| Part one Expression of HNF1A in esophageal cancer and its correlation with clinicopathologic characteristics and prognosisObjective:Definitive chemoradiotherapy is an important treatment modality for patients with unresectable esophageal squamous cell carcinoma(ESCC).However,some patients have poor prognosis after radiotherapy due to radio-resistance.This study analyzed the expression of HNF1A in esophageal cancer tissues and explored its relationship with clinicopathologic characteristics and prognosis,providing new ideas for improving radiosensitivity and targeted therapy.Methods:Based on bioinformatics,the expression level of HNF1A gene in esophageal cancer tissue specimens was comprehensively analyzed to explore the correlation between HNF1A gene and clinicopathological characteristics,prognosis and survival.Results:1.Sublocalization of HNF1A protein expression in cellsIn human normal esophageal squamous epithelial tissue,HNF1A protein was located in the cell nucleus and cytoplasm.In human colorectal adenocarcinoma cell line(CACO-2),human hepatoma cell line(Hep G2)and human osteosarcoma cell line(U-2 OS),HNF1A protein was mainly expressed in the cell nucleus and partly in cytoplasm.2.The relationship between the HNF1A gene and protein expression level and clinicopathological characteristics of patients with ESCCHNF1A gene and protein expression level was associated with age(χ2=8.919,P=0.003),weight(χ2=57.744,P=0.000),BMI(χ2=29.539,P=0.000),tumor location(χ2=43.539,P=0.000),N stage(χ2=8.526,P=0.004)and pathological grade(χ2=7.230,P=0.007),and the differences were statistically significant.3.Expression of HNF1A gene in esophageal cancer tissues and its relationship with prognosis of patients with ESCCHNF1A gene was highly expressed in esophageal cancer tissues,and the results showed that the expression level of HNF1A gene had no significant effect on the overall survival(OS)and progression-free survival(PFS)of patients with esophageal cancer(P>0.05).The PFS was significantly lower in high expression of HNF1A gene patients with ESCC than that in low expression patients(P=0.039),while OS of the two groups with ESCC were no significantly differences(P=0.1).Conclusion:1.HNF1A protein was mainly expressed in the cell nucleus and little pert in cytoplasm.2.The expression of HNF1A gene was closely related with age,weight,BMI,tumor location,N stage and pathological grade of esophageal cancer patients.3.The expression of HNF1A gene was highly expressed in esophageal carcinoma tissues,and patients with high expression of HNF1A gene had a poor PFS.Part two Overexpression of HNF1A gene and protein effect on radiosensitivity of ESCC cellsObjective:To observed the effect of overexpression of HNF1A gene and protein on the proliferation activity,migration and invasion ability,cell cycle distribution,apoptosis and radiosensitivity of ESCC cells before and after irradiation(IR),and to explore the molecular mechanism of HNF1A regulating the radiosensitivity.In order to provide a theoretical basis for improving the radiosensitivity of ESCC cells by targeting HNF1A.Methods:1.The expression levels of HNF1A protein in ECA109,TE1,TE13,KYSE150 and KYSE30 ESCC cells and human normal esophageal squamous epithelial cells(HEEC)were detected by Western blotting assay,and the cell lines with low expression of HNF1A were screened.2.TE1 and KYSE150 cell lines were constructed with stable overexpression of HNF1A and negative control(NC),transfection efficiency was observed by fluorescence microscope.3.The effects of HNF1A overexpression on proliferation activity and radiosensitivity of ESCC cells were observed by CCK-8 assay and clone formation assay,respectively.4.The effects of HNF1A overexpression on the migration and invasion of ESCC cells were detected by scratch assay and transwell assay,respectively.5.Flow cytometry was used to check the effect of overexpression of HNF1A on apoptosis and cell cycle distribution of ESCC cells before and after IR.6.Western blotting assay was used to detect the effect of HNF1A on cell invasion related protein MMP2/MMP9,EMT-related protein E-cadherin/N-cadherin/Vimentin,and apoptosis related protein BCL2/BAX and cell cycles-related Cyclin D1/CDK4 expression levels.Results:1.Expression of HNF1A protein in different ESCC cell linesIn HEEC cells and 5 different ESCC cell lines ECA109,TE1,TE13,KYSE150 and KYSE30,Western