前期研究结果表明,乙型肝炎病毒X蛋白结合蛋白(hepatitis B virus X-interacting protein,HBXIP)具有促进细胞增殖的作用.为了进一步阐明其分子机制,观察了HBXIP对核因子κB(NF-κB)转录活性的影响.实验中通过基因共转染将NF-κB报告基...前期研究结果表明,乙型肝炎病毒X蛋白结合蛋白(hepatitis B virus X-interacting protein,HBXIP)具有促进细胞增殖的作用.为了进一步阐明其分子机制,观察了HBXIP对核因子κB(NF-κB)转录活性的影响.实验中通过基因共转染将NF-κB报告基因质粒pNF-κB-Luc和HBXIP真核表达载体pcDNA3-hbxip导入人肝癌H7402细胞系中,进行荧光素酶活性分析.结果显示:H7402细胞过表达HBXIP后NF-κB的转录活性明显增强;此外,基因转染后经免疫印迹检测显示,与NF-κB二聚体结合的抑制亚基IκBα的磷酸化水平明显增加;同时,提取H7402细胞的核蛋白,然后应用免疫印迹检测细胞核中p65/NF-κB的水平.结果显示,H7402细胞中HBXIP过表达后细胞核中p65/NF-κB的水平明显增加.当应用RNA干扰技术抑制了细胞内源性的HBXIP基因表达后,则出现与上述结果相反的效果.上述结果提示,HBXIP可增加核内p65/NF-κB蛋白水平,进而发挥NF-κB促转录调控的作用.因此,HBXIP可通过调控NF-κB信号途径而促进细胞增殖.展开更多
乙型肝炎病毒X蛋白结合蛋白(hepatitis B virus X-interacting protein,HBXIP)可与乙肝病毒X蛋白(HBX)的C端结合,它具有抑制HBX活性的作用.为进一步阐明HBXIP对细胞增殖的作用及其分子机制,构建了HBXIP的真核表达载体,并将其稳定转染至...乙型肝炎病毒X蛋白结合蛋白(hepatitis B virus X-interacting protein,HBXIP)可与乙肝病毒X蛋白(HBX)的C端结合,它具有抑制HBX活性的作用.为进一步阐明HBXIP对细胞增殖的作用及其分子机制,构建了HBXIP的真核表达载体,并将其稳定转染至正常人肝细胞系L-O2细胞中,建立了稳定表达HBXIP蛋白的肝细胞系,命名为L-O2-hbxip.然后,应用MTT、BrdU标记实验和流式细胞术等方法,发现HBXIP过表达后,L-O2细胞的生长速度明显加快,可促进细胞由G1期进入到S期,表明HBXIP具有促进L-O2-hbxip细胞增殖的作用.应用免疫印迹对有关细胞周期相关蛋白进行了检测.结果显示,HBXIP过表达时可上调细胞周期蛋白D1、细胞周期蛋白E的表达,并下调p21和p27的表达,从而调节细胞周期,产生对细胞增殖的影响.展开更多
Objective:The oncoprotein,hepatitis B X-interacting protein(HBXIP),has been reported to play an important role in human malignancies.However,its functions in non-small cell lung cancer(NSCLC)are poorly understood.The ...Objective:The oncoprotein,hepatitis B X-interacting protein(HBXIP),has been reported to play an important role in human malignancies.However,its functions in non-small cell lung cancer(NSCLC)are poorly understood.The goal of the present study was to identify the role of HBXIP in the regulation of NSCLC development.Methods:The level of HBXIP expression in NSCLC tissue was assessed by immunohistochemical and Western blot analyses,and its relationships with clinicopathological features and outcomes were statistically evaluated.The effects of HBXIP on NSCLC cell progression were assessed through cell viability,colony formation,and flow cytometry analyses in vitro.The mechanism by which HBXIP regulated the MAPK pathway was studied by Western blot,immunofluorescence,and immunoprecipitation assays.In addition,in vivo experiments were performed to evaluate the progression of NSCLC and ERK signaling pathway activation after HBXIP knockdown.Results:HBXIP