BACKGROUND Esophageal squamous cell carcinoma(ESCC),the predominant type of esophageal cancer,has a 5-year survival rate less than 20%.Although the cause of poor prognosis is the high incidence and mortality of ESCC,t...BACKGROUND Esophageal squamous cell carcinoma(ESCC),the predominant type of esophageal cancer,has a 5-year survival rate less than 20%.Although the cause of poor prognosis is the high incidence and mortality of ESCC,the high rate of metastasis after esophageal cancer surgery is the main cause of death after the surgery.Bromodomain-containing protein 4(BRD4),an epigenetic reader of chromatinacetylated histones in tumorigenesis and development,plays an essential role in regulating oncogene expression.BRD4 inhibition and BRD4 inhibition-based treatment can potentially suppress ESCC growth.However,the effects and mechanisms of action of BRD4 on ESCC cell migration remain unclear.AIM To explore the effect of BRD4 on cell migration of ESCC in vitro and its possible molecular mechanism.METHODS Human ESCC cell lines KYSE-450 and KYSE-150 were used.The 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay was performed to examine cell proliferation,and the transwell migration assay was conducted to test ESCC cell migration.JQ1,a BRD4 inhibitor,was applied to cells,and BRD4 siRNA was transfected into ESCC cells to knockdown endogenous BRD4.GFP-RFP-LC3 adenovirus was infected into ESCC cells to evaluate the effect of JQ1 on autophagy.Western blotting was performed to determine the protein levels of BRD4,E-cadherin,vimentin,AMP-activated protein kinase(AMPK),and p-AMPK.RESULTS BRD4 was either downregulated by small interfering RNA or pretreated with JQ1 in ESCC cells,leading to increased tumor migration in ESCC cells in a dose-and time-dependent manner.Inhibition of BRD4 not only significantly suppressed cell proliferation but also strongly increased cell migration by inducing epithelial-mesenchymal transition(EMT).The protein expression of vimentin was increased and E-cadherin decreased in a dose-dependent manner,subsequently promoting autophagy in KYSE-450 and KYSE-150 cells.Pretreatment with JQ1,a BRD4 inhibitor,inhibited BRD4-induced LC3-II activation and upregulated AMPK phosphorylation in a dosedependent manner.Additionally,an increased number of autophagosomes and autolysosomes were observed in JQ1-treated ESCC cells.The autophagy inhibitor 3-methyladenine(3-MA)reversed the effects of BRD4 knockdown on ESCC cell migration and blocked JQ1-induced cell migration.3-MA also downregulated the expression of vimentin and upregulation E-cadherin.CONCLUSION BRD4 inhibition enhances cell migration by inducing EMT and autophagy in ESCC cells via the AMPK-modified pathway.Thus,the facilitating role on ESCC cell migration should be considered for BRD4 inhibitor clinical application to ESCC patients.展开更多
BACKGROUND Esophageal squamous cell carcinoma(ESCC)is one of the most common malignancies.A total of 45 kinesin superfamily proteins(KIFs)have been identified in humans,among which several family members have demonstr...BACKGROUND Esophageal squamous cell carcinoma(ESCC)is one of the most common malignancies.A total of 45 kinesin superfamily proteins(KIFs)have been identified in humans,among which several family members have demonstrated varied functions in tumor pathobiology via different mechanisms,including regulation of cell cycle progression and metastasis.KIFC3 has microtubule motor activity and is involved in cancer cell invasion and migration,as well as survival.However,the role of KIFC3 in ESCC is still unknown.AIM To evaluate the role of KIFC3 in ESCC and the underlying mechanisms.METHODS Expression of KIFC3 was evaluated in ESCC tissues and adjacent normal esophageal tissues.The prognostic value of KIFC3 was analyzed using Kaplan-Meier Plotter.Colony formation,EdU assays,cell cycle analysis,Transwell assay,immunofluorescence,and western blotting were performed in ESCC cell lines after transfection with pLVX-Puro-KIFC3-shRNA-and pLVX-Puro-KIFC3-expressing lentiviruses.A xenograft tumor model in nude mice was used to evaluate the role of KIFC3 in tumorigenesis.Inhibitor ofβ-catenin,XAV-939,was used to clarify the mechanism of KIFC3 in ESCC.To analyze the differences between groups,t test and nonparametric tests were used.P<0.05 was considered statistically significant.RESULTS Immunohistochemical staining indicated that KIFC3 was upregulated in ESCC tissues compared with adjacent normal tissues.Kaplan-Meier Plotter revealed that overexpressed KIFC3 was associated with poor prognosis in ESCC patients.Colony formation and EdU assay showed that KIFC3 overexpression promoted cell proliferation,while KIFC3 knockdown inhibited cell proliferation in ESCC cell lines.In addition,cell cycle analysis showed that KIFC3 overexpression promoted cell cycle progression.KIFC3 knockdown suppressed ESCC tumorigenesis in vivo.Transwell assay and western blotting revealed that KIFC3 overexpression promoted cell migration and invasion,as well as epithelial-mesenchymal transition(EMT),while KIFC3 knockdown showed the opposite results.Mechanistically,KIFC3 overexpression promoted β-catenin signaling in KYSE450 cells;however,the role of KIFC3 was abolished by XAV-939,the inhibitor of β-catenin signaling.CONCLUSION KIFC3 was overexpressed in ESCC and was associated with poor prognosis.Furthermore,KIFC3 promoted proliferation,migration and invasion of ESCC via β-catenin signaling and EMT.展开更多
