目的探讨乙型肝炎病毒DNA聚合酶反式调节蛋白1(HBV DNA polymerase trans activated protein 1,HBVDNAPTP1)在羧酸酯酶1(carboxylesterase 1,CES1)介导的单核细胞凋亡信号通路中的作用机制。方法利用Phyre2在线工具预测得到CES1三级结构...目的探讨乙型肝炎病毒DNA聚合酶反式调节蛋白1(HBV DNA polymerase trans activated protein 1,HBVDNAPTP1)在羧酸酯酶1(carboxylesterase 1,CES1)介导的单核细胞凋亡信号通路中的作用机制。方法利用Phyre2在线工具预测得到CES1三级结构,分别用pCMV-Myc载体构建HBVDNAPTP1基因、pCMV-HA载体构建CES1基因及其截短体219-264aa和265-303aa的真核细胞表达质粒。分别转染细胞后通过免疫荧光和免疫共沉淀法确定HBVDNAPTP1和CES1互相作用的区段。后通过HBVDNAPTP1和CES1单独转染及共转染,检测其对细胞凋亡率的影响;同时在分离人外周血单个核细胞(Peripheral blood mononuclear cell,PBMC)上转染上述质粒,以确定CES1和HBVDNAPTP1对单核细胞形态的影响。利用qRT-PCR和Western blot检测不同质粒转染后下游Janus激酶-1(Janus kinase,JAK1)和信号传导及转录激活蛋白3(Signal transducer and activator of transcription 3,STAT3)的基因和蛋白的表达情况。结果HBVDNAPTP1与CES1有靶向关系,其中HBVDNAPTP1与CES1全长靶向结合效果最好,故后续实验以CES1全长序列为对象开展。CCK-8和PBMC电镜实验结果表明,与Control组和NC组相比,ov-HBVDNAPTP1组、ov-CES1组和ov-HBVDNAPTP1+ov-CES1组THP-1细胞的凋亡率均升高(P<0.001),细胞受损;与ov-HBVDNAPTP1组和ov-CES1组相比,ov-HBVDNAPTP1+ov-CES1组细胞凋亡率进一步升高(P<0.01),凋亡细胞、坏死细胞的数量增多。qRT-PCR和Western blot结果显示,HBVDNAPTP1和CES1均可以在转录水平和翻译水平上显著上调JAK1和STAT3的表达,且二者同时作用时上调趋势更加明显。结论本研究证实HBVDNAPTP1与CES1二者可协同抑制THP-1细胞增殖并诱导细胞损伤;还可协同上调JAK1/STAT3信号通路在转录和蛋白水平的表达来调控单核细胞凋亡。该发现为深入理解HBV感染相关免疫调控机制提供了新的理论依据。展开更多
Background:Head and neck squamous cell carcinoma(HNSCC)is a prevalent form of cancer globally,with chemoresistance posing a major challenge in treatment outcomes.The efficacy of the commonly used chemotherapeutic agent...Background:Head and neck squamous cell carcinoma(HNSCC)is a prevalent form of cancer globally,with chemoresistance posing a major challenge in treatment outcomes.The efficacy of the commonly used chemotherapeutic agent,cisplatin,is diminished in patients with poor prognoses.Methods:Various bioinformatics databases were utilized to examine Carboxylesterase 1(CES1)gene expression,clinicopathologic features,patient survival analysis,and gene function.An organoid model of HNSCC was established,along with the induction of drug-resistant HNSCC in the organoid model.CES1 expression was assessed using qRT-PCR and Western Blot,and differential markers were identified through transcriptome sequencing.Knockdown and overexpression models of CES1 were created in SCC-9 and patient-derived organoid(PDO)cells using shRNA and lentivirus to investigate the tumor biology and cisplatin resistance associated with CES1.Results:Research in bioinformatics has uncovered a strong correlation between the expression level of CES1 and the prognosis of HNSCC.The data suggests a significant link between CES1 expression and tobacco smoking.RNA-sequencing revealed a notable increase in CES1 expression in HNSCC-PDOcis-R cells compared to the parental PDO cells.Subsequently,we performed in vitro studies by HNSCC-PDO and SCC-9 and found that CES1-overexpressing cells exhibited reduced sensitivity to cisplatin and stronger tumor malignant biological behavior compared with CES1-knockdown cells.Conclusion:The observed association between CES1 expression and tobacco smoking implies a potential influence of smoking on the efficacy of cisplatin-based chemotherapy in HNSCC through the regulation of CES1 expression.展开更多