was overexpressed in human NSCLC and was correlated with the invasiveness of NSCLC.The high expression of HBXIP in NSCLC was significantly correlated with gender(P=0.033),N stage(P=0.002),and tumor-node-metastasis stage(P=0.008).In vitro experiments using an NSCLC cell line revealed that HBXIP knockdown resulted in the suppression of cell proliferation and colony formation,which was consistent with the enhanced cell cycle arrest in G1 phase.The results of a mechanistic investigation suggested that binding of HBXIP to MEK1 protein promoted MAPK/ERK signaling pathway activation in NSCLC by preventing the proteasome-mediated degradation of MEK1.In addition,the results obtained using in vivo subcutaneous tumor xenografts confirmed that HBXIP deficiency decreased MEK1 protein levels and NSCLC tumor growth.Conclusions:Taken together,our results showed that the HBXIP-MEK interaction promoted oncogenesis via the MAPK/ERK pathway,which may serve as a novel therapeutic target for cancers in which MAPK/ERK signaling is a dominant feature.展开更多
Tumor metastasis depends on the dynamic balance of the actomyosin cytoskeleton.As a key component of actomyosin filaments,non-muscle myosin-ⅡA disassembly contributes to tumor cell spreading and migration.However,its...Tumor metastasis depends on the dynamic balance of the actomyosin cytoskeleton.As a key component of actomyosin filaments,non-muscle myosin-ⅡA disassembly contributes to tumor cell spreading and migration.However,its regulatory mechanism in tumor migration and invasion is poorly understood.Here,we found that oncoprotein hepatitis B X-interacting protein(HBXIP) blocked the myosin-ⅡA assemble state promoting breast cancer cell migration.Mechanistically,mass spectrometry analysis,co-immunoprecipitation assay and GST-pull down assay proved that HBXIP directly interacted with the assembly-competent domain(ACD) of non-muscle heavy chain myosin-ⅡA(NMHC-ⅡA).The interaction was enhanced by NMHC-ⅡA S1916 phosphorylation via HBXIP-recruited protein kinase PKCβⅡ.Moreover,HBXIP induced the transcription of PRKCB,encoding PKCβⅡ,by coactivating Sp1,and triggered PKCβⅡ kinase activity.Interestingly,RNA sequencing and mouse metastasis model indicated that the anti-hyperlipidemic drug bezafibrate(BZF) suppressed breast cancer metastasis via inhibiting PKCβⅡ-mediated NMHC-ⅡA phosphorylation in vitro and in vivo.We reveal a novel mechanism by which HBXIP promotes myosin-ⅡA disassembly via interacting and phosphorylating NMHC-ⅡA,and BZF can serve as an effective anti-metastatic drug in breast cancer.展开更多
