| Part one The expression level of HMGB1 in plasma exosomes of patients with ESCC and its relationship with radiosensitivityObjective:Radioresistance is the main cause of radiotherapy failure in patients with esophageal squamous cell carcinoma(ESCC)the underlying mechanism of which remains unclear.The purpose of this study was to analyze the expression of high mobility group boxl(HMGB1)in plasma exosomes of patients with esophageal squamous cell carcinoma before radiotherapy and to explore the relationship between HMGB1 and radiosensitivity in order to find a new target for improving the radiotherapy efficacy of ESCC.Methods:1.The plasma of healthy volunteers and patients with ESCC was separated by low temperature and high-speed centrifuge,and plasma exosomes were extracted by the exosome kit.2.Enzyme linked immunosorbent assay(ELISA)was used to detect HMGB1 expression in plasma exosomes of healthy volunteers and patients with ESCC before radiotherapy.3.The relationship between plasma exosomal HMGB1 expression level and radiosensitivity of patients with ESCC was analyzed statistically.Results:1.Expression of HMGB1 in plasma exosomes of healthy volunteers and patients with ESCCPeripheral blood of 24 healthy volunteers and 21 patients with pathological ESCC were collected,and exosomes were successfully extracted by the kit.ELISA results showed that the average level of plasma exosomal HMGB1 in patients with ESCC was 1821 ±386.6 pg/mL higher than that in healthy volunteers 868.7±119.3 pg/mL(P=0.0169).2.Relationship between plasma exosomal HMGB1 expression level and radiotherapy efficacy in patients with ESCCIn this study,all patients with ESCC received intensity modulated radiotherapy(IMRT)to evaluate their response of radiotherapy.In 12 radiosensitive patients(57.1%)had partial response(PR)or complete response(CR),and 9 radioresistant patients(42.9%)had stable disease(SD)or progressive disease(PD).The mean plasma HMGB1 level in radioresistant patients was 2659±641.9pg/mL higher than that in radiosensitive patients 899.3±369.9pg/mL(P=0.0197).ROC analysis was used to determine the optimal critical value of exosomal HMGB1 for predicting radiotherapy efficacy.The results showed that the optimal cutoff value of plasma exosomal HMGB1 was 1418 and the AUC was 0.80(95%CI 0.6105～0.9895,P=0.0201).Conclusion:1.Compared with healthy volunteers,exosomal HMGB1 was highly expressed in the plasma of patients with ESCC.2.Patients with high expression of plasma exosomal HMGB1 have a significantly increased rate of radioresistance,which may provide a new biomarker for judging the efficacy of radiotherapy in patients with ESCC.Part two Irradiation induced ESCC cell to promote the release of HMGB1 through exosomes,thus affecting the radiosensitivity of recipient cellsObjective:The supernatant of ESCC was collected before and after X-ray irradiation,and exosomes were extracted to observe the differences in physicochemical properties and the changes in HMGB1 expression level.The effects of exosome phagocytosis on the proliferation ability and radiosensitivity of human ESCC receptor cells before and after irradiation were studied.Methods:1.The Western blot assay was used to detect HMGB1 expression levels in 6 types of human ESCC cell lines(KYSE450,TE1,KYSE150,KYSE410,KYSE30 and ECA109),and select high-expression cell lines for subsequent experiments.2.Exosomes were successfully extracted with the kit from the supernatant of KYSE150 and ECA109 cells before and after irradiation,and the existence of exosomes was confirmed and their physicochemical properties were characterized by transmission electron microscopy(TEM),nanoparticle tracking analysis(NTA)and Western blot.3.Fluorescent dye DIL was used to stain exosomes,and laser scanning confocal microscopy was used to observe the successful uptake of tumorigenic exosomes by ESCC,and to observe the co-localization of exosomes and HMGB1,and further observe the position changes of exosomes and HMGB1 after irradiation induction.4.The effects of exosomes on the proliferative ability and radiosensitivity of KYSE150 and ECA109 after irradiation were investigated by cell proliferation assay CCK-8 and clonal formation assay.Results:1.Screening ESCC cell lines with high expression of HMGB1The HMGB1 expression levels were detected in 6 different ESCC cell lines(KYSE450,TE1,KYSE150,KYSE410,KYSE30,and ECA109)by Western blot assay.The detection results showed that the expression level of HMGB1 in KYSE150 and ECA109 was significantly higher than in other cell lines.Therefore,KYSE150,and ECA109 cell lines were selected for follow-up experiments.2.Isolation and identification of exosomesExosomes were successfully extracted with the kit from the supernatant of KYSE150 and ECA109 cells before and after irradiation.The morphological characteristics,size distribution and presence of exosome markers were evaluated by TEM,NTA and Western blot,respectively.TEM showed that the exosomes in both cell lines were round vesicular