Radiotherapy-induced Exogenous HMGB1/mtDNA Promotes Residual Pancreatic Cancer Cells Invasion And Migration

Objective:The complex tumor microenvironment(TME)after radiotherapy(RT)is closely revelant to the metastasis and recurrence of pancreatic cancer(PC),while the specific mechanism remains unclear.This article aims to investigate the effects of the RT-released high-mobility group box 1/mitochondrial DNA(HMGB1/mtDNA)on the invasion and migration of residual PC cells and its exact mechanism;we further explore the regulation of immune globulin(IgG)in TME on the release of mtDNA into the extracellular space of PC cells.Methods:Different doses of X-ray(0 Gy,8 Gy)were treated with PC cell lines and then co-cultured with Pa Tu8988,Panc1 and Bx PC3 cells respectively,Transwell were utilized to detect the number of invased and migrated PC cells;Enzyme linked immunosorbent assay(ELISA)were used to detect the concentration of HMGB1protein in the supernatant of Pa Tu8988 cells treated with different doses of X-ray(0Gy,8 Gy);Quantitative-real time polymerase chain reaction(q RT-PCR)were served to detect the relative mtDNA copy number in the supernatant of Pa Tu8988 cells after different doses of X-ray irradiation;Exogenous recombinant human high mobility group protein B1(rh HMGB1)and extracted mtDNA were co-cultured with Pa Tu8988cells,and Transwell detected the number of invased and migrated cells;Fluorescence microscope and Western blot detected the interference efficiency of HMGB1 protein in Pa Tu8988 cells;Transwell detected the effects of stable interference with HMGB1 and exogenous rh HMGB1 on the invasion and migration of Pa Tu8988 cells;Ethidium bromide(Et Br,EB)were used to construct mtDNA partial deletion(ρ~0)Pa Tu8988 cells,and q RT-PCR detected the mtDNA deletion rate;Cloning experiment detected the proliferation ability of mtDNA partial deletion(ρ~0)Pa Tu8988 cells;PC cell lines were treated with different doses of X-ray(0 Gy,8 Gy)and be co-cultured with Pa Tu8988and Bx PC3 cells,then Western blot were used to detect the protein expression levels of cyclic guanylate-adenylate synthase(c GAS),stimulator of interferon genes(STING),TANK binding kinase 1(TBK1)and interferon regulatory factor 3(IRF3);Pa Tu8988cells were cultured with exogenous rh HMGB1 and mtDNA,and the protein expression levels of c GAS,STING,p-TBK1(Ser172)/TBK1,p-IRF3(Ser396)/IRF3 were detected by Western blot;Pa Tu8988 cells with lower expression of HMGB1(sh-HMGB1)and partial deletion of mtDNA(ρ~0)treated with radiation were co-cultured with Pa Tu8988cells,and Western blot were performed to detect the phosphorylation level of TBK1 in Pa Tu8988 cells;Exogenous rh HMGB1 and mtDNA were used to culture Pa Tu8988and Panc1 cells,respectively,and the q RT-PCR were used to detect the changes of interferon-β(interferon-β,IFN-β)transcription level;GEPIA2 website analyzed the expression differences of IgG-related cell surface receptors FCGR1A and FCGR2A in normal tissues and PC tissues,and their correlation with the survival rate of PC patients;Cloning experiment detected the effects of IgG treatment on the proliferation of Pa Tu8988,Panc1 and Bx PC3 cells;q RT-PCR detected the effects of IgG and RT treatment on the release of mtDNA into the extracellular space of Pa Tu8988,Panc1 and Bx PC3 cells;Western blot were utilized to detect the protein expression levels of TFAM and the c GAS-STING pathway related indicators in Pa Tu8988,Panc1 and Bx PC3 cells by IgG treatment.Results:ELISA test showed that RT could induce the extracellular release of HMGB1 in Pa Tu8988 cells;q RT-PCR proved that RT promoted the extracellular release of mtDNA in Pa Tu8988 cells;Transwell performed that radiation-induced exogenous HMGB1/mtDNA promoted the invasion and migration of residual PC cell lines;Fluorescence microscope and Western blot showed that the interference plasmid of HMGB1(sh-HMGB1)have been successfully and stably transferred into the Pa Tu8988 cells with superior interference efficiency;Transwell confirmed that the invasion and migration ability were weakened in the Pa Tu8988 cells which were stably interfered with HMGB1,besides the invasion and migration ability of Pa Tu8988 cells could be enhanced by exogenous rh HMGB1;q RT-PCR confirmed that EB could effectively knock out the mtDNA in Pa Tu8988 cells,and Cloning experiment showed that the proliferation ability ofρ~0 cells were stronger than the normal PC cells;Western blot confirmed that the protein expression levels of c GAS,STING,p-TBK1(Ser172)/TBK1,p-IRF3(Ser396)/IRF3 in the the residual Pa Tu8988 and Bx PC3 cells were inhibited after co-cultured with irradiated PC cell lines;Western blot showed that either exogenous rh HMGB1 or mtDNA could up-regulate the protein expression levels of c GAS,STING,p-TBK1(Ser172)/TBK1 and p-IRF3(Ser396)/IRF3 in different degrees in Pa Tu8988 cells,while the aboved protein expression levels were inhibited by treated with exogenous rh HMGB1 and mtDNA simultaneously;Western blot confirmed that the irradiated PC cells with stably interfered of HMGB1 or partial knockdown of mtDNA which were co-cultured with Pa Tu8988 cells could up-regulate the pho-sphorylation level of TBK1 in Pa Tu8988 cells;q RT-PCR showed that both exogenous rh HMGB1 and mtDNA were able to up-regulate the transcription level of IFN-βin Pa Tu8988 and Panc1 cells,however,the transcription level of IFN-βin Pa Tu8988 and Panc1 cells were down-regulated by treated with exogenous rh HMGB1 and mtDNA together;Bioinformatics analysis of GEPIA2 website proved that the expression of IgG-related cell surface receptors FCGR1A and FCGR2A in PC tissues were higher than that in normal tissues,and the expression levels of FCGR1A and FCGR2A were negatively correlated with the survival rate of PC patients;Cloning experiment showed that the proliferation of Pa Tu8988,Panc1 and Bx PC3 cells were enhanced by IgG treatment;q RT-PCR showed that IgG treatment of Pa Tu8988,Panc1 and Bx PC3 cells restricted the extracellular release of mtDNA after RT;Western blot confirmed that after IgG treatment of Pa Tu8988,Panc1 and Bx PC3 cells,the protein expression level of TFAM was up-regulated,and the protein expression levels of related indicators in the c GAS-STING pathway were decreased.Conclusions:Radiotherapy promoted the release of HMGB1 and mtDNA to the extracellular of human PC cells,moreover,HMGB1/mtDNA enhanced the invasion and migration of residual PC cells by inhibiting the activation of c GAS-STINGpathway and the production of IFN-β.Besides,the IgG in TME limited the extracellular release of mtDNA after RT.