Objective:Hepatitis B X-interacting protein(HBXIP)plays an important role in breast tumorigenesis,tumor growth and metastasis,but its functional contribution in radioresistance remains poorly understood.As radiotherap...Objective:Hepatitis B X-interacting protein(HBXIP)plays an important role in breast tumorigenesis,tumor growth and metastasis,but its functional contribution in radioresistance remains poorly understood.As radiotherapy served as an essential adjuvant treatment,uncovering the role of HBXIP as well as its downstream molecular XIAP in radioresistance could benefit for the development of individual therapy strategy.Methods:Immunohistochemistry of 42 breast cancer tissue samples and Western blot analysis of proteins from MCF-7 and MDA-MB-231 cells exposed to fractioned doses(γ-rays)were used to identify the expression of HBXIP/XIAP in breast cancer.To verify the radioresistance effects and potential mechanism,the cells were treated with designed pCMV and siRNA of targeting genes,and then measured with MTT assay,clonogenic survival assay and flow cytometry.Furthermore,a subcutaneous xenotransplanted tumor model of breast cancer was established in nude mice to validate the radioresistization effect of HBXIP in vivo.Results:HBXIP and XIAP expression levels in breast cancer tissues were positively correlated with chemoradiotherapy resistance of breast cancer.Overexpression of HBXIP could desensitize MCF-7 and MDA-MB-231 cells to irradiation by inhibiting radiation-induced cell apoptosis,and knockdown of HBXIP in these cells had the converse response.Moreover,up-regulation of HBXIP resulted in the increase of XIAP and NF-κB levels in vitro and in vivo,while down-regulation of HBXIP led to the opposite effects.In addition,inhibition of XIAP and NF-κB abrogated the HBXIP overexpression induced radioresistization and increased cell apoptosis(25.8%augment for siRNA XIAP and 28.1%for NF-κB in MDA-MB-231 cells;25.4%augment for siRNA XIAP and 27.2%for NF–κB in MCF-7 cells).Conclusions:HBXIP enhances radioresistance of human breast cancer cells via upregulating XIAP,and targeting the HBXIP–NF–κB-XIAP pathway may be a potentially effective strategy to enhance the efficacy of radiotherapy for human breast cancer.展开更多
