Background Dysregulated metallothionein 2A （MT2A） has been implicated in carcinogenesis. The purpose of this study was to investigate the expression of MT2A in gastric cancer （GC） and its correlation with prognosi...Background Dysregulated metallothionein 2A （MT2A） has been implicated in carcinogenesis. The purpose of this study was to investigate the expression of MT2A in gastric cancer （GC） and its correlation with prognosis. Methods Reverse transcription-polymerase chain reaction and real-time polymerase chain reaction were used to detect the mRNA expression of MT2A in 12 GC cell lines, normal gastric epithelial GES-1 cells, and 36 GC and adjacent normal tissues. MT2A protein expression was determined in 258 GC tissues and 171 adjacent normal tissues by immunohistochemistry. Results MT2A mRNA expression was lower in GC cells and primary tumors than in GES-1 cells and adjacent normal tissues, respectively, High protein expression of MT2A was present in 130 of 171 normal tissues （76.0%） and in 56 of 258 GC tissues （21,7%; P 〈0.001）. MT2A protein expression was higher in well/moderately differentiated GC （22/54; 40.7%） than in poorly differentiated GC （34/204; 16.7%; P 〈0.001）. Moreover, the protein expression of MT2A was lower in diffuse-type GC （6/82; 7.3%） than in intestinal-type GC （50/176; 28.4%; P=0.0001）. Importantly, MT2A expression was an independent prognostic factor for GC, and decreased MT2A expression was associated with poor clinical outcome （P 〈0.001）. The expression status of MT2A could predict prognosis in intestinal and diffuse-type GCs. Conclusion Expression status of MT2A might be a useful prognostic biomarker for GC, especially when used in combination with Lauren＇s classification.展开更多
【目的】通过对猪(Sus Scrofa)金属硫蛋白1A(metallothionein-1A of S.Scrofa,SsMT-1A)和2A(Metallothionein-2A of S.Scrofa,SsMT-2A)的生物信息学分析、原核表达和纯化,研究它们与Zn(Ⅱ)和Cu(Ⅰ)的结合特性。为饲料中添加Zn和Cu在促...【目的】通过对猪(Sus Scrofa)金属硫蛋白1A(metallothionein-1A of S.Scrofa,SsMT-1A)和2A(Metallothionein-2A of S.Scrofa,SsMT-2A)的生物信息学分析、原核表达和纯化,研究它们与Zn(Ⅱ)和Cu(Ⅰ)的结合特性。为饲料中添加Zn和Cu在促进猪生产性能的作用机制研究提供理论基础。【方法】首先,从NCBI中获得SsMT-1A和SsMT-2A的基因序列,利用ClustalX2和ExPASy分析两者的蛋白序列和结构差异,利用MEGA-X构建两者与其他物种MT蛋白分子的进化树。其次,构建pET-28a-SUMO-SsMT-1A/SsMT-2A原核表达载体,转化BL21(DE3)plysS,用IPTG诱导表达。利用Ni-NTA柱亲和层析和葡聚糖凝胶层析纯化重组蛋白。最后,利用大肠杆菌金属耐受性实验、圆二色光谱(circular dichroism,CD)、基质辅助激光解析电离飞行时间质谱(matrix assisted laser analytic ionization time of flight mass spectrometry,MALDI-TOF-MS)和等温微量热仪(isothermal micrometer calorimetry,ITC)分析SsMT-1A和SsMT-2A与Zn(Ⅱ)和Cu(Ⅰ)的结合特性。【结果】生物信息学分析表明:SsMT-1A和SsMT-2A蛋白分子的同源性较高,半胱氨酸(Cysteine,Cys)含量和排列基序完全一致,仅有8个非Cys位点差异。经原核表达、Ni-NTA柱和Superdex-75柱纯化、SUMO酶酶切,成功获得了SsMT-1A和SsMT-2A。金属耐受性试验表明:与SsMT-2A相比,转SsMT-1A大肠杆菌具有较强的Zn和Cu耐受性。CD光谱表明:SsMT-1A和SsMT-2A均可与Zn(Ⅱ)和Cu(Ⅰ)结合,两者均展示了Zn(Ⅱ)结合偏好性,然而,SsMT-1A较SsMT-2A具有较强的Zn(Ⅱ)和Cu(Ⅰ)结合能力。MALDI-TOF-MS表明:apo-SsMT-1A和apo-SsMT-2A的分子量分别为6047.5 Da和6048 Da,SsMT-1A和SsMT-2A与Zn(Ⅱ)的结合稳定,与Cu(Ⅰ)的结合不稳定。ITC表明:SsMT-1A和SsMT-2A与Zn(Ⅱ)的结合不稳定,与Cu(Ⅰ)的结合稳定,两者结合Cu(Ⅰ)的化学计量数均为7,SsMT-1A结合Zn(Ⅱ)的化学计量数为2。【结论】虽然SsMT-1A和SsMT-2A具有较高的同源性,然而,8个非Cys位点的差异决定了两者金属结合特性的差别。尽管SsMT-1A和SsMT-2A共享了高度一致的Zn(Ⅱ)和Cu(Ⅰ)结合行为,然而,两者与Zn(Ⅱ)和Cu(Ⅰ)的结合特性存在细微差别,两者不应被认为是完全生理等价分子。在生理条件下:SsMT-1A可能发挥重金属离子的解毒作用,SsMT-2A可能发挥Zn内稳态的调控作用。本研究为进一步阐明SsMT-1A和SsMT-2A调节金属离子内稳态,发挥促进猪生产性能的作用研究奠定了基础。展开更多
