| Esophageal cancer(EC)is an aggressive gastrointestinal malignant tumor,which is divided into two categories according to the histological classification:Esophageal Squamous Cell carcinoma(ESCC)and Esophageal adenocarcinoma(EAC).In China,ESCC is the main one.The early symptoms of ESCC are often insidious and difficult to detect.When symptoms such as progressive dysphagia appear,most patients had been accompanied by local infiltration and metastasis.At this time,the course of the disease has entered the middle and late stages,and the current treatment effect is poor.At present,radiotherapy is still the main and effective treatment for ESCC,especially in the comprehensive treatment of patients with early esophageal cancer and patients with advanced esophageal cancer that cannot be surgically removed.However,along with radiotherapy,the radiosensitivity of some tumor cells decreases,which further leads to the incomplete response of ESCC to radiotherapy.Acetyl-CoA Synthetase 2(ACSS2)is a key metabolic enzyme known to convert free acetate to acetyl-CoA,which has been confirmed in a variety of tumors.Currently,ACSS2 plays a key role in biological behaviors such as cell proliferation and gene expression regulation,and is considered as a potential target for cancer therapy.However,the expression intensity and specific function of ACSS2 in ESCC remain unclear.Based on the results of previous studies,we focused on the role of ACSS2 in the formation of radiotherapy resistance in ESCC.Purposes:1.To clarify the expression of ACSS2 in ESCC and to analyze the relationship between the expression of ACSS2 and the clinicopathology and prognosis of ESCC patients.2.To explore the specific molecular mechanism of ACSS2 involved in ESCC resistance to radiotherapy.Part Ⅰ Analysis of the expression of ACSS2 and the clinicopathology and prognosis of ESCC.Methods:(1)Bioinformatics analysis of ACSS2 expression in esophageal cancer was performed through Oncomine and TCGA databases.Immunohistochemical analysis of the relationship between ACSS2 expression and clinicopathological characteristics of patients.The mRNA and protein levels of ACSS2 in ESCC tissue samples,ESCC cell lines and esophageal epithelial cells Het-1A were detected by RT-PCR and Western Blot.Kaplan-Meier survival curves were used to analyze the relationship between ACSS2 expression and prognosis of ESCC patients.(2)The relationship between ACSS2 expression intensity and microvessel density in ESCC tissue was analyzed by immunohistochemistry and immunofluorescence co-localization,and the distribution of blood vessels in the tumors of mice with high ACSS2 expression was detected by doppler color ultrasound.Results:(1)The expression intensity of ACSS2 in ESCC tissues was higher than that in adjacent tissues.Compared with the ACSS2-positive cells in the adjacent normal tissues,which were mainly cells near the basal layer,most tumor cells expressed strong ACSS2 and accompanied invasion.ACSS2 accounted for 37/60(61.66%)of strongly positive and positive sections in esophageal cancer tissue,while 18/60(30%)were ACSS2 positive or weakly positive in adjacent tissue.(2)The expression intensity of ACSS2 was not significantly correlated with the patient’s gender,age,tumor differentiation,and tumor volume;it was positively correlated with tumor stage,lymph node metastasis and recurrence.The mRNA and protein expression levels of ACSS2 in ESCC tissues were higher than those in adjacent tissues and the high expression of ACSS2 in ESCC was associated with lymph node metastasis and distant metastasis of patients.ESCC patients with high expression of ACSS2 have a relatively poor prognosis.(3)The expression intensity of ACSS2 was negatively correlated with the tumor microvessel density.The distribution of blood vessels in the tumor body of the overexpression ACSS2 group was less than that of the empty esophagus cancer cell group.Conclusion:ACSS2 is highly expressed in ESCC tissues and is associated with clinicopathological features,tumor microvessel density and prognosisPart Ⅱ The mechanism of ionizing radiation up-regulating ACSS2 expression in ESCC cellsMethods:(1)Immunohi stochemi stry,RT-PCR and Western Blot methods were used to detect the expression of ACSS2 in ESCC tissues and tumor tissues of tumor-bearing mice before and after radiotherapy.(2)Western Blot and RT-PCR were used to detect the mRNA and protein expression of ACSS2 in ESCC cell lines after different doses and different times of ionizing radiation.(3)KYSE150 cells were irradiated with 10 Gy ionizing radiation,and the expression of 90 nuclear transcription factors was detected by nuclear transcription factor chip.Western Blot was used to detect the phosphorylation level of STAT3 protein at different times of ionizing radiation.RT-PCR was used to detect the expression level of ACSS2 mRNA in ESCC cells after using STAT3 inhibitor Stattic combined with ionizing radiation.Using dual-luciferase reporter gene and chromatin immunoprecipitation assay(ChIP),we tested whether STAT3 can directly bind to the ACSS2 promoter region and promote ACSS2 gene expression.Results:(1)The expression level of ACSS2 in ESCC tissues and tumor-bearing mice after radiotherapy was higher than that in non-radiotherapy tissues.Ionizing radiation promotes the up-regulation of ACSS2 mRNA and protein expression and the accumulation of ACSS2 in ESCC nucleus.