| Part one Pan-cancer analysis of HMGB1 expression and its prognostic and clinicopatholgical significance in patients with esophageal squamous cell carcinomaObjective:The objective was to perform a pan-cancer analysis of the expression of high mobility group box protein 1(HMGB1)and its relationship with the clinicopathological characteristics of esophageal squamous cell carcinoma(ESCC).The study further elucidated the effect of HMGB1 overexpression on the survival of patients with ESCC who were treated with radiotherapy.Methods:1.The pan-cancer expression of HMGB1 was analyzed using bioinformatics.The relationship between HMGB1 and the clinicopathological characteristics of patients was investigated,as well as the effect of HMGB1 on patient survival.2.The study includes patients with a pathological diagnosis of ESCC and treated with radiotherapy.The expression of HMGB1 protein in ESCC tissues was determined immunohistochemically(IHC).3.The relationship between the expression of HMGB1 protein and the clinicopathological characteristics of 39 patients was analyzed with a two-sided Fisher exact test.The relationship between the expression of HMGB1 protein and the overall survival(OS)of patients was analyzed using the Kaplan-Meier method.Results:1.Pan-cancer analyses of HMGB1 expression and intracellular localization analyses of HMGB1 proteinBased on the analysis of the UALCAN database and the HPA database,the HMGB1 was differentially expressed in a variety of tumor tissues compared with the corresponding normal tissues.HMGB1 protein was mainly localized in the nucleus,with only a tiny amount expressed in the cytoplasm.2.Effect of HMGB1 gene expression on patient survivalThe results of the pan-cancer survival analysis in the TISIDB database showed that patients with esophageal carcinoma(EC)and lung adenocarcinoma(LUAD)with higher levels of HMGB1 expression had poor prognosis than patients with low HMGB1 expression(EC,P = 0.028;LUAD,P = 0.033).3.Differential expression of HMGB1 m RNA in tumor and paired non-tumor tissues and the correlation between expression of HMGB1 and clinicopathological characteristics of EC patientsThe analysis of UCSC XENA database showed that the expression of HMGB1 m RNA was significantly higher in EC tissues than in paired non-tumor tissues(P < 0.001).No difference was found in the expression of HMGB1 m RNA between LUAD and paired non-tumor tissues(P > 0.05).The analysis of TCGA database showed that patients with EC aged ≤ 60 years expressed higher levels of HMGB1 m RNA than those above 60 years old(P <0.05).No statistical differences were found in the expression of HMGB1 m RNA in patients based on gender,body mass index,and other characteristics(P > 0.05).4.Correlation between HMGB1 protein expression and clinicopathological characteristics of patients with ESCCA total of 39 patients diagnosed with ESCC were included in this study.We found that the expression of HMGB1 protein was strongly correlated with the TNM stage(P = 0.01).No significant correlation existed between the expression of HMGB1 protein and patients’ gender,age,tumor location,lesion length,gross tumor volume,and T and N stages(P > 0.05).5.Effect of HMGB1 protein expression on ESCC patient survivalThe results of survival analysis showed that among patients with ESCC who were treated with radiotherapy alone,those with negative HMGB1 protein expression had better survival than those with positive expression(P =0.02).Conclusions:1.Compared with corresponding normal tissues,HMGB1 was differentially expressed in a variety of tumor tissues.HMGB1 protein was mainly localized in the nucleus.Patients with EC expressing high levels of HMGB1 had poorer survival than those with low expression.2.The expression of HMGB1 protein correlated with TNM stage in patients with ESCC.Patients with positive expression of HMGB1 protein had poorer long-term survival than those with negative expression.Part two Effect of HMGB1 gene overexpression on radiosensitivity of ESCC cellsObjective:The cell lines used for HMGB1 gene overexpression experiments were screened based on HMGB1 protein expression in ESCC cell lines.The study analyzed the effects of HMGB1 on migration,proliferation,apoptosis,and cell cycle distribution of ESCC cells.The possible regulatory mechanism was also analyzed to provide a theoretical basis for improving the radiosensitivity of ESCC cells.Methods:1.Western blotting analysis was used to detect the