Micro RNA-491-5 p(miR-491-5 p) plays an important role in regulating cell proliferation and migration;however,the effect of miR-491-5 p on neovascularization after traumatic brain injury remains poorly understood.In t...Micro RNA-491-5 p(miR-491-5 p) plays an important role in regulating cell proliferation and migration;however,the effect of miR-491-5 p on neovascularization after traumatic brain injury remains poorly understood.In this study,a controlled cortical injury model in C57 BL/6 mice and an oxygen-glucose deprivation model in microvascular endothelial cells derived from mouse brain were established to simulate traumatic brain injury in vivo and in vitro,respectively.In the in vivo model,quantitative real-time-polymerase chain reaction results showed that the expression of miR-491-5 p increased or decreased following the intracerebroventricular injection of an miR-491-5 p agomir or antagomir,respectively,and the expression of miR-491-5 p decreased slightly after traumatic brain injury.To detect the neuroprotective effects of miR-491-p,neurological severity scores,Morris water maze test,laser speckle techniques,and immunofluorescence staining were assessed,and the results revealed that miR-491-5 p downregulation alleviated neurological dysfunction,promoted the recovery of regional cerebral blood flow,increased the number of lectin-stained microvessels,and increased the survival of neurons after traumatic brain injury.During the in vitro experiments,the potential mechanism of miR-491-5 p on neovascularization was explored through quantitative real-time-polymerase chain reaction,which showed that miR-491-5 p expression increased or decreased in brain microvascular endothelial cells after transfection with an miR-491-5 p mimic or inhibitor,respectively.Dual-luciferase reporter and western blot assays verified that metallothionein-2 was a target gene for miR-491-5 p.Cell counting kit 8(CCK-8) assay,flow cytometry,and 2′,7′-dichlorofluorescein diacetate(DCFH-DA) assay results confirmed that the downregulation of miR-491-5 p increased brain microvascular endothelial cell viability,reduced cell apoptosis,and alleviated oxidative stress under oxygen-glucose deprivation conditions.Cell scratch assay,Transwell assay,tube formation assay,and western blot assay results demonstrated that miR-491-5 p downregulation promoted the migration,proliferation,and tube formation of brain microvascular endothelial cells through a metallothionein-2-dependent hypoxia-inducible factor-1α/vascular endothelial growth factor pathway.These findings confirmed