目的:探讨乙肝病毒X蛋白结合蛋白(HBXIP)在乳腺癌组织中的表达及预后评估的临床病理学意义。方法:采用免疫组化En Vision法检测60例乳腺癌、15例乳腺导管内原位癌(ductal carcinoma in situ,DCIS)及27例癌旁正常乳腺组织中HBXIP蛋白的表...目的:探讨乙肝病毒X蛋白结合蛋白(HBXIP)在乳腺癌组织中的表达及预后评估的临床病理学意义。方法:采用免疫组化En Vision法检测60例乳腺癌、15例乳腺导管内原位癌(ductal carcinoma in situ,DCIS)及27例癌旁正常乳腺组织中HBXIP蛋白的表达,并结合临床病理指标及生存时间进行统计学分析。结果:乳腺肿瘤组织中HBXIP蛋白阳性表达率为88.3%(53/60),明显高于乳腺导管内原位癌组织和癌旁正常乳腺组织(分别为46.7%和29.6%,均P<0.01);卡方检验结果显示,HBXIP蛋白表达与乳腺癌患者临床分期、淋巴结转移及HER-2表达密切相关(P值分别为0.003、0.027和0.009),而与性别、原发灶大小、临床分级、ER及PR表达等参数无关。结论:HBXIP蛋白过表达可以预示乳腺癌患者的不良预后,而且可以作为早期诊断的重要辅助指标和乳腺癌分子靶向治疗的新候选基因靶点。展开更多
乙型肝炎病毒X蛋白(hepatitis B virus X protein,HBx)对肝癌的发生发展具有十分重要的作用.HBx具有促进肝癌迁移的作用,但其作用的分子机制不清.本研究对HBx促进肝癌细胞迁移的分子机制进行了探讨.伤口愈合和Boyden’s chamber结果表明...乙型肝炎病毒X蛋白(hepatitis B virus X protein,HBx)对肝癌的发生发展具有十分重要的作用.HBx具有促进肝癌迁移的作用,但其作用的分子机制不清.本研究对HBx促进肝癌细胞迁移的分子机制进行了探讨.伤口愈合和Boyden’s chamber结果表明,HBx可明显促进肝癌Hep G2细胞迁移.在稳定转染HBx的Hep G2(Hep G2-X)细胞中转染HBx结合蛋白(hepatitis B X-interacting protein,HBXIP)的RNA干扰片段,可明显抑制HBx的促迁移作用.免疫组化和实时定量PCR结果表明,HBXIP在肝癌组织中显著高表达,并且与HBx表达成正相关.荧光素酶报告基因和免疫印迹结果表明,HBx显著增强HBXIP的启动子活性和蛋白质表达水平.应用HBx的RNA干扰处理Hep G2-X细胞,HBXIP的启动子活性和蛋白质表达水平明显下降.将HBXIP启动子区的c AMP效应元件结合因子(CREB)结合位点突变后,HBx上调HBXIP的作用消失.应用CREB的RNA干扰处理肝癌细胞,在启动子水平和蛋白质水平上,HBx对HBXIP的上调作用被显著抑制.染色质免疫共沉淀结果表明,HBx能够通过CREB结合到HBXIP的启动子上,进而发挥激活HBXIP的功能.本研究结果表明,HBx促进肝癌细胞迁移的作用是通过CREB上调HBXIP实现的.这一发现对进一步揭示HBx促进肝癌细胞迁移的分子机制具有重要意义.展开更多
文摘前期研究结果表明,乙型肝炎病毒X蛋白结合蛋白(hepatitis B virus X-interacting protein,HBXIP)具有促进细胞增殖的作用.为了进一步阐明其分子机制,观察了HBXIP对核因子κB(NF-κB)转录活性的影响.实验中通过基因共转染将NF-κB报告基因质粒pNF-κB-Luc和HBXIP真核表达载体pcDNA3-hbxip导入人肝癌H7402细胞系中,进行荧光素酶活性分析.结果显示:H7402细胞过表达HBXIP后NF-κB的转录活性明显增强;此外,基因转染后经免疫印迹检测显示,与NF-κB二聚体结合的抑制亚基IκBα的磷酸化水平明显增加;同时,提取H7402细胞的核蛋白,然后应用免疫印迹检测细胞核中p65/NF-κB的水平.结果显示,H7402细胞中HBXIP过表达后细胞核中p65/NF-κB的水平明显增加.当应用RNA干扰技术抑制了细胞内源性的HBXIP基因表达后,则出现与上述结果相反的效果.上述结果提示,HBXIP可增加核内p65/NF-κB蛋白水平,进而发挥NF-κB促转录调控的作用.因此,HBXIP可通过调控NF-κB信号途径而促进细胞增殖.
文摘乙型肝炎病毒X蛋白结合蛋白(hepatitis B virus X-interacting protein,HBXIP)可与乙肝病毒X蛋白(HBX)的C端结合,它具有抑制HBX活性的作用.为进一步阐明HBXIP对细胞增殖的作用及其分子机制,构建了HBXIP的真核表达载体,并将其稳定转染至正常人肝细胞系L-O2细胞中,建立了稳定表达HBXIP蛋白的肝细胞系,命名为L-O2-hbxip.然后,应用MTT、BrdU标记实验和流式细胞术等方法,发现HBXIP过表达后,L-O2细胞的生长速度明显加快,可促进细胞由G1期进入到S期,表明HBXIP具有促进L-O2-hbxip细胞增殖的作用.应用免疫印迹对有关细胞周期相关蛋白进行了检测.结果显示,HBXIP过表达时可上调细胞周期蛋白D1、细胞周期蛋白E的表达,并下调p21和p27的表达,从而调节细胞周期,产生对细胞增殖的影响.