文摘目的探讨乙型肝炎病毒DNA聚合酶反式调节蛋白1(HBV DNA polymerase trans activated protein 1,HBVDNAPTP1)在羧酸酯酶1(carboxylesterase 1,CES1)介导的单核细胞凋亡信号通路中的作用机制。方法利用Phyre2在线工具预测得到CES1三级结构,分别用pCMV-Myc载体构建HBVDNAPTP1基因、pCMV-HA载体构建CES1基因及其截短体219-264aa和265-303aa的真核细胞表达质粒。分别转染细胞后通过免疫荧光和免疫共沉淀法确定HBVDNAPTP1和CES1互相作用的区段。后通过HBVDNAPTP1和CES1单独转染及共转染,检测其对细胞凋亡率的影响;同时在分离人外周血单个核细胞(Peripheral blood mononuclear cell,PBMC)上转染上述质粒,以确定CES1和HBVDNAPTP1对单核细胞形态的影响。利用qRT-PCR和Western blot检测不同质粒转染后下游Janus激酶-1(Janus kinase,JAK1)和信号传导及转录激活蛋白3(Signal transducer and activator of transcription 3,STAT3)的基因和蛋白的表达情况。结果HBVDNAPTP1与CES1有靶向关系,其中HBVDNAPTP1与CES1全长靶向结合效果最好,故后续实验以CES1全长序列为对象开展。CCK-8和PBMC电镜实验结果表明,与Control组和NC组相比,ov-HBVDNAPTP1组、ov-CES1组和ov-HBVDNAPTP1+ov-CES1组THP-1细胞的凋亡率均升高(P<0.001),细胞受损;与ov-HBVDNAPTP1组和ov-CES1组相比,ov-HBVDNAPTP1+ov-CES1组细胞凋亡率进一步升高(P<0.01),凋亡细胞、坏死细胞的数量增多。qRT-PCR和Western blot结果显示,HBVDNAPTP1和CES1均可以在转录水平和翻译水平上显著上调JAK1和STAT3的表达,且二者同时作用时上调趋势更加明显。结论本研究证实HBVDNAPTP1与CES1二者可协同抑制THP-1细胞增殖并诱导细胞损伤;还可协同上调JAK1/STAT3信号通路在转录和蛋白水平的表达来调控单核细胞凋亡。该发现为深入理解HBV感染相关免疫调控机制提供了新的理论依据。
基金supported by the National Natural Science Foundation of China(No.82160386)the Guangxi Natural Science Foundation of China(No.2024GXNSFDA010032,2023GXNSFAA026189).
文摘Background:Head and neck squamous cell carcinoma(HNSCC)is a prevalent form of cancer globally,with chemoresistance posing a major challenge in treatment outcomes.The efficacy of the commonly used chemotherapeutic agent,cisplatin,is diminished in patients with poor prognoses.Methods:Various bioinformatics databases were utilized to examine Carboxylesterase 1(CES1)gene expression,clinicopathologic features,patient survival analysis,and gene function.An organoid model of HNSCC was established,along with the induction of drug-resistant HNSCC in the organoid model.CES1 expression was assessed using qRT-PCR and Western Blot,and differential markers were identified through transcriptome sequencing.Knockdown and overexpression models of CES1 were created in SCC-9 and patient-derived organoid(PDO)cells using shRNA and lentivirus to investigate the tumor biology and cisplatin resistance associated with CES1.Results:Research in bioinformatics has uncovered a strong correlation between the expression level of CES1 and the prognosis of HNSCC.The data suggests a significant link between CES1 expression and tobacco smoking.RNA-sequencing revealed a notable increase in CES1 expression in HNSCC-PDOcis-R cells compared to the parental PDO cells.Subsequently,we performed in vitro studies by HNSCC-PDO and SCC-9 and found that CES1-overexpressing cells exhibited reduced sensitivity to cisplatin and stronger tumor malignant biological behavior compared with CES1-knockdown cells.Conclusion:The observed association between CES1 expression and tobacco smoking implies a potential influence of smoking on the efficacy of cisplatin-based chemotherapy in HNSCC through the regulation of CES1 expression.