expression of HMGB1 protein in ESCC cell lines KYSE180,ECA109,TE1,KYSE150,KYSE510,and KYSE30.The cell lines were screened for HMGB1 gene overexpression.2.HMGB1 gene overexpression and negative control(NC)cells were constructed using lentivirus,and stably transfected cell lines were screened with puromycin.The overexpression of HMGB1 m RNA was detected by reverse transcription-quantitative polymerase chain reaction(RT-q PCR).Western blotting was used to detect the overexpression of HMGB1 protein.3.The altered HMGB1 protein localization in ESCC cells was determined via confocal immunofluorescence microscopy before and after irradiation.4.The effect of HMGB1 gene overexpression on the migration of ESCC cells was detected using a scratch assay.5.The effects of HMGB1 gene overexpression on the proliferation and radiosensitivity of ESCC cells were analyzed using a Cell Counting Kit-8(CCK-8)assay and clone formation assay,respectively.6.The effect of HMGB1 gene overexpression on apoptosis and cell cycle distribution of ESCC cells was detected by flow cytometry.Western blotting was used to analyze the effects of HMGB1 on the expression of apoptosis-related proteins,Bcl-2 and Bax,as well as P16 protein,the cell cycle-related protein.7.Changes in AKT and phospho-AKT(p-AKT)protein expression before and after irradiation were detected by Western blotting analysis.Results:1.Expression of HMGB1 protein in ESCC cell linesThe expression of HMGB1 protein in ESCC cell lines(KYSE180,ECA109,TE1,KYSE150,KYSE510,and KYSE30)was analyzed via Western blotting.The expression of HMGB1 protein was relatively low in KYSE30 and KYSE510 cells,which were selected for experimental analysis of HMGB1 gene overexpression.2.Construction of ESCC cell lines overexpressing HMGB1 geneKYSE30 and KYSE510 cells transfected with HMGB1-overexpressing or null-loaded lentivirus were found in good condition with a broad green fluorescence.The RT-q PCR results showed that the expression of HMGB1 m RNA was significantly increased in the group overexpressing HMGB1 gene compared with the NC group(P < 0.01).The Western blotting results suggested that the expression of HMGB1 protein was significantly higher in the group with HMGB1 gene overexpression compared with the NC group(P< 0.01).3.The localization changes of HMGB1 protein in ESCC cells after irradiationThe results of confocal immunofluorescence assays suggested that HMGB1 protein in ESCC cells was mainly localized in the unirradiated nucleus,and only a tiny amount of green fluorescence was observed in the cytoplasm.Following irradiation,the distribution of green fluorescence of HMGB1 protein was increased in the cytoplasm of KYSE30 and KYSE510 cells,suggesting cytoplasmic translocation of HMGB1 protein in ESCC cells.4.Effect of HMGB1 gene overexpression on ESCC cell migrationThe results of scratch assays showed that the healing area of the cells in the HMGB1 gene overexpression group was larger than that of the NC group at 24 h and 48 h after scratching without irradiation,and the difference was statistically significant(P < 0.05).The healing area of irradiated ESCC cells in the group with HMGB1 gene overexpression was still larger than in the corresponding NC group at 24 h and 48 h,and the difference was statistically significant(P < 0.05),which suggests that HMGB1 gene overexpression enhanced the migration ability of ESCC cells.The healing area of cells in the corresponding groups was significantly reduced at 24 h and 48 h after irradiation compared with that of the unirradiated groups(P < 0.05).5.Effect of HMGB1 gene overexpression on the proliferation and radiosensitivity of ESCC cellsThe results of CCK-8 assays showed that the absorbance of cells overexpressing HMGB1 gene was significantly higher at 450 nm than in NC groups without irradiation after 24 h,48 h,and 72 h of culture(P < 0.05).Under irradiation,the absorbance of ESCC cells overexpressing HMGB1 gene at 450 nm after 24 h,48 h,and 72 h of culture was also significantly higher than in the NC groups(P < 0.05),which suggests that HMGB1 gene overexpression increased the proliferation ability of ESCC cells.Excluding irradiated KYSE30 cells overexpressing the HMGB1 gene at 24 h,where no significant difference was observed compared with the corresponding unirradiated cells(P > 0.05),the absorbance values at 450 nm in irradiated groups at 24 h,48 h,and 72 h were significantly lower than in the corresponding unirradiated