blotting results showed that the expression of HNF1A protein in 5 ESCC cell lines was significantly higher than those of HEEC cells.TE1 and KYSE150 ESCC cell lines with relatively low HNF1A protein expression were selected for subsequent experiments.2.Overexpression of HNF1A gene and protein after lentivirus transfection in ESCC cellsTE1 and KYSE150 cells line were transfected with lentivirus to construct HNF1A overexpressing cell lines and negative control cell lines.The expression levels of HNF1A mRNA and protein were detected by RT-qPCR and western blotting assay,respectively.This result showed that compared with NC group of two cell lines,the HNF1A mRNA and protein expression in overexpression HNF1A group was significantly increased(P<0.01).The above results indicated that TE1 and KYSE150 cell lines with overexpressing HNF1A had been successfully constructed.3.Effect of HNF1A gene and protein overexpression on proliferation and radiosensitivity of ESCC cellsCCK-8 assay was used to check the proliferation ability of ESCC cells.The results showed that the proliferation ability of HNF1A overexpression group in TE1 and KYSE150 cells was significantly increased compared with NC group before and after IR(P<0.05).It was indicated that overexpression of HNF1A could promote proliferation and reduce the inhibitory effect of IR on cell proliferation of ESCC cells.The radiosensitivity of ESCC cells in each group was detected by clone formation assay.A single-hit-multi-target model was used to fit the cell survival curves.The results showed that the radiobiological parameters(D0,Dq and SF2)of HNF1A overexpression group were significantly higher than those of NC group(P<0.05).It was suggested that the overexpression of HNF1A reduced radiosensitivity of ESCC cells,that is,radio-resistance occurs.4.Effect of HNF1A gene and protein overexpression on migration and invasion in ESCC cellsThe migration ability of ESCC cells was explored by scratch assay.We observed that the scratch healing area of HNF1A overexpression group was significantly increased compared with NC group before and after IR(P<0.05).At the same time,the scratch healing ability of each group was weakened after IR(P<0.05).However,the degree of attenuation in overexpression of HNF1A group was lower than those of NC group.These results indicated that radiation reduced the migration ability of ESCC cells,while the degree of reduction was lower in HNF1A overexpression group.5.Effect of HNF1A gene and protein overexpression on apoptosis of ESCC cellsFlow cytometry was used to detect the occurrence of apoptosis in each group.The results showed that the apoptosis rate of HNF1A overexpression group was significantly lower than that of NC group before IR(P<0.05).After IR,apoptosis rate of all groups was significantly increased compared with corresponding unirradiated group(P<0.05),but apoptosis rate of HNF1A overexpression group were significantly lower than those of NC group(P<0.05).These results showed that HNF1A overexpression could counteract the radiation-induced apoptosis of ESCC cells.6.Effect of HNF1A gene and protein overexpression on cycle distribution in ESCC cellsFlow cytometry was used to detect cell cycle distribution in all groups.Compared with the unirradiated group,the proportion of G0/G1 phase in all groups was significantly increased after IR(P<0.05).The proportion of G0/G1 phase in HNF1A overexpression group was significantly decreased than those in NC group(P<0.05),that is,lesser degree of arrest in G0/G1 phase.Which presented that radiation induced ESCC cells arrest in G0/G1 phase,and overexpression of HNF1A could weaken cycle arrest phenomenon.7.Effect of HNF1A gene and protein overexpression on the expression levels of invasion related protein MMP2/MMP9,EMT-related protein E-cadherin/N-cadherin/Vimentin,apoptosis related protein BAX/BCL2 and cell cycle related protein Cyclin D1/CDK4 in ESCC cells1)Effects of HNF1A gene and protein overexpression on the expression of invasion related proteins MMP2 and MMP9 in ESCC cellsWestern blotting results showed that expression levels of MMP2 and MMP9 proteins in HNF1A overexpression group were significantly higher than those in NC group before and after IR.These results suggested that HNF1A could promote migration and invasion by upregulating MMP2 and MMP9 proteins of ESCC cells.2)Effect of HNF1A gene and protein overexpression