that miR-491-5 p downregulation promotes neovascularization,restores cerebral blood flow,and improves the recovery of neurological function after traumatic brain injury.The mechanism may be mediated through a metallothionein-2-dependent hypoxia-inducible factor-1α/vascular endothelial growth factor signaling pathway and the alleviation of oxidative stress.All procedures were approved by Ethics Committee of the First Affiliated Hospital of Chongqing Medical University,China(approval No.2020-304) on June 22,2020.展开更多
目的探讨金属硫蛋白2A(MT-2A)、E钙黏蛋白(E-cadherin)、白细胞介素6(IL-6)、细胞周期蛋白E(cyclin E)、增殖细胞核抗原(PCNA)和抗凋亡蛋白bcl-2在前列腺癌组织中的表达及其与前列腺癌生化复发之间的相关性。方法收集2012年10月至2017...目的探讨金属硫蛋白2A(MT-2A)、E钙黏蛋白(E-cadherin)、白细胞介素6(IL-6)、细胞周期蛋白E(cyclin E)、增殖细胞核抗原(PCNA)和抗凋亡蛋白bcl-2在前列腺癌组织中的表达及其与前列腺癌生化复发之间的相关性。方法收集2012年10月至2017年10月于山西大医院接受前列腺癌根治性手术的128例患者的前列腺癌组织标本,记录患者的临床数据。制作组织芯片,采用免疫组织化学生物素-亲和素复合物(ABC)染色法检测芯片组织MT-2A、E-cadherin、IL-6、cyclin E、PCNA和bcl-2的表达水平,并分析不同分子标志物与前列腺癌生化复发之间的相关性。结果 128例前列腺癌患者生化复发率为30.5%(39/128),低危、中危和高危前列腺癌患者生化复发率分别是14.8%(8/54)、38.7%(24/62)和58.3%(7/12)。前列腺癌患者危险分级和病理学T分期与MT-2A、cyclin E、IL-6、E-cadherin表达均有关(均P<0.05)。多因素Cox风险模型结果显示,高危险分级(HR=1.81,95% CI 1.56~2.19,P=0.042)、MT-2A阳性表达(HR=2.01,95% CI 1.08~3.15,P=0.005)、cyclin E阳性表达(HR=1.79,95% CI 1.08~2.21,P=0.042)和E-cadherin阴性表达(HR=1.92,95% CI 1.22~2.45,P=0.020)是前列腺癌生化复发的独立危险因素。结论前列腺癌组织中MT-2A、cyclin E和E-cadherin是预测前列腺癌生化复发的重要分子标志物。展开更多
文摘Background Dysregulated metallothionein 2A （MT2A） has been implicated in carcinogenesis. The purpose of this study was to investigate the expression of MT2A in gastric cancer （GC） and its correlation with prognosis. Methods Reverse transcription-polymerase chain reaction and real-time polymerase chain reaction were used to detect the mRNA expression of MT2A in 12 GC cell lines, normal gastric epithelial GES-1 cells, and 36 GC and adjacent normal tissues. MT2A protein expression was determined in 258 GC tissues and 171 adjacent normal tissues by immunohistochemistry. Results MT2A mRNA expression was lower in GC cells and primary tumors than in GES-1 cells and adjacent normal tissues, respectively, High protein expression of MT2A was present in 130 of 171 normal tissues （76.0%） and in 56 of 258 GC tissues （21,7%; P 〈0.001）. MT2A protein expression was higher in well/moderately differentiated GC （22/54; 40.7%） than in poorly differentiated GC （34/204; 16.7%; P 〈0.001）. Moreover, the protein expression of MT2A was lower in diffuse-type GC （6/82; 7.3%） than in intestinal-type GC （50/176; 28.4%; P=0.0001）. Importantly, MT2A expression was an independent prognostic factor for GC, and decreased MT2A expression was associated with poor clinical outcome （P 〈0.001）. The expression status of MT2A could predict prognosis in intestinal and diffuse-type GCs. Conclusion Expression status of MT2A might be a useful prognostic biomarker for GC, especially when used in combination with Lauren＇s classification.