groups(P < 0.05),suggesting that irradiation decreased the proliferation ability of ESCC cells.Based on the results of clone formation assays,we found that the cell survival curves of HMGB1 gene overexpression groups were shifted upward compared with those of NC groups,suggesting that cells overexpressing HMGB1 gene were more radioresistant than those of NC groups.The results of clone formation assays suggested a significant increase in the D0,Dq,SF2,and N values of HMGB1 gene-overexpressing groups(P < 0.05),which suggests that the radiosensitivity of cells overexpressing HMGB1 gene were significantly decreased.6.Effect of HMGB1 gene overexpression on the apoptosis of ESCC cellsFlow cytometry results showed no significant difference in apoptotic rates between the HMGB1 gene-overexpressing groups and the NC groups(P > 0.05)in the absence of radiation exposure.Under irradiation,the apoptotic rates of cells overexpressing the HMGB1 gene were significantly decreased compared with those of the NC groups(P < 0.05).The apoptotic rates in the irradiated groups were significantly increased compared with those of the corresponding unirradiated groups(P < 0.05).Western blotting results showed that the expression of apoptosis inhibitory protein,Bcl-2,was increased in the HMGB1 gene-overexpressing groups compared with that of the corresponding NC groups before and after irradiation.The expression of the pro-apoptotic protein,Bax,was decreased in the groups overexpressing HMGB1 gene,resulting in radio-resistance of ESCC cells.The expression of apoptosis inhibitory protein(Bcl-2)decreased,and the expression of pro-apoptotic protein,Bax,increased after irradiation higher than in the unirradiated groups,indicating that irradiation increased apoptosis.7.Effect of HMGB1 gene overexpression on cell cycle distribution of ESCC cellsThe cell cycle distribution was analyzed by flow cytometry.The proportion of G0/G1 phase cells overexpressing HMGB1 gene without irradiation was decreased when compared with that of NC(P < 0.05).Under irradiation,the proportion of G0/G1 phase cells overexpressing HMGB1 gene was still decreased when compared with that of NC(P < 0.05).The proportion of cells in G2/M phase overexpressing HMGB1 gene was significantly increased compared with that of NC(P < 0.05).Compared with the unirradiated groups,the proportion of G0/G1 phase cells was significantly increased(P < 0.01),and the proportion of cells in the G2/M phase was significantly decreased(P < 0.01)in the corresponding irradiated groups.The results of Western blotting analysis showed that the expression of the cell cycle-related protein(P16)was increased in irradiated groups compared with that of unirradiated groups,suggesting that HMGB1 gene overexpression attenuated G0/G1 phase arrest.The phenomenon of G0/G1 phase arrest induced by irradiation was accompanied by increased P16 protein expression.8.Effect of HMGB1 gene overexpression on p-AKT protein expression in ESCC cellsWestern blotting results showed no significant change in the expression of AKT protein in ESCC cells before and after irradiation.The expression of p-AKT protein was significantly increased in irradiated ESCC cells compared with unirradiated cells.The expression of p-AKT protein was increased higher in ESCC cells overexpressing HMGB1 gene than in the NC group,suggesting that HMGB1 affected the radiosensitivity of ESCC cells by regulating p-AKT protein expression.Conclusions:1.HMGB1 protein is mainly localized in the nucleus of ESCC cells.HMGB1 gene overexpression promoted ESCC cell migration and cell proliferation,which persisted after irradiation.HMGB1 gene overexpression decreased apoptosis in ESCC cells after irradiation.2.HMGB1 gene overexpression attenuated G0/G1 phase arrest in ESCC.HMGB1 gene overexpression reduced the radiosensitivity of ESCC cells by up-regulating expression of p-AKT protein.Part three Clustering analyses of HMGB1-HNF1 A co-regulatory genes in ECObjective:This study aimed to screen the differential gene expression of HMGB1 and hepatocyte nuclear factor 1 alpha(HNF1A)in EC and further analyze the biological processes and molecular functions of HMGB1-HNF1 A co-regulatory genes involved in the development of EC via gene ontology(GO)enrichment analyses and Kyoto encyclopedia of genes and genomes(KEGG)-based pathway analysis.Methods:1.The bioinformatics data was used to analyze the relationship between the survival of patients with EC and