on expression of EMT-related proteins E-cadherin,N-cadherin and Vimentin in ESCC cellsWestern blotting results showed that expression levels of N-cadherin and Vimentin in HNF1A overexpression group were significantly increased compared with NC group,while expression level of E-cadherin was significantly decreased.It was suggested that HNF1A activated EMT to promote migration and invasion ability of ESCC cells.3)Effect of HNF1A gene and protein overexpression on expression of apoptosis-related proteins BAX and BCL2 in ESCC cellsWestern blotting results showed that radiation induced the increasing expression of BAX protein and weakened the expression of BCL2 protein,which promoted the occurrence of apoptosis,thereby achieving the purpose of killing tumor cells.However,compared with the two cell lines in NC groups,the expression of BAX protein was decreased and the expression of BCL2 protein was increased in the overexpressed HNF1A group.The results showed that overexpression of HNF1A counteracted radiation-induced apoptosis by down-regulating expression of BAX protein and up-regulating the expression of BCL2 protein.4)Effect of HNF1A gene and protein overexpression on the expression of Cyclin D1 and CDK4 in ESCC cellsWestern blotting results showed that expression of Cyclin D1 and CDK4 in HNF1A overexpression group was significantly lower than that in NC group,and expression of Cyclin D1 and CDK4 proteins significantly decreased in IR group compared with unirradiated group.These results indicated that radiation down-regulated expression of Cyclin D1 and CDK4 proteins to promote ESCC cell cycle arrest in G0/G1 phase,but HNF1A overexpression cells line could reduce the occurrence of this phenomenon.Conclusion:1.Overexpression of HNF1A gene and protein could relieve the occurrence of radiation-induced cycle arrest in G0/G1 phase and apoptosis of ESCC cells lines,thus promoting cell proliferation and reducing the radiosensitivity of ESCC cells.2.Overexpression of HNF1A gene and protein activated EMT procession and upregulated the expression level of MMP2/MMP9 proteins,and to promote the migration and invasion of ESCC cells lines.3.HNF1A affected cell cycle distribution and apoptosis of ESCC cells by regulating the expression of apoptosis-related proteins BAX/BCL2 and cell cycle related protein Cyclin D1/CDK4.Part three HNF1A regulated yH2AX protein expression by activating PI3K/AKT signal pathway and influenced radiosensitivity of ESCC cellsObjective:The molecular mechanism of radiation killing tumor cells is mainly to induce DNA strand breaks,including DNA single-strand breaks and double-strand breaks(DSBs),resulting in the phosphorylation of histone H2AX to form yH2AX.It is not only a marker of DNA damage,but also recruits more repair factors of DNA damage,which has become a hot spot in the study of radiosensitivity.This study explored the relationship between HNF1A and yH2AX proteins in ESCC cells,analyzed their possible molecular regulatory pathways,and provided a theoretical basis for improving the radiosensitivity of ESCC cells.Methods:1.The correlation between HNF1A and H2AX gene,gene expression and cell sublocation in esophageal cancer tissues were comprehensively analyzed based on bioinformatics.2.Western blotting assay was used to detect the relationship between HNF1A and yH2AX proteins in ESCC cells and irradiated time and irradiated dose,and to analyze their possible molecular regulatory pathways.3.Immunofluorescence(IF)assay was used to detect cell sublocalization of HNF1A and yH2AX proteins in ESCC cells before and after IR.Results:1.Correlation between HNF1A and H2AX gene expression in esophageal carcinoma tissues and cell sublocalization.In simple TCGA database of esophageal cancer(182 cases in tumor tissues and 13 cases in adjacent tissues),H2AX mRNA expression was significantly increased in tumor tissues compared with adjacent tissues(P<0.05),the difference was statistically significant.In addition,in TCGA and GTEx databases(182 tumor tissues,13 paracancer tissues,and 273 normal tissues),H2AX mRNA expression was significantly increased in tumor tissues compared with paracancer and normal tissues(P<0.05),the difference was statistically significant.The online analysis of esophageal cancer unpaired samples through the Xiantao Academic website showed that expression of H2AX mRNA in tumor tissue was also