Radiotherapy-induced HMGB1 Regulates Stemness Of Pancreatic Cancer Stem Cell

Objective:To investigate the HMGB1 which released from dead cell of post-radiation effect on pancreatic cancer stem cell,and the molecular mechanisms of HMGB1-TLR2/4signal axis.To provides a new point for the research of radiation sensitization,molecular therapy and biological target therapy for pancreatic cancer.Methods:(1)Flow cytometry(FCM)was used to detect the rate of CD133+cells in pancreatic cancer cells after different doses of radiation.The expression changes of CD133,Nanog,Oct4 and Sox2 in pancreatic cancer cells were measured by Western-Blot.(2)The expression levels of HMGB1 protein and m RNA in pancreatic cancer cell lines(SW1990,PANC-1,Aspc-1)were detected by Western-Blot and q RT-PCR.The expression of HMGB1 in pancreatic cancer cells and the content of HMGB1 in supernatant were measured by Western-Blot.Immune fluorescence(IF)was used to detect the release of HMGB1 from pancreatic cancer cells after radiotherapy.(3)Radiotherapy was performed on pancreatic cancer cells which expressed and interfered with HMGB1 respectively,and SUP was obtained for subsequent experiments.The stem cell culture system was used to induce pancreatic cancer cells,and CD133+ cells were isolated by flow separation.Pancreatic CD133+ cells were treated with recombinant human HMGB1 protein(rh HMGB1)at different doses and treated with cell radiotherapy.The size and number of spheres were observed and recorded under microscope.The m RNA expression levels of Oct4,Sox2 and Nanog were detected by q RT-PCR.Cell proliferation changes of CD133+ cells under different treatments were detected with Cell Countingkit-8(CCK-8).(4)The expression of HMGB1 receptor TLR2/TLR4 in pancreatic cancer cells was detected by Western-Blot and q RT-PCR.Knock-out TLR2 and overexpression TLR4 in SW1990,overexpression TLR4 and Knock-out TLR2 in Aspc1.Western-Blot were used to detect the expression of stemness-related proteins in cell treated with rhHMGB1.The variation of the sphere forming ability was measured by clone formation assay.(5)The expression of Wnt signal proteins in pancreatic cancer CD133 + cells treated with rh HMGB1 and SUP were detected by Western-Blot.Results:(1)The results of FCM and Western-Blot showed that x-ray irradiation induced the stemness of survival cancer cells in vitro and there was correlation with dose and time.(2)The results of Western-Blot and QRT-PCR showed that the levels of HMGB1 in pancreatic cancer cells were different,and there HMGB1 was released after radiotherapy.(3)Dying cells derived HMGB1 regulates CD133+ cancer cells stemness,self-renewal,and proliferation.(4)HMGB1 maintain and enhance the stemness of CD133+ cancer cells depending on TLR2 and Wnt/?-catenin.Conclusion:(1)This study demonstrated that HMGB1 could be released from pancreatic cancer cells after radiotherapy in vitro.(2)This study demonstrated that the extracellular HMGB1 could dramatically promote stemness of pancreatic cancer stem cells in vitro.(3)This study confirmed that HMGB1 regulates the Wnt / beta-catenin signal pathway through TLR2,and promotes the ability of self-renewal in pancreatic cancer stem cells.