(2)Ionizing radiation promotes the increase of STAT3 protein phosphorylation,and inhibition of STAT3 protein phosphorylation reduces the expression of ACSS2 mRNA.STAT3(Y705D-S727D)can directly bind to the 322-455 bp region of ACSS2 promoter to promote ACSS2 gene expression.Conclusion:STAT3 protein Y705D-S727D is phosphorylated after ionizing radiation,and phosphorylated STAT3 directly binds to the ACSS2 promoter region and promotes the expression of ACSS2 gene.Part Ⅲ ACSS2 participates in the antitumor effect of ionizing radiation and the related molecular mechanismMethods:(1)RT-PCR and Western Blot methods were used to detect the expression levels of ACSS2 mRNA and protein that were stably knocked down and overexpressed by ACSS2 in ESCC cells.CCK8 assay,clone formation assay,Transwell invasion assay,ELISA and flow cytometry were used to detect the proliferation,invasion,apoptosis and ROS production of ESCC cells after knockdown or overexpression of ACSS2 combined with ionizing radiation.(2)KYSE150 cells with knockdown and overexpression of ACSS2 were used to construct subcutaneous xenografts to verify the radioresistance effect of ACSS2.Results:(1)Knockdown of ACSS2 combined with ionizing radiation inhibited the proliferation,colony formation and invasion ability of ESCC cells and increased apoptosis and ROS production.Knockdown of ACSS2 combined with radiotherapy inhibits the growth of subcutaneous xenografts.(2)Overexpression of ACSS2 partially reversed the proliferation and invasion abilities of ionizing radiation-suppressed ESCC cells and reduced apoptosis and ROS production.Overexpression of ACSS2 partially reversed the effect of ionizing radiation on tumor cell proliferation.Overexpression of ACSS2 combined with radiotherapy can reverse the volume reduction of subcutaneous xenografts caused by radiotherapy alone.Conclusion:ACSS2 is involved in ESCC cell radioresistance through cell proliferation,invasion,apoptosis and ROS productionPart Ⅳ ACSS2 regulates HMOX1 expression and participates in the molecular mechanism of ESCC cells radioresistanceMethods:(1)Using oxidative stress and anti-oxidative stress chips to detect the changes of stress-related molecules after knockdown of ACSS2 combined with ionizing radiation.(2)The expression level of HMOX1 mRNA in ESCC cells after knockdown of ACSS2 combined with ionizing radiation was detected by RT-PCR and Western Blot.After ESCC cells expressed ACSS2 combined with knockdown of HMOX1,flow cytometry was used to detect the apoptosis and ROS production of ESCC cells after ionizing radiation.(3)Using hTFtarget,UCSC,PROMO and SPP database to jointly analyze the transcription factors that can bind HMOX1,and analyze the correlation between transcription factors and ACSS2 through the GEPIA website.(4)The mRNA and protein expressions of HMOX1 and ACSS2 after knockdown of USF1 were detected by RT-PCR and Western Blot.ChIP tested whether USF1 could promote HMOX1 gene expression by directly binding to the HMOX1 promoter region.RT-PCR and Western Blot were used to detect the expression of USF1 mRNA,total protein and nucleoplasmic protein after knockdown of ACSS2.After overexpression of ACSS2 combined with knockdown of USF1,the mRNA and protein expression levels of HMOX1 were detected by RT-PCR and Western Blot methods.(5)KYSE150 cells were treated with 5 μM TSA(Trichostatin A)for 6 hours,and the cell lysate was subjected to co-immunoprecipitation(Co-Immunoprecipitation,IP)with control IgG,anti-USF1 or anti-acetylated lysine(Ac-K)antibodies.assay to verify whether USF1 can be acetylated.After KYSE150 cells overexpressed ACSS2,cell lysates were subjected to IP assay with anti-Ac-K antibody,followed by immunoblot analysis with anti-USF1 antibody to verify whether ACSS2 could promote USF1 acetylation.(6)Mass spectrometry analysis of proteins interacting with ACSS2 after 10 Gy dose of ionizing radiation in KYSE150 cells.IP verifies that NAT10 can interact with ACSS2.Colocalization of NAT10 and ACSS2 was detected using confocal lasers.IP assay using anti-Ac-K antibody after overexpression of ACSS2 and knockdown of NAT10,followed by immunoblot analysis with anti-USF1 antibody,was performed to verify whether ACSS2 could promote USF1 acetylation through NAT10.(7)RT-PCR and Western Blot were used to detect the expression levels of USF1 mRNA and protein after treatment with different concentrations of NAT 10-specific plasmids.The cells were treated with cycloheximide(CHX),and the protein expression level of USF1 was detected by Western Blot method.Results:(1)The oxidative stress and anti-oxidative stress chip screened out the HMOX1 molecule regulated by ACSS2,and ACSS2 unidirectionally regulates the expression of HMOX mRNA and protein.Knockdown of HMOX1 partially reversed the apoptosis inhibitory effect and the reduction of ROS levels caused by overexpression of ACSS2 combined with ionizing radiation.(2)USF1 can directly bind to the HMOX1 promoter to promote the expression of HMOX1.ACSS2 regulates HMOX1 expression by up-regulating USF1 protein expression.(3)Mass spectrometry identified a protein NAT10 that interacts with ACSS2,and ACSS2 promotes USF1 acetylation by binding to NAT10(4)NAT10 stabilizes the expression of USF1.Conclusion:ACSS2 promotes USF1 expression and acetylation by binding to NAT10,thereby regulating HMOX1 expression and participating in radioresistance effect... |
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