基金supported by the National Natural Science Foundation of China(Grant Nos.81502322,81872169,and 81702629)the Tianjin Key Research and Development Program of Science and Technology Support Key Projects(Grant No.17YFZCSY00690)the XI SI KE--CSPC Cancer Research Fund。
文摘Objective:The oncoprotein,hepatitis B X-interacting protein(HBXIP),has been reported to play an important role in human malignancies.However,its functions in non-small cell lung cancer(NSCLC)are poorly understood.The goal of the present study was to identify the role of HBXIP in the regulation of NSCLC development.Methods:The level of HBXIP expression in NSCLC tissue was assessed by immunohistochemical and Western blot analyses,and its relationships with clinicopathological features and outcomes were statistically evaluated.The effects of HBXIP on NSCLC cell progression were assessed through cell viability,colony formation,and flow cytometry analyses in vitro.The mechanism by which HBXIP regulated the MAPK pathway was studied by Western blot,immunofluorescence,and immunoprecipitation assays.In addition,in vivo experiments were performed to evaluate the progression of NSCLC and ERK signaling pathway activation after HBXIP knockdown.Results:HBXIP was overexpressed in human NSCLC and was correlated with the invasiveness of NSCLC.The high expression of HBXIP in NSCLC was significantly correlated with gender(P=0.033),N stage(P=0.002),and tumor-node-metastasis stage(P=0.008).In vitro experiments using an NSCLC cell line revealed that HBXIP knockdown resulted in the suppression of cell proliferation and colony formation,which was consistent with the enhanced cell cycle arrest in G1 phase.The results of a mechanistic investigation suggested that binding of HBXIP to MEK1 protein promoted MAPK/ERK signaling pathway activation in NSCLC by preventing the proteasome-mediated degradation of MEK1.In addition,the results obtained using in vivo subcutaneous tumor xenografts confirmed that HBXIP deficiency decreased MEK1 protein levels and NSCLC tumor growth.Conclusions:Taken together,our results showed that the HBXIP-MEK interaction promoted oncogenesis via the MAPK/ERK pathway,which may serve as a novel therapeutic target for cancers in which MAPK/ERK signaling is a dominant feature.
基金supported by the grants from National Natural Science Foundation of China(82072929,82072943,and 31870752,China).
文摘Tumor metastasis depends on the dynamic balance of the actomyosin cytoskeleton.As a key component of actomyosin filaments,non-muscle myosin-ⅡA disassembly contributes to tumor cell spreading and migration.However,its regulatory mechanism in tumor migration and invasion is poorly understood.Here,we found that oncoprotein hepatitis B X-interacting protein(HBXIP) blocked the myosin-ⅡA assemble state promoting breast cancer cell migration.Mechanistically,mass spectrometry analysis,co-immunoprecipitation assay and GST-pull down assay proved that HBXIP directly interacted with the assembly-competent domain(ACD) of non-muscle heavy chain myosin-ⅡA(NMHC-ⅡA).The interaction was enhanced by NMHC-ⅡA S1916 phosphorylation via HBXIP-recruited protein kinase PKCβⅡ.Moreover,HBXIP induced the transcription of PRKCB,encoding PKCβⅡ,by coactivating Sp1,and triggered PKCβⅡ kinase activity.Interestingly,RNA sequencing and mouse metastasis model indicated that the anti-hyperlipidemic drug bezafibrate(BZF) suppressed breast cancer metastasis via inhibiting PKCβⅡ-mediated NMHC-ⅡA phosphorylation in vitro and in vivo.We reveal a novel mechanism by which HBXIP promotes myosin-ⅡA disassembly via interacting and phosphorylating NMHC-ⅡA,and BZF can serve as an effective anti-metastatic drug in breast cancer.
基金This study was supported by the grants from the National Natural Science Foundation of China (No.31770910 and No.81730086).