the expression of HMGB1 and HNF1 A m RNA.2.The bioinformatics data was used to identify the key genes of the HMGB1-HNF1 A Group in EC.3.GO and KEGG pathway analyses were performed based on the key genes obtained from the HMGB1-HNF1 A Group.4.The confocal immunofluorescence assay was used to detect the intracellular localization of HMGB1 protein and HNF1 A protein with or without irradiation,and determine the co-localization of HMGB1 and HNF1 A proteins in ESCC cells.5.Effect of HNF1 A gene overexpression on the proliferation and clonogenic ability of ESCC cells1)The expression of HNF1 A protein in ESCC cell lines(KYSE180,ECA109,TE1,KYSE150,KYSE510,and KYSE30)was determined via Western blotting to screen the cell lines for experimental analysis of HNF1 A gene overexpression.2)The cells of HNF1 A gene overexpression and NC were both constructed using lentivirus.Stably transfected cells were obtained by puromycin selection.RT-q PCR and Western blotting assays were used to analyze the overexpression of HNF1 A m RNA and HNF1 A protein,respectively.3)The effect of HNF1 A gene overexpression on ESCC cell proliferation was analyzed by CCK-8 assay.4)The effect of HNF1 A gene overexpression on the clone-forming ability of ESCC cells was assayed.Results:1.Effect of HMGB1 and HNF1 A m RNA expression on the prognosis of patients with ECThe results based on the analysis of Oncolnc database showed that compared with EC patients with low expression of HMGB1 m RNA,those with high expression of HMGB1 m RNA had poor outcomes(P = 0.032).The expression of HNF1 A m RNA did not affect the survival of patients with EC(P > 0.05).Among patients with high levels of HNF1 A m RNA expression,the survival was better in those with low expression of HMGB1 m RNA than in patients with high expression of HMGB1 m RNA(P = 0.049).No difference in survival was detected between low and high expression of HMGB1 m RNA in patients with low expression of HNF1 A m RNA(P > 0.05).2.The identification of key genes of HMGB1-HNF1 A Group in ECA total of 832 HMGB1 and HNF1 A differential genes in EC were screened using bioinformatics data in the Linked Omics database,and 842 genes of EC disease targets with “correlation scores” > 10 were screened in the Gene Cards database.The intersectional genes of the two gene sets(“HMGB1-HNF1 A Group” and “EC disease targets”),designated“HMGB1-HNF1 A Group Key Genes”,were identified by plotting the Venn diagram.The results of protein-protein interaction(PPI)analysis in the String database suggested that the proteins of the 36 key genes were closely linked to each other,and the proteins formed a closely connected PPI network.3.GO and KEGG signaling pathway enrichment analyses of HMGB1-HNF1 A Group Key GenesThe results of GO analysis revealed three components: biological process(BP),cellular component(CC),and molecular function(MF),enriched with523,30,and 22 corresponding entries,respectively.KEGG signaling pathway was enriched in 19 entries.The results of GO and KEGG enrichment analysis suggested that HMGB1-HNF1 A Group Key Genes regulated the oxygen level,DNA synthesis and recombination,position maintenance,and other GO-BP terms in EC development.The GO-CC component involved the endoplasmic reticulum-Golgi intermediate region and others.The GO-MF component included protease and adhesin binding,and others.The KEGG analysis involved the PI3K-Akt and Erb B signaling pathways,and others.4.Co-localization of HMGB1 and HNF1 A proteins in ESCC cells before and after irradiationThe results of the confocal immunofluorescence assays suggested that both HMGB1 and HNF1 A proteins were mainly localized in the nucleus without irradiation.Following irradiation,the cytoplasmic distribution of both the green fluorescence of HMGB1 protein and the red fluorescence of HNF1 A protein was increased.HMGB1 and HNF1 A proteins showed co-localization.5.The effect of HNF1 A gene expression on the proliferation and clonogenic abilities of ESCC cells1)The expression of HNF1 A protein in ESCC cell linesThe expression of HNF1 A protein in ESCC cell lines(KYSE150,KYSE510,KYSE30,ECA109,KYSE180,and TE1)was detected by Western blotting analysis.The results suggested that the expression of HNF1 A protein was relatively low in KYSE150 and TE1 cells.KYSE150 and TE1 cells were selected for HNF1 A gene overexpression experiments.2)Construction of ESCC cell lines overexpressing HNF1 A geneKYSE150 and TE1 cells transfected with HNF1 A