significantly higher than that in adjacent tissue(P<0.001),and the difference was statistically significant.In paired samples from 8 patients with esophageal cancer,H2AX mRNA expression in tumor tissues was also significantly increased compared with that in adjacent tissues(P<0.001),the difference was statistically significant.All the above results suggested that H2AX mRNA was highly expressed in esophageal cancer tissues.HPA database was used to observe that subcellular localization of H2AX protein was in the nucleus of human normal esophageal squamous epithelium and other tumor cell lines.GEPIA 2.0 database analysis showed a weak positive correlation between HNF1A and H2AX mRNA expression(R=0.14,P=0.043),which was statistically significant.2.The relationship between expression of HNF1A and yH2AX in ESCC and related to irradiated time and irradiated dose.Western blotting results showed that expression levels of HNF1A and yH2AX protein in ESCC cells reached the peak at 2h after IR,and were positively correlated with irradiated dose.And IF assay results showed that HNF1A and yH2AX protein were co-localized and expressed in nucleus of ESCC after IR.HNF1A protein was translocated from nucleus to cytoplasm.3.HNF1A activated PI3K/AKT signaling pathway to regulate expression of yH2AX proteinIF assay showed that number of yH2AX foci in HNF1A overexpression group was significantly increased compared with NC group after IR.Western blotting results showed that expression levels of PI3K,p-AKT and yH2AX proteins in HNF1A overexpression group were also significantly increased compared with NC group.These results indicated that HNF1A regulated expression of yH2AX protein by activating PI3K/AKT signal pathway,thus initiating DNA damage repair process.Conclusion:1.H2AX gene expression was high in esophageal cancer tissues,H2AX protein was located in cell nucleus,and HNF1A was positively correlated with H2AX gene.2.The protein expression of HNF1A and yH2AX in ESCC cells reached the peak at 2h after IR,and was positively correlated with irradiated dose.3.Overexpression of HNF1A regulated expression of yH2AX protein by activating PI3K/AKT signal pathway,result in initiating DNA damage repair procession of ESCC cells.Part four Exosome releasing HSPD1 protein assists HNF1A in regulating the invasion ability of ESCC cells via activating EMTObjective:Heat Shock Protein 60(HSP60),also known as HSPD1,has molecular chaperone activity,assists in protein translation,synthesis and folding,and participates in cell immune response,antigen presenting,cell growth,cell apoptosis and cell invasion biological functions.It plays an important role in the development of normal and tumor tissues.The purpose of this study was to explore interaction between HSPD1 and HNF1A protein on the invasion ability of ESCC cells,and to find out the possible molecular regulation mechanism,so as to provide new ideas for targeted therapy of ESCC.Methods:1.Immunoprecipitation(IP)assay and liquid chromatography-tandem mass spectrometry(LC-MS/MS)were used to analyze HNF1A correlated protein in ESCC cells,and bioinformatics was used to screen them.2.The interaction between HSPD1 and HNF1A protein in ESCC was verified by coimmunoprecipitation(CO-IP)assay and IF assay.3.The expression of HSPD1 in esophageal cancer tissues was analyzed based on bioinformatics to explore its effect on prognosis of patients with esophageal cancer.4.Plasmids were constructed to knockdown HSPD1 and NC group.Lipofectamine 2000 was used to transfect ESCC cells,and knockdown efficiency was verified by RT-qPCR and western blotting method.5.The invasive ability of ESCC cells was detected by transwell assay.6.Western blotting assay was performed to check changes of HSPD1 protein in ESCC cells after intervention with exosome inhibitor GW4869.7.Western blotting assay was used to detect changes of HSPD1 protein after IR,the regulation of HNF1A and HSPD1 protein,expression of EMT-related proteins and expression of HSPD1 protein in cell and exosome.Results:1.HNF1A protein correlated proteins in ESCC cellsIP and LC-MS/MS assays results revealed that HNF1A may interact with HSPD1,TCP1 and hnRNPA1/A3 proteins in TE1 cells.Further bioinformatics analysis showed that HNF1A gene was significantly correlated with HSPD1 gene in esophageal cancer tissues(P<0.001).CO-IP assay confirmed the interaction between HNF1A protein and HSPD1 