文摘Objective:Hepatitis B X-interacting protein(HBXIP)plays an important role in breast tumorigenesis,tumor growth and metastasis,but its functional contribution in radioresistance remains poorly understood.As radiotherapy served as an essential adjuvant treatment,uncovering the role of HBXIP as well as its downstream molecular XIAP in radioresistance could benefit for the development of individual therapy strategy.Methods:Immunohistochemistry of 42 breast cancer tissue samples and Western blot analysis of proteins from MCF-7 and MDA-MB-231 cells exposed to fractioned doses(γ-rays)were used to identify the expression of HBXIP/XIAP in breast cancer.To verify the radioresistance effects and potential mechanism,the cells were treated with designed pCMV and siRNA of targeting genes,and then measured with MTT assay,clonogenic survival assay and flow cytometry.Furthermore,a subcutaneous xenotransplanted tumor model of breast cancer was established in nude mice to validate the radioresistization effect of HBXIP in vivo.Results:HBXIP and XIAP expression levels in breast cancer tissues were positively correlated with chemoradiotherapy resistance of breast cancer.Overexpression of HBXIP could desensitize MCF-7 and MDA-MB-231 cells to irradiation by inhibiting radiation-induced cell apoptosis,and knockdown of HBXIP in these cells had the converse response.Moreover,up-regulation of HBXIP resulted in the increase of XIAP and NF-κB levels in vitro and in vivo,while down-regulation of HBXIP led to the opposite effects.In addition,inhibition of XIAP and NF-κB abrogated the HBXIP overexpression induced radioresistization and increased cell apoptosis(25.8%augment for siRNA XIAP and 28.1%for NF-κB in MDA-MB-231 cells;25.4%augment for siRNA XIAP and 27.2%for NF–κB in MCF-7 cells).Conclusions:HBXIP enhances radioresistance of human breast cancer cells via upregulating XIAP,and targeting the HBXIP–NF–κB-XIAP pathway may be a potentially effective strategy to enhance the efficacy of radiotherapy for human breast cancer.
文摘目的:探讨乙肝病毒X蛋白结合蛋白(HBXIP)在乳腺癌组织中的表达及预后评估的临床病理学意义。方法:采用免疫组化En Vision法检测60例乳腺癌、15例乳腺导管内原位癌(ductal carcinoma in situ,DCIS)及27例癌旁正常乳腺组织中HBXIP蛋白的表达,并结合临床病理指标及生存时间进行统计学分析。结果:乳腺肿瘤组织中HBXIP蛋白阳性表达率为88.3%(53/60),明显高于乳腺导管内原位癌组织和癌旁正常乳腺组织(分别为46.7%和29.6%,均P<0.01);卡方检验结果显示,HBXIP蛋白表达与乳腺癌患者临床分期、淋巴结转移及HER-2表达密切相关(P值分别为0.003、0.027和0.009),而与性别、原发灶大小、临床分级、ER及PR表达等参数无关。结论:HBXIP蛋白过表达可以预示乳腺癌患者的不良预后,而且可以作为早期诊断的重要辅助指标和乳腺癌分子靶向治疗的新候选基因靶点。
文摘乙型肝炎病毒X蛋白(hepatitis B virus X protein,HBx)对肝癌的发生发展具有十分重要的作用.HBx具有促进肝癌迁移的作用,但其作用的分子机制不清.本研究对HBx促进肝癌细胞迁移的分子机制进行了探讨.伤口愈合和Boyden’s chamber结果表明,HBx可明显促进肝癌Hep G2细胞迁移.在稳定转染HBx的Hep G2(Hep G2-X)细胞中转染HBx结合蛋白(hepatitis B X-interacting protein,HBXIP)的RNA干扰片段,可明显抑制HBx的促迁移作用.免疫组化和实时定量PCR结果表明,HBXIP在肝癌组织中显著高表达,并且与HBx表达成正相关.荧光素酶报告基因和免疫印迹结果表明,HBx显著增强HBXIP的启动子活性和蛋白质表达水平.应用HBx的RNA干扰处理Hep G2-X细胞,HBXIP的启动子活性和蛋白质表达水平明显下降.将HBXIP启动子区的c AMP效应元件结合因子(CREB)结合位点突变后,HBx上调HBXIP的作用消失.应用CREB的RNA干扰处理肝癌细胞,在启动子水平和蛋白质水平上,HBx对HBXIP的上调作用被显著抑制.染色质免疫共沉淀结果表明,HBx能够通过CREB结合到HBXIP的启动子上,进而发挥激活HBXIP的功能.本研究结果表明,HBx促进肝癌细胞迁移的作用是通过CREB上调HBXIP实现的.这一发现对进一步揭示HBx促进肝癌细胞迁移的分子机制具有重要意义.
