Exogenous HMGB1 Promotes Dedifferentiation Of Pancreatic Cancer Cells

Objective:In this study,we investigate the HMGB1(high mobility group box-1 protein),in the pancreatic cancer cells supernatant after irradiation,promotes pancreatic cancer cells reprogramming towards a cancer stem cell phenotype in vitro,and to further explore its potential molecular mechanisms of the reprogramming,and then provide a certain experimental basis and theoretical basis for clinical treatment and prevention of relapse on pancreatic cancer.Methods:(1)Flow cytometry was used to sort out three subpopulations of CD 133-negative cells of pancreatic cancer cells.(2)Western blot was used to detect the sternness related protein(CD133?OCT4?SOX2?C-MYC?Nanog)and HIFla protein in human pancreatic cancer cells after different treatment.(3)The morphology and the capability of sphere-forming of the human pancreatic cancer cells(SW1990?Patu8988)differently changed after different treatment by optical microscope.(4)Flow cytometry was used to measure the proportion of CD133 positive cells in human pancreatic cancer cells(SW1990,Panc-1).(5)Western blot was used to detect the expression of the sternness related protein(CD133?OCT4?SOX2?C-MYC?Nanog)and HIF1? protein in human pancreatic cancer cells(SW1990,Panc-1)after interfering with sh-TLR2 and inhibitor knockdown TLR2 receptor,and co-culture with HMGB1.(6)Western blot was used to detect the change of p-YAP/YAP and HIF1? in human pancreatic cancer cells with HMGB1 co-culture.(7)Nucleocytoplasmic separation,immunofluorescence(IF)and luciferase reporter assay were used to detect the effects of HMGB1 on the nuclear import of YAP and HIF1?.(8)Immunoprecipitation(IP)technology was used to detect the effect of HMGB1 on the interaction between YAP and HIF1.(9)Chromatin immunoprecipitation(ChIP)was used to detect the effect of HMGB1 and hypoxia on the binding of HIFla to the Nanog promoter region.Results:(1)Western blot analysis revealed bioactive HMGB1(HMGB1),oxidized HMGB1(D-HMGB1),reduced HMGB1(R-HMGB1),and radiotherapy supernatant(SUP)in human pancreatic cancer cells(SW1990 CD133-,Patu8988 CD133-).All of them can promote the expression of sternness related proteins(CD133,OCT4,SOX2,C-MYC,Nanog)and HIF1? to varying degrees,and the HMGB1 effect is the most obvious,but it has no effect on the Panc-1 CD 133-cell line.(2)Light microscopic observations revealed that the human pancreatic cancer cell lines(SW1990 CD133-,Patu8988 CD133-)were treated with HMGB1,D-HMGB1,R-HMGB1,and SUP and the cell morphology became round to varying degrees,and the number of cell-forming cells increased significantly.,increase in size.(3)The percentage of CD133 positive cells in pancreatic cancer cell lines(SW1990 CD133-)treated with HMGB1,D-HMGB1,R-HMGB1,and SUP was increased by flow cytometry,and the difference was statistically significant,while that of Panc-1 CD 133-no effect.(4)After co-incubation of interfering RNA(sh-TLR2)or TLR2 receptor inhibitors with HMGB1,there was no significant increase in stem-associated proteins(CD 133,OCT4,SOX2,C-MYC,Nanog)and HIFla protein in pancreatic cancer.(5)Western blot analysis showed that the ratio of p-YAP/YAP decreased and HIFla protein increased in human pancreatic cancer cell line(SW1990 CD133-)after co-culture with HMGB1.(6)Nucleocytoplasmic separation,immunofluorescence(IF)and luciferase reporter assay have showed that HMGB1 can increase cytoplasmic YAP,HIF1? protein,and YAP fluorescence activity.(7)Immunoprecipitation(IP)technology was used to find that HMGB1 can increase the binding of YAP and HIF1.(8)The chromatin immunoprecipitation(ChIP)demonstrated that HMGB1 and anoxic environment could enhance the binding of HIFla to the Nanog promoter region.Conclusion:(1)After radiotherapy of human pancreatic cancer cells,passive release or active secretion of HMGB1 in the cell supernatant can promote dedifferentiation of residual cells in radiotherapy.(2)The binding of HMGB1 to the surface receptor TLR2 of pancreatic cancer cells promotes the process of cell dedifferentiation.(3)HMGB1 promotes YAP and HIF1? as central molecules in the process of dedifferentiation of human pancreatic cancer cells,that is,HMGB1 promotes the binding of YAP and HIFla and promotes the transcriptional activity of the two genes into the nucleus and initiates the transcription of stem-related proteins(such Nanog),promote the dedifferentiation of tumor cells.