overexpressing or null lentivirus were in good condition and showed extensive green fluorescence under fluorescence microscopy.The results of the RT-q PCR assay suggested that the expression of HNF1 A m RNA was significantly increased in the group with HNF1 A gene overexpression compared with the NC group(P < 0.01).Further,the Western blotting results suggested that the expression of HNF1 A protein was significantly higher in the HNF1 A gene overexpression group than that in the NC group(P < 0.05).3)The effect of HNF1 A gene overexpression on ESCC cell proliferationThe results of CCK-8 assays showed that the absorbance values at 450 nm in the groups with HNF1 A gene overexpression were significantly higher than in NC groups after culturing for 24 h,48 h,and 72 h(P < 0.05),which suggests that HNF1 A gene overexpression improved the proliferation ability of ESCC cells.4)The effect of HNF1 A gene overexpression on the clonogenic ability of ESCC cellsThe results of clone formation assays showed that the clonogenic ability of cells with HNF1 A gene overexpression was significantly increased compared with that of the NC group(P < 0.01),which suggests that HNF1 A gene overexpression improved the clonogenic ability of ESCC cells.Conclusions:1.The HMGB1-HNF1 A Group Key Genes are involved in the regulation of specific biological processes during the development of EC.The combination of HMGB1 and HNF1 A affects the survival of EC patients.2.HMGB1 and HNF1 A proteins were co-localized in ESCC cells before and after irradiation.The effects of HNF1 A and HMGB1 genes on the proliferation of ESCC cells were consistent.The overexpression of the two genes promoted the proliferation of ESCC cells.Part four Altered expression of HMGB1 and HNF1 A protein in ESCC tissues before and after neoadjuvant chemoradiotherapy and the effects of neoadjuvant chemoradiotherapy on patient survivalObjective:We retrospectively investigated the changes in the expression of HMGB1 and HNF1A proteins in ESCC tissues before and after neoadjuvant chemoradiotherapy,explored the relationship between them and clinicopathological characteristics of ESCC patients,and analyzed the effects of neoadjuvant chemoradiotherapy on survival in patients with stage II-III ESCC to provide reliable predictive indices and reference standards for the accurate assessment of patient survival after neoadjuvant chemoradiotherapy.Methods:1.IHC was used to determine the expression of HMGB1 and HNF1 A proteins in ESCC tissues before and after neoadjuvant therapy.The differences in HMGB1 and HNF1 A protein expression before and after radiotherapy and their correlations were analyzed.2.We used a two-sided Fisher exact test to analyze the relationship between altered HMGB1 or HNF1 A protein expression in ESCC tissues after radiotherapy and the clinicopathological characteristics of patients.The independent sample t-test was used to analyze the differences in peripheral blood indices of patients with differential expression of HMGB1 and HNF1 A proteins.3.The Kaplan-Meier method was used to analyze the effect of altered HMGB1 and HNF1 A protein expression in ESCC tissues after radiotherapy on patient survival.4.Patients diagnosed with stage II-III(AJCC 7th)ESCC between 2010 and 2015 were screened in the “Surveillance,Epidemiology and End Results(SEER)” database and included in the study.5.Subgroup analyses were performed according to different T stages.Univariate and multivariate Cox regression analyses were used to determine the factors affecting patient prognosis,and disease-specific survival(DSS)was assessed via Kaplan-Meier method.6.Subgroup analyses were performed according to different N stages.The factors affecting patient prognoses were determined via univariate and multivariate Cox regression analyses.DSS was assessed by the Kaplan-Meier method.Results:1.Changes in the expression of HMGB1 and HNF1 A protein in ESCC tissues before and after neoadjuvant therapy and correlation analysisThe HMGB1 and HNF1 A protein expression in ESCC tissues of 15 patients was determined by IHC.The results suggest that both HMGB1 and HNF1 A protein expression levels were significantly increased in ESCC tissues after radiotherapy.The IHC scores(mean ± SD)of HMGB1 protein were3.533 ± 1.06 before radiotherapy and 4.467 ± 1.552 after radiotherapy.The IHC scores of HNF1 A protein were 3.133 ± 1.187 and 4.2 ± 1.521,respectively.Paired t-test results suggested that the