western blotting and clone formation test were used to detect the expression of genes related to apoptosis of liver cancer cell of over-expressed Egr-1 and its cell?lar clone formation ability.Res?lt: 1.the HepG2 and SMMC-7721 cell line were radiated by radiotherapy ray with dosage of 0,2,4 and 8 Gy respectively,and the expression level of Egr-1 was detected,western blotting revealed that,the expression level of Egr-1 was significantly elevated when the radiation dosage was 8Gy.2.SMMC-7721 and HepG2 cell were infected by adenovirus empty carrier(Ad-GFP)and Ad-DN-Egr-1 virus respectively.In comparison to the blank control group,the liver cancer cells that were infected with Ad-DN-Egr-1 were significantly reduced in cell survival rate when they were exposed to 8Gy ionized radiation.SMMC-7721 cells were exposed to 8Gy ionized radiation,on hour 72,the survival rate of cells in control group was 74.9%,the cells infected by Ad-DN-Egr-1 in the experiment group had a survival rate of 49.4%.Similarly,the survival rate among HepG2 cells was respectively 61.3% and 38.2%;3.while the res?lt of clone formation test revealed that,the liver cancer cells infected by Ad-DN-Egr-1 and exposed to ionized radiation were far inferior to the cells in control group in clone formation ability,the res?lt of transwell test revealed that,the liver cancer cells infected by Ad-DN-Egr-1 were clearly superior to the cells in control group in migration ability.On a condition of 4.8Gy radiotherapy radiation,with the Egr-1 gene inhibited,the res?lts of western blotting test of SMMC-7721 and HepG2 cells both revealed that,the expression of anti-apoptosis protein Bcl-2 was reduced,meanwhile the expression of apoptosis protein Bax and cleaved caspase-2 was elevated.In terms of expression of protein related to cell autophagy after exposure to radiotherapy radiation detected by Western blotting.Compared with the untreated group,the protein expression of Atg4 B and LC3 II were increased in the radiation-treated group,while expression of autophagy bottom protein p62/SQSTM1 was reduced,which was especially evident when the radiation dosage was 8Gy.6.the 3MA and CQ autophagy inhibitors were used to close the cell?lar autophagy in the experiment group,the res?lt of clone formation test revealed that,HepG2 and SMMC-7721 cell were significantly attenuated in proliferation ability in comparison to the cells in control group that were equally exposed to the radiation,similarly,a significant decline in expression level of Bcl-2 of liver cancer cells inhibited by the autophagy inhibitor CQ and 3MA had been detected by western blotting,while the expression of Bax and cleaved caspase-3 was increased.Luciferase test res?lt revealed that,the group radiated by radiotherapy had a significant enhancement in their activity of luciferase,while the activity of luciferase was reduced significantly when the expression of Egr-1 was inhibited by DN-Egr-1;the res?lt of CHIP test revealed that,the three potential binding loci in the atg4 b promoter region were all able to bind to Egr-1,on a condition of exposure to radiotherapy,Egr-1's antibody respectively precipitated the sequence target spot of three promoters of atg4 b.8.processed by autophagy inhibitor,HepG2 and SMMC-7721 cells showed an overexpression of Egr-1,the res?lt of western blotting revealed that,the expression of apoptosis protein of liver cancer cells was reduced,the clone formation test prompted that,the cell clone formation ability was elevated as compared to the control group.Conclusion: 1)the radiotherapy radiation can induce the liver cancer cells to immediately produce lots of Egr-1,which causes an autophagy in the cells,thus leading to radioresistance,while inhibiting Egr-1 can partly reverse this process;2)transcription factor Egr-1 can reg?late the occurrence of autophagy of liver cancer cells by binding to the autophagy-related protein atg4 b.this study has proposed a new molec?lar mechanism that,when exposed to radiotherapy,the liver cancer cells reg?late the cell autophagy by producing lots of Egr-1 and promote the radioresistance of liver cancer cells,providing a theoretical basis for the clinical inhibition of the expression of this gene so as to reduce the incidence rate of radioresistance and improve the sensitivity to radiotherapy of liver cancer and its curative effect. |
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