protein.IF assay results also indicated that HNF1A protein and HSPD1 protein were located in cell nucleus and cytoplasm before IR,while HNF1A and HSPD1 proteins co-located in cell nucleus and cytoplasm after IR.2.Relationship between HNF1A protein and HSPD1 protein and irradiated time,and their regulatory relationshipWestern blotting results showed that expression of HSPD1 protein in ESCC cells was the lowest at 4h after IR.Further analysis showed that expression of HSPD1 protein in HNF1A overexpression group was significantly decreased compared with NC group(P<0.01).After IR,expression of HSPD1 protein in HNF1A overexpression group was significantly increased compared with NC group(P<0.01).These results suggested that there was no obvious regulatory relationship between HNF1A and HSPD1.3.Expression of HSPD1 gene in esophageal cancer tissues and its effect on prognosis of patients with esophageal cancer.Bioinformatics analysis showed that HSPD1 gene was significantly increased in esophageal carcinoma tissues compared with normal tissues(P<0.05),and patients with high expression of HSPD1 gene had lower OS than those with low expression patients(P<0.05).4.Construct and screen effective HSPD1 knockdown plasmids and detect the effect of HSPD1 knockdown on invasion ability of ESCC cellsThe results of RT-qPCR and western blotting showed that knockdown efficiency of HSPD1 gene and protein in ShRNAl and ShRNA2 groups was higher than that in ShRNA3 group.Further transwell assay presented that cells number crossing membrane in HSPD1 ShRNAl and ShRNA2 group were decreased compared with Sh-NC group(P<0.05),the difference was statistically significant.In addition,western blotting results also showed that expression of E-cadherin protein in ShRNAl group was increased compared with Sh-NC group before and after IR,while expression of N-cadherin and Vimentin protein was decreased.These results suggested that HSPD1 reduced invasion ability of ESCC cells by inhibiting EMT procession.5.Effect of HSPD1 gene and protein knockdown on invasion ability of HNF1A overexpression ESCC cell linesHSPD1 gene was knocked down in overexpressed HNF1A ESCC cells,and invasion ability of ESCC cells was detected by transwell assay.The results showed that cells number crossing membrane in HNFIA-Sh-HSPD1 group was significantly lower than HNF1A-Sh-NC group and HNF1A overexpression group(P<0.05).In addition,Western blotting results also showed that expression of E-cadherin protein in HNFIA-Sh-HSPD1 group was significantly increased compared with HNF1 A-Sh-NC group and HNF1A overexpression group,while expression of N-cadherin and Vimentin protein was decreased.These results suggested that HSPD1 knockdown could inhibit that overexpression of HNF1A activated EMT process,result in enhancing invasion ability of ESCC cells.6.Effect of HNF1A overexpression gene and protein on radiation-induced cell releasing HSPD1 protein from exosomes to extracellularIntracellular total protein was extracted from ESCC cells after intervention with exosome inhibitor GW4869.Western blotting results revealed that expression of HSPD1 in GW4869 group was significantly increased than that in DMSO group(P<0.05).Exosomes secreted by TE1 cells were extracted with exosome extraction kit.Western blotting results showed that no obvious changes were observed expression of HSPD1 protein in HNF1A overexpression group and NC group without IR.Furthermore,expression of HSPD1 protein in NC group was significantly reduced compared with HNF1A overexpression group after IR(P<0.05).These results indicated that overexpression of HNF1A could inhibit that radiation-induced cells released HSPD1 protein into tumor microenvironment via exosome in ESCC cells.Conclusion:1.HNF1A protein binds to HSPD1 protein,but there was no finding obviously regulatory relationship between them.2.HSPD1 gene was highly expressed in esophageal cancer tissues,and high expression of HSPD1 patients with esophageal cancer had a poor OS than those with low expression patients.3.Knockdown of HSPD1 gene and protein expression could weaken that overexpression of HNF1A activated EMT,and promoted invasion ability of ESCC cells.4.Overexpression of HNF1A gene and protein could counteract that radiation-induced cells released HSPD1 protein into tumor microenvironment via exosome in ESCC cells. |
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