expression of HMGB1 and HNF1 A protein in ESCC tissues was significantly altered after radiotherapy,with statistically significant differences(HMGB1: t =-2.432,P = 0.029;HNF1A: t =-2.359,P = 0.033).A significant positive correlation existed between HMGB1 and HNF1 A protein levels before radiotherapy(r = 0.542,P= 0.037).Following radiotherapy,no significant correlation was observed between HMGB1 and HNF1 A protein levels(r = 0.089,P = 0.751).2.Relationship between clinicopathological characteristics of patients and altered HMGB1 or HNF1 A protein expression in ESCC tissues after radiotherapy and statistical analysis of peripheral blood indices in patients with different levels of HMGB1 or HNF1 A protein expressionNo significant correlation was found between the clinicopathological characteristics of patients and the altered HMGB1 or HNF1 A protein expression in ESCC tissues after radiotherapy(P > 0.05).Notably,patients with elevated HMGB1 protein expression in ESCC tissues after treatment had higher prognostic nutrition index(PNI,t = 2.428,P = 0.03),lower monocyte-to-lymphocyte ratio(MLR,t =-2.215,P = 0.045),lower systemic immune-inflammation index(SII,t =-2.839,P = 0.014)and lower systemic inflammation response index(SIRI,t =-2.503,P = 0.026)than those with unelevated HMGB1 protein expression.No significant differences were found between peripheral blood indices of patients with different levels of HNF1 A protein expression(P > 0.05).3.Effects of altered expression of HMGB1 and HNF1 A proteins in ESCC tissues after radiotherapy on patient survivalBefore radiotherapy,the IHC scores of HMGB1 protein were(mean ± SD)3.67 ± 1.37 and 3.44 ± 0.88 in the groups with elevated and unelevated HMGB1 protein expression,respectively,and the IHC scores of HNF1 A protein were 3.29 ± 0.95 and 3 ± 1.41,respectively,which suggested that patients with elevated HMGB1 or HNF1 A protein expression had relatively low pre-treatment HMGB1 or HNF1 A protein expression in the corresponding tumor tissues.The results of survival analysis showed that the altered HMGB1 protein expression after radiotherapy did not significantly affect patient survival(P > 0.05).However,patients with elevated HMGB1 protein expression after treatment(patients with relatively low HMGB1 protein expression before treatment)had better chances of survival.4.Selection of subjects in the SEER databaseA total of 1160 patients with stage II-III(AJCC 7th)ESCC were included in this study.Among the included subjects,289 received neoadjuvant radiotherapy and 871 did not.5.Univariate and multivariate analyses and survival analyses of patients with different T stagesWe found that neoadjuvant radiotherapy correlated with a survival benefit for patients at stage T2-T4(P < 0.05).However,neoadjuvant radiotherapy was not found to improve the prognosis of patients at T1 stage(P > 0.05).The results of survival analyses were consistent with the results of univariate and multivariate analyses,which also suggested that neoadjuvant radiotherapy correlated with a significant survival benefit for patients at T2-T4 stage(P <0.01),and did not improve survival in patients at T1 stage(P = 0.34).6.Univariate and multivariate analyses and survival of patients at different N stagesPatients at N0,N1,and N2-3 stages who received neoadjuvant radiotherapy showed a significant survival benefit compared with those who did not receive neoadjuvant radiotherapy(P < 0.05).The results of survival analyses were consistent with the results of univariate and multivariate analyses,suggesting that the effect of neoadjuvant radiotherapy on patient survival was not different among patients at different N stages.Conclusions:1.There was a significant positive correlation between HMGB1 and HNF1 A protein expression in ESCC tissues before neoadjuvant radiotherapy.The expression of both proteins was significantly elevated after neoadjuvant radiotherapy.Further,significant differences were found between different values of peripheral blood indices(PNI,MLR,SII,and SIRI)in patients with different levels of alter HMGB1 protein expression after neoadjuvant radiotherapy.2.Neoadjuvant radiotherapy correlated with superior survival in patients with stage II-III ESCC at stages T2-T4,while the survival benefit was not significant in patients at stage T1.Patients at different N stages experienced a survival benefit due to neoadjuvant radiotherapy. |
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