AIM To explore the effect of miR-382 on esophageal squamous cell carcinoma (ESCC) in vitro and its possible molecular mechanism. METHODS Eca 109 cells derived from human ESCC and Het-1A cells derived from human normal...AIM To explore the effect of miR-382 on esophageal squamous cell carcinoma (ESCC) in vitro and its possible molecular mechanism. METHODS Eca 109 cells derived from human ESCC and Het-1A cells derived from human normal esophageal epithelium were used. Lentivirus-mediated miR-382 was overexpressed in Eca109 cells. The effect of miR-382 on cell proliferation was evaluated by MTT and colony formation assay. For cell cycle analysis, cells were fixed and stained for 30 min with propidium iodide (PI) staining buffer containing 10 mg/mL PI and 100 mg/mL RNase A, and analyzed by BD FACSCalibur (TM) flow cytometer. For cell apoptosis assay, cells were stained with an Annexin V-FITC/PI Apoptosis Detection Kit according to the manufacturer's instructions and analyzed by a dual-laser flow cytometer. Cell invasion and migration abilities were determined through use of transwell chambers, non-coated or pre-coated with matrigel. Levels of proteins related to cell growth and migration were examined by western blotting. RESULTS Endogenous miR-382 was down-regulated in Eca109 cells compared with Het-1A. Introduction of miR-382 not only significantly inhibited proliferation and colony formation, but also arrested cell cycle at the G2/M phase, as well as promoted apoptosis and autophagy in Eca109 cells. Migration, invasion and epithelial-mesenchymal transition of Eca109 cells were suppressed by overexpressing miR-382. Western blotting results showed that miR-382 inhibited the phosphorylation of mTOR and 4E-BP1. CONCLUSION miR-382 functions as a tumor suppressor against ESCC development and metastasis, and could be considered as a potential drug source for the treatment of ESCC patients.展开更多
基金the Key Project of Science and Technology of Xinxiang,No.GG2020027the Health Commission of Henan Province of China,No.SBGJ202102188.
文摘BACKGROUND Esophageal squamous cell carcinoma(ESCC),the predominant type of esophageal cancer,has a 5-year survival rate less than 20%.Although the cause of poor prognosis is the high incidence and mortality of ESCC,the high rate of metastasis after esophageal cancer surgery is the main cause of death after the surgery.Bromodomain-containing protein 4(BRD4),an epigenetic reader of chromatinacetylated histones in tumorigenesis and development,plays an essential role in regulating oncogene expression.BRD4 inhibition and BRD4 inhibition-based treatment can potentially suppress ESCC growth.However,the effects and mechanisms of action of BRD4 on ESCC cell migration remain unclear.AIM To explore the effect of BRD4 on cell migration of ESCC in vitro and its possible molecular mechanism.METHODS Human ESCC cell lines KYSE-450 and KYSE-150 were used.The 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay was performed to examine cell proliferation,and the transwell migration assay was conducted to test ESCC cell migration.JQ1,a BRD4 inhibitor,was applied to cells,and BRD4 siRNA was transfected into ESCC cells to knockdown endogenous BRD4.GFP-RFP-LC3 adenovirus was infected into ESCC cells to evaluate the effect of JQ1 on autophagy.Western blotting was performed to determine the protein levels of BRD4,E-cadherin,vimentin,AMP-activated protein kinase(AMPK),and p-AMPK.RESULTS BRD4 was either downregulated by small interfering RNA or pretreated with JQ1 in ESCC cells,leading to increased tumor migration in ESCC cells in a dose-and time-dependent manner.Inhibition of BRD4 not only significantly suppressed cell proliferation but also strongly increased cell migration by inducing epithelial-mesenchymal transition(EMT).The protein expression of vimentin was increased and E-cadherin decreased in a dose-dependent manner,subsequently promoting autophagy in KYSE-450 and KYSE-150 cells.Pretreatment with JQ1,a BRD4 inhibitor,inhibited BRD4-induced LC3-II activation and upregulated AMPK phosphorylation in a dosedependent manner.Additionally,an increased number of autophagosomes and autolysosomes were observed in JQ1-treated ESCC cells.The autophagy inhibitor 3-methyladenine(3-MA)reversed the effects of BRD4 knockdown on ESCC cell migration and blocked JQ1-induced cell migration.3-MA also downregulated the expression of vimentin and upregulation E-cadherin.CONCLUSION BRD4 inhibition enhances cell migration by inducing EMT and autophagy in ESCC cells via the AMPK-modified pathway.Thus,the facilitating role on ESCC cell migration should be considered for BRD4 inhibitor clinical application to ESCC patients.