structures.NTA results showed that the diameter of KYSE150 and ECA109 exosomes before irradiation was 137.5 nm and 125.0 nm,and the concentration was 3.3 × 106 particles/mL and 2.5 × 106 particles/mL,respectively.In contrast,the diameter of KYSE150 and ECA109 exosomes induced by radiation was 145.1 nm and 132.4 nm,and the concentration was 2.5×106 particles/mL and 2.3 ×106 particles/mL,respectively.The exosome types of the two cell lines before and after irradiation were characterized by the expression of related biomarkers CD9,TSG101,HSP70 and the absence of Calnexin.The results further indicated that there were no significant differences in exosome morphology,diameter and related biomarkers before and after irradiation.3.Effects of exosome uptake experiment and irradiation on HMGB1 expressionCompared with before irradiation,HMGB 1 was highly expressed in KYSE150(P=0.0387)and ECA109(P=0.025)cells after irradiation.Intracellular HMGB1 expression was significantly increased after treatment with exosome inhibitor GW4869,which showed that GW4869 weakened the extracellular release of HMGB 1 from irradiation induced KYSE150(P=0.0379)and ECA109(P=0.0186)cell lines.The uptake of exosomes by ESCC cell lines was confirmed by fluorescent dye DIL staining.The expression and co-localization of HMGB 1 and exosomes in KYSE150 and ECA109 cell lines before and after irradiation were observed by laser confocal microscopy.The results of this study specifically showed that HMGB1 expression was increased in the cytoplasm 2 hours after irradiation,suggesting that irradiation could induce HMGB1 migration from the nucleus to the cytoplasm.Colocalization experiments of exosomal HMGB 1 revealed that HMGB 1 is released from the cytoplasm to the extracellular through the exosomes.In addition,the Western blot verified the increased expression of HMGB1 in KYSE150 and ECA109 exosomes induced by irradiation.These results suggest that irradiation may be an important pathway to induce the migration and extracellular release of HMGB1 in exosomes.4.Effects of irradiation-induced exosomes on proliferation ability and radiosensitivity of ESCC cellsThe proliferation ability of KYSE150 and ECA109 cell lines was detected by CCK-8 method.The results showed that the proliferation rate of irradiation-induced exosomes treated group was significantly higher than that of non-irradiation-induced exosomes treated group at 48 h,72 h and 96 h(P<0.05).In addition,the effects of two exosomes on the radiosensitivity of KYSE150 and ECA109 cell lines were detected by cloning assay.According to the number of clones,the cell survival curve was drawn using a single multi-target model,and the radiobiological parameters of each group were statistically analyzed.The results showed that the radioresistance of the irradiation-induced exosome group was significantly higher than that of the non-radiation-induced exosome group(P<0.05).Conclusion:1.By examining the morphology,diameter,and related biomarkers of exosomes exposure before and after irradiation.We found that irradiation exposure did not change the typical characterization of exosomes.2.Irradiation induces the expression of HMGB1 in ESCC cell lines and migrates from the nucleus to the cytoplasm.In addition,irradiation may be an important pathway to induce the release of exosomal HMGB1 from the nucleus to the cytoplasm and then to the extracellular.3.Irradiation of ESCC cell lines induced extracellular release of exosomes,which were absorbed by recipient ESCC cells and enhanced the proliferation and radiation resistance of ESCC cells.Part three Effects of HNMGB1 silencing exosomes on radiosensitivity of ESCC receptor cell linesObjective:By observing the changes of exosome contents after HMGB1 gene silencing,the effects of cell proliferation ability and radiosensitivity of KYSE150 and ECA109 before and after irradiation were observed.At the same time,the relationship between exosome HMGB1 and cell cycle distribution and apoptosis was explored,and relevant pathways of action were sought to further clarify the potential mechanism of regulating the radiosensitivity of human ESCC cells by exosomal HMGB1.This study aimed to provide a new research idea and theoretical basis for enhancing the radiosensitivity of ESCC.Methods:1.Lentivirus was used to construct ESCC cell lines with stable silencing HMGB1 and negative control(NC),and fluorescence microscopy was used to observe the transfection efficiency.The transfection was verified by Real-time quantitative polymerase chain reaction(RT-qPCR)and Western blot.2.The effects of exosomes secreted by HMGB1 silenced cell lines on the proliferative ability and radiosensitivity of ESCC cell lines were detected by cell proliferation assay CCK-8 and clonal formation assay.3.Flow cytometry was used to detect the effects of exosomes after silencing HMGB1 gene expression on the cycle distribution and apoptosis level of ESCC cell lines before and after irradiation.Results:1.Changes in protein expression of ESCC