The Effect Of Ubiquitin-specific Protease 53 On Radiosensitivity Of Esophageal Cancer Eca109 Cells

Objective: Radiation therapy is one of the main treatments for esophageal cancer,but some patients still have radiotherapy resistance,which affects the therapeutic effect;the nuclear factor E2 related factor(Nrf2)is an important antioxidant protein in the cell,It indicates that Nrf2 has a protective effect on cells after ionizing radiation.Nrf2 overactivation can increase the antioxidant capacity of cells and reduce the radiosensitivity of esophageal cancer.Ubiquitin-Specific Protease 53(USP53)is a number of Deubiquitinating enzymes(DUBs),Which can reverse the degradation of substrate proteins and regulate the expression of substrate proteins.This study mainly studies the regulation of the expression of Nrf2 in cells by USP53,and observes the effect of USP53 expression on the sensitivity of esophageal cancer Eca109 cells.Methods1.Eca109 cells were irradiated with 2Gy,4Gy,8Gy,12 Gy,20Gy radiation doses,and cultured in the incubator for 24 h.The survival rate of the cells was detected by CCK8,Real-Time PCR was used to detect the mRNA expression of Nrf2,and the appropriate irradiation dose was screened.Then the protein expression of Nrf2 and USP53 in the cells was detected by Western Blot.2.The USP53 full-length gene fragment was cloned from the purchased plasmid containing the USP53 full-length gene sequence,and then ligated with the enzyme-cut lentiviral vector plasmid,and the constructed USP53 recombinant lentiviral expression plasmid was ligated with the viral packaging plasmid.The 293 T cells were infected to obtain virus particles,and the USP53 esophageal cancer Eca109 cell line was stably expressed after infection of esophageal cancer cells.3.Western Blot and Real-Time PCR methods were used to verify the successful construction of USP53 stably overexpressing cell lines.4.Western Blot method was used to detect the protein expression of Nrf2 in Eca109 cells overexpressing USP53,extracting mRNA from overexpressing USP53 cells,detecting whether the mRNA expression of Nrf2 was changed,and detecting the cell line overexpressing USP53.mRNA expression of anti-oxidative stress genesdownstream of Nrf2.5.USP53-siRNA was transfected into cells by lipo3000 transfection method to interfere with the expression of USP53 in esophageal cancer cells.The interference efficiency of USP53 was detected by real-time PCR,and the protein expression of USP53 and Nrf2 was detected by Western Blot assay.The mRNA expression of proteins downstream of the Nrf2 signaling pathway was detected by real-time fluorescent quantitative PCR.6.The method of colony formation assay was used to detect the change of colony formation rate by over-expression of USP53 in cells and interference after irradiation.Results1.When the dose of cells was 0-4Gy,the mRNA expression of Nrf2 in the cells increased with the increase of irradiation dose.When the dose increased to 8Gy,the mRNA expression of Nrf2 began to decrease,and the experimental results of CCK8 showed that the radiation The cell survival rate was significantly reduced when the dose reached 8 Gy(SF < 50%),so subsequent experiments chose to irradiate the cells with a 4 Gy dose of radiation.2.Western Blot assay showed that the expression of antioxidant protein Nrf2 and USP53 protein increased after 4Gy irradiation.3.The Lenti-CMV-USP53-Puro recombinant expression plasmid was constructed and the USP53 stably expressed Eca109 cell line was successfully screened.4.Western Blot and real-time PCR results showed that USP53 overexpressing esophageal cancer cell line Eca109 was successfully constructed.Due to the deubiquitination of USP53,the expression of intracellular Nrf2 protein was increased when USP53 was overexpressed(P < 0.05).However,the mRNA expression of Nrf2 was unchanged(P > 0.05),and the mRNA expression levels of anti-oxidation genes HO-1 and NQO-1 downstream of Nrf2 were increased(P < 0.05).5.After the USP53 was interfered in esophageal cancer cells,the protein expression of Nrf2 was decreased(P <0.05),but the mRNA expression of Nrf2 was not changed(P > 0.05),and the downstream related antioxidant stress was detected.The mRNAexpression levels of the genes HO-1 and NQO-1 were decreased,confirming that USP53 affects the expression level of Nrf2 in cells.6.The clone formation assay showed that the cell formation rate of USP53 high expression in cells was higher than that of normal cells under the irradiation of radiation(P <0.05),while the cell type with low expression of USP53 in the cell was cloned under irradiation.Reduced(P <0.05)Conclusion1.The increase of Nrf2 in cells after irradiation,in addition to the effect of ionizing radiation,the increase in the expression of USP53 is also the reason;2.The level of intracellular USP53 affects the expression of Nrf2 protein in cells;3.The increase of Nrf2 protein expression caused by USP53 can activate and affect the expression of downstream antioxidant stress genes HO-1 and NQO-1;4.USP53 can enhance the expression of Nrf2 in esophageal cancer Eca109 cells by deubiquitination,which leads to the resistance of esophageal cancer Eca109 cells to radiotherapy.