文摘BACKGROUND Esophageal squamous cell carcinoma(ESCC)is one of the most common malignancies.A total of 45 kinesin superfamily proteins(KIFs)have been identified in humans,among which several family members have demonstrated varied functions in tumor pathobiology via different mechanisms,including regulation of cell cycle progression and metastasis.KIFC3 has microtubule motor activity and is involved in cancer cell invasion and migration,as well as survival.However,the role of KIFC3 in ESCC is still unknown.AIM To evaluate the role of KIFC3 in ESCC and the underlying mechanisms.METHODS Expression of KIFC3 was evaluated in ESCC tissues and adjacent normal esophageal tissues.The prognostic value of KIFC3 was analyzed using Kaplan-Meier Plotter.Colony formation,EdU assays,cell cycle analysis,Transwell assay,immunofluorescence,and western blotting were performed in ESCC cell lines after transfection with pLVX-Puro-KIFC3-shRNA-and pLVX-Puro-KIFC3-expressing lentiviruses.A xenograft tumor model in nude mice was used to evaluate the role of KIFC3 in tumorigenesis.Inhibitor ofβ-catenin,XAV-939,was used to clarify the mechanism of KIFC3 in ESCC.To analyze the differences between groups,t test and nonparametric tests were used.P<0.05 was considered statistically significant.RESULTS Immunohistochemical staining indicated that KIFC3 was upregulated in ESCC tissues compared with adjacent normal tissues.Kaplan-Meier Plotter revealed that overexpressed KIFC3 was associated with poor prognosis in ESCC patients.Colony formation and EdU assay showed that KIFC3 overexpression promoted cell proliferation,while KIFC3 knockdown inhibited cell proliferation in ESCC cell lines.In addition,cell cycle analysis showed that KIFC3 overexpression promoted cell cycle progression.KIFC3 knockdown suppressed ESCC tumorigenesis in vivo.Transwell assay and western blotting revealed that KIFC3 overexpression promoted cell migration and invasion,as well as epithelial-mesenchymal transition(EMT),while KIFC3 knockdown showed the opposite results.Mechanistically,KIFC3 overexpression promoted β-catenin signaling in KYSE450 cells;however,the role of KIFC3 was abolished by XAV-939,the inhibitor of β-catenin signaling.CONCLUSION KIFC3 was overexpressed in ESCC and was associated with poor prognosis.Furthermore,KIFC3 promoted proliferation,migration and invasion of ESCC via β-catenin signaling and EMT.