cell cells silenced HMGB1 gene after transfection with lentivirusESCC cell lines KYSE150 and ECA109 with high HMGB1 expression were selected for lentivirus transfection,and low HMGB1 expression cell lines were constructed.The expression levels of HMGB1 mRNA and protein were detected by RT-qPCR and Western blot,respectively.The results showed that the expression level of HMGB1 mRNA in the two cell knockdown groups was significantly decreased compared with that in the NC group(P<0.05).The expression of HMGB1 in the HMGB1 knockdown group was also significantly decreased compared with that in the NC group(P<0.05).In conclusion,the results indicate that HMGB1 ESCC cell lines KYSE150 and ECA109 have been successfully constructed.2.The effect of exosomes secreted after HMGB1 gene silencing on the proliferative ability and radiosensitivity of ESCC cellsThe proliferation ability of KYSE150 and ECA109 cell lines cocultured with different exosomes was detected by CCK-8 assay.The results showed that the proliferation ability of the two groups of ESCC cells cocultured with the secretion of exosomes by knockdown HMGB1 genome cells was significantly decreased compared with the NC group before and after irradiation(P<0.05).These results indicated that co-culture of exosomes secreted by cells after HMGB 1 gene knockdown could reverse the proliferative activity of ESCC cells and enhance the effect of irradiation on cell proliferation.In addition,the radiosensitivity of KYSE150 and ECA109 cell lines in each group was detected by cloning formation assay,and cell survival curves were fitted using a click-multitarget model.The results showed that the values of N,D0,Dq and SF2 of the two co-cultured cells secreted exosomes after HMGB 1 gene knockdown were significantly lower than those in the NC group(P<0.05),suggesting that the co-cultured cells secreted exosomes after HMGB1 gene knockdown improved the tumor microenvironment of radiation resistance.The radiosensitivity of ESCC cells was increased.3.Effect of exosomes secreted by esophageal squamous cell cells after HMGB1 gene silencing on the cycle distribution of recipient ESCC cellsFlow cytometry was used to detect the cycle distribution of ESCC cells in each group.Compared with before radiotherapy,the G2/M phase of cell cycle in all groups was significantly increased after radiotherapy(P<0.05).In addition,the proportion of G2/M phase in the exosome coculture group secreted by the two HMGB1 knockdown cells was significantly significantly increased compared with that of the NC exosomes co-culture group(P<0.05),that is,the degree of arrest in the G2/M phase was increased.These results indicated that the cycle arrest of ESCC cells was in G2/M phase induced by irradiation,and the co-culture group of exosomes secreted by cells after HMGB1 gene knockdown could increase the cell arrest in G2/M phase to a certain extent,thus increasing the radiosensitivity.4.Effect of exosomes secreted by knockdown HMGB1 ESCC cells on apoptosis level of recipient ESCC cellsFlow cytometry was used to detect the apoptosis level of ESCC cells in each group,and it was found that there was no significant difference in the apoptosis rate of the two groups of cells before irradiation(P>0.05).After irradiation,the apoptosis level of the two NC groups co-cultured with secreting exosome was slightly increased compared with that before irradiation,but no statistical difference was found(P>0.05),while the apoptosis rate of the ESCC cells co-cultured with secreting exosomes of knockdown HMGB1 was significantly increased compared with the NC group and the groups before irradiation(P<0.05).These results all indicated that the HMGB 1 contained in exosomes could weaken the ability to induce apoptosis of ESCC cells by irradiation,and the expression of HMGB1 secreted by cells in exosomes was also reduced after HMGB1 gene knockdown,thus improving the ability of irradiated cells to resist apoptosis.Conclusion:1.After HMGB1 gene knockdown,exosomes secreted by ESCC cells were co-cultured with recipient ESCC cells,which could reverse the proliferation ability of recipient ESCC cells and prevent the occurrence of radiation resistance.2.After HMGB1 gene knockdown,the co-culture of exosome secreted by ESCC cells with recipient ESCC cells can promote the cycle arrest of ESCC cells in G2/M phase and the occurrence of cell apoptosis,thus improving the radiosensitivity of ESCC cells.Part four The mechanism of exosomal HMGB1 affecting the radio sensitivity of ESCC cellsObjective:The molecular mechanism of radiation killing tumor cells is mainly through affecting cell cycle and apoptosis related proteins and inducing DNA double-strand breaks.In this study,the effects of exosomal HMGB1 on the expressions of Cyclin B1 and CDK1 and apoptosis related proteins Bax and Bcl2 in ESCC cells were analyzed,and the possible molecular regulatory signaling pathways were explored to provide theoretical basis for improving the radiosensitivity of ESCC cells.Methods:1.Western