基金Supported by Key Technologies R&D Program of Science and Technology Commission of Henan Province,No.152102310110 to Zhao BSKey Science and Technique Fund of Xinxiang,No.ZG15018 to Zhao BS
文摘AIM To explore the effect of miR-382 on esophageal squamous cell carcinoma (ESCC) in vitro and its possible molecular mechanism. METHODS Eca 109 cells derived from human ESCC and Het-1A cells derived from human normal esophageal epithelium were used. Lentivirus-mediated miR-382 was overexpressed in Eca109 cells. The effect of miR-382 on cell proliferation was evaluated by MTT and colony formation assay. For cell cycle analysis, cells were fixed and stained for 30 min with propidium iodide (PI) staining buffer containing 10 mg/mL PI and 100 mg/mL RNase A, and analyzed by BD FACSCalibur (TM) flow cytometer. For cell apoptosis assay, cells were stained with an Annexin V-FITC/PI Apoptosis Detection Kit according to the manufacturer's instructions and analyzed by a dual-laser flow cytometer. Cell invasion and migration abilities were determined through use of transwell chambers, non-coated or pre-coated with matrigel. Levels of proteins related to cell growth and migration were examined by western blotting. RESULTS Endogenous miR-382 was down-regulated in Eca109 cells compared with Het-1A. Introduction of miR-382 not only significantly inhibited proliferation and colony formation, but also arrested cell cycle at the G2/M phase, as well as promoted apoptosis and autophagy in Eca109 cells. Migration, invasion and epithelial-mesenchymal transition of Eca109 cells were suppressed by overexpressing miR-382. Western blotting results showed that miR-382 inhibited the phosphorylation of mTOR and 4E-BP1. CONCLUSION miR-382 functions as a tumor suppressor against ESCC development and metastasis, and could be considered as a potential drug source for the treatment of ESCC patients.
文摘目的探究DDX解旋酶39A(DEAD-box RNA helicases 39A,DDX39A)对食管鳞癌KYSE-150和TE-1细胞生物学行为的影响及作用机制。方法利用癌症基因组图谱(the cancer genome atlas,TCGA)和高通量基因表达数据库(gene expression omnibus,GEO)分析DDX39A在食管鳞癌肿瘤组织和正常组织中的表达差异。进一步在TCGA数据库中,采用Spearman相关分析(设置r>0.5,P<0.05)得到与DDX39A表达最相关基因,通过京都基因与基因组百科全书(Kyoto encyclopedia of genes and genomes,KEGG)和基因集富集分析(gene set enrichment analysis,GSEA),注释DDX39A在食管鳞癌中的生物学功能。通过慢病毒介导的短发夹RNA(shRNA)干扰技术下调DDX39A在食管鳞癌KYSE-150和TE-1细胞中的表达,并将细胞分为对照(shCtrl)组和DDX39A敲减(shDDX39A)组。利用qPCR和Western blot法检测敲减效率;利用Celigo法、克隆形成实验和MTT法检测KYSE-150和TE-1细胞的增殖能力和活力;利用流式细胞术检测KYSE-150和TE-1细胞的凋亡水平;利用Transwell实验检测KYSE-150和TE-1细胞的迁移和侵袭能力;利用裸鼠成瘤实验检测敲减DDX39A对体内肿瘤的作用。利用Western blot法筛选敲减DDX39A后KYSE-150细胞内蛋白表达水平显著变化的肿瘤相关经典通路分子。通过慢病毒转染法构建已筛选的分子CDH2(Cadherin 2)过表达的稳转细胞系,并将KYSE-150细胞分为shDDX39A+NC-OE组(转染shDDX39A慢病毒和过表达空载对照病毒)和shDDX39A+CDH2-OE组(转染shDDX39A慢病毒和过表达CDH2病毒)。利用Celigo法、MTT法和Transwell实验分别检测过表达CDH2对DDX39A敲减细胞增殖能力、活力以及转移能力的影响。结果与正常组织相比,食管鳞癌组织中DDX39A的表达显著上调(P<0.001)。DDX39A及其相关基因主要作用于遗传信息的翻译与加工和细胞凋亡等过程。与shCtrl组相比,shDDX39A组KYSE-150和TE-1细胞中DDX39A的mRNA和蛋白表达显著降低(P<0.01),KYSE-150和TE-1细胞增殖和细胞活力显著下降(P<0.001),KYSE-150和TE-1细胞的克隆形成能力显著降低(P<0.01),KYSE-150和TE-1细胞的凋亡水平显著升高(P<0.01),KYSE-150和TE-1细胞的迁移和侵袭能力显著下降(P<0.001)。裸鼠成瘤实验提示DDX39A敲减后瘤体的体积和质量均明显减少(P<0.001)。与shDDX39A+NC-OE组相比,shDDX39A+CDH2-OE组KYSE-150细胞的增殖、细胞活力以及转移能力均显著提高(P<0.001)。结论DDX39A可作为食管鳞癌诊治的新靶点,上调其表达可能通过诱导上皮间充质转化促进食管鳞癌细胞生长、迁移与侵袭。