blot was used to detect the effects of exosomal HMGB1 on the expression levels of cycle and apoptosis related proteins Cyclin B1,CDK1,Bax,and Bcl2 in ESCC cells.2.Western blot was used to detect the expression changes of yH2AX protein at different irradiation times and doses,and the effects of exosomal HMGB1 on this protein,and to explore the possible molecular regulatory signaling pathways.3.The intracellular localization and migration of yH2AX protein in recipient ESCC cells treated with extracellular released exosomes after HMGB 1 gene silencing were detected by laser confocal microscopy before and after irradiation.Results:1.Effects of exosomal HMGB1 on the expression levels of cycle and apoptosis related proteins Cyclin B1,CDK1,Bax,and Bcl2 in ESCC cells1.1 Effects of exosomal HMGB1 on the expression of Cyclin B1 and CDK1 in ESCC cellsAccording to the results of Western blot experiment,the expressions of Cyclin B1 and CDK1 in the two HMGB1 knockdown exosome coculture groups were significantly lower than those in the NC exosome coculture groups.It was also found that the expressions of cyclin related proteins Cyclin B1 and CDK1 in all groups of the two strains were significantly decreased after irradiation compared with those in the unirradiated group,indicating that irradiation down regulated the expressions of cyclin related proteins Cyclin B1 and CDK1 and promoted the cycle arrest of ESCC cells in G2/M phase.However,the G2/M phase arrest was increased and the radiosensitivity was enhanced in the co-culture group of exosomes secreted by HMGB1 gene knockdown.1.2 Effects of exosomal HMGB1 on expression of apoptosis related proteins Bax and Bcl2 in ESCC cellsThe results of Western blot experiments showed that irradiation induced the increase of the expression level of pro-apoptosis related protein Bax in the two strains of all groups of ESCC cells,and reduced the expression level of apoptosis related protein Bcl2,promoting the occurrence of apoptosis and achieving the purpose of killing tumor cells.However,compared with the two NC secreted exosome co-culture groups,the apoptotic proapoptotic protein Bax expression increased and apoptotic inhibitory protein Bcl2 expression decreased in the HMGB1 knockdown exosome co-culture group.These results further indicated that the HMGB1 knockdown exosome co-culture group can enhance the occurrence of cell apoptosis induced by irradiation by up-regulating the expression of apoptosis promoting protein Bax and down regulating the expression of apoptosis inhibiting protein Bcl2.2.The expression of yH2AX protein at different irradiation time and dose and the effect of exosomal HMGB1 on the expression of yH2AX protein.According to the results of Western blot experiments,the expression of yH2AX protein in KYSE150 and ECA109 cell lines reached its peak 2h after irradiation,and was positively correlated with the dose of radiation.Immunofluorescence experiments showed that yH2AX protein was localized and expressed in the nucleus of ESCC cells after irradiation,and the number of fluorescent spots was significantly increased compared with that before irradiation.Compared with the NC secreted exosome group,the expression of yH2AX protein was decreased in the exosomes secreted by the HMGB1 gene knockdown group after irradiation.These results suggest that irradiation induced co-culture of exosomal HMGB1 can promote the recruitment of yH2AX protein in the recipient cancer cells to promote DNA damage repair,while the co-culture of exosome secreted by the ESCC cell line and the recipient ESCC cells after HMGB1 gene knockdown can reverse this phenomenon and reduce DNA damage repair.3.Effects of exosomes secreted by cells after HMGB1 gene knockdown on PI3K/AKT/FOXO3A signaling pathwayThe results of Western blot showed that the protein expressions of PI3K,p-AKT,p-FOXO3A,and yH2AX in the co-culture group of exosomes secreted by the two ESCC cell lines with HMGB1 gene knockdown were also lower than those in the NC exosomes co-culture group.These results indicated that irradiation induced exosomal HMGB1 affected the activity of these related proteins by activating the PI3K/AKT/FOXO3Apathway,and thus affects the proliferative ability and radiosensitivity of ESCC cells.By regulating the expression of yH2AX protein and intracellular translocation,the repair process after DNA damage is initiated.Exosomes secreted by ESCC cells after HMGB1 gene knockdown can reverse this trend,increase radiosensitivity and change the microenvironment of radioresistance.Conclusion:1.Irradiation-induced tumor derived exosomal HMGB 1 affects the cycle distribution and apoptosis level of ESCC cells by regulating the expressions of Cyclin B1 and CDK1 and apoptosis related proteins Bax and Bcl2.2.Irradiation-induced tumor-derived exosomal HMGB1 recruits yH2AX by activating the PI3K/AKT/FOXO3A signaling pathway,thereby initiating DNA repair in ESCC cells exposed to irradiation. |
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