文摘【目的】通过对猪(Sus Scrofa)金属硫蛋白1A(metallothionein-1A of S.Scrofa,SsMT-1A)和2A(Metallothionein-2A of S.Scrofa,SsMT-2A)的生物信息学分析、原核表达和纯化,研究它们与Zn(Ⅱ)和Cu(Ⅰ)的结合特性。为饲料中添加Zn和Cu在促进猪生产性能的作用机制研究提供理论基础。【方法】首先,从NCBI中获得SsMT-1A和SsMT-2A的基因序列,利用ClustalX2和ExPASy分析两者的蛋白序列和结构差异,利用MEGA-X构建两者与其他物种MT蛋白分子的进化树。其次,构建pET-28a-SUMO-SsMT-1A/SsMT-2A原核表达载体,转化BL21(DE3)plysS,用IPTG诱导表达。利用Ni-NTA柱亲和层析和葡聚糖凝胶层析纯化重组蛋白。最后,利用大肠杆菌金属耐受性实验、圆二色光谱(circular dichroism,CD)、基质辅助激光解析电离飞行时间质谱(matrix assisted laser analytic ionization time of flight mass spectrometry,MALDI-TOF-MS)和等温微量热仪(isothermal micrometer calorimetry,ITC)分析SsMT-1A和SsMT-2A与Zn(Ⅱ)和Cu(Ⅰ)的结合特性。【结果】生物信息学分析表明:SsMT-1A和SsMT-2A蛋白分子的同源性较高,半胱氨酸(Cysteine,Cys)含量和排列基序完全一致,仅有8个非Cys位点差异。经原核表达、Ni-NTA柱和Superdex-75柱纯化、SUMO酶酶切,成功获得了SsMT-1A和SsMT-2A。金属耐受性试验表明:与SsMT-2A相比,转SsMT-1A大肠杆菌具有较强的Zn和Cu耐受性。CD光谱表明:SsMT-1A和SsMT-2A均可与Zn(Ⅱ)和Cu(Ⅰ)结合,两者均展示了Zn(Ⅱ)结合偏好性,然而,SsMT-1A较SsMT-2A具有较强的Zn(Ⅱ)和Cu(Ⅰ)结合能力。MALDI-TOF-MS表明:apo-SsMT-1A和apo-SsMT-2A的分子量分别为6047.5 Da和6048 Da,SsMT-1A和SsMT-2A与Zn(Ⅱ)的结合稳定,与Cu(Ⅰ)的结合不稳定。ITC表明:SsMT-1A和SsMT-2A与Zn(Ⅱ)的结合不稳定,与Cu(Ⅰ)的结合稳定,两者结合Cu(Ⅰ)的化学计量数均为7,SsMT-1A结合Zn(Ⅱ)的化学计量数为2。【结论】虽然SsMT-1A和SsMT-2A具有较高的同源性,然而,8个非Cys位点的差异决定了两者金属结合特性的差别。尽管SsMT-1A和SsMT-2A共享了高度一致的Zn(Ⅱ)和Cu(Ⅰ)结合行为,然而,两者与Zn(Ⅱ)和Cu(Ⅰ)的结合特性存在细微差别,两者不应被认为是完全生理等价分子。在生理条件下:SsMT-1A可能发挥重金属离子的解毒作用,SsMT-2A可能发挥Zn内稳态的调控作用。本研究为进一步阐明SsMT-1A和SsMT-2A调节金属离子内稳态,发挥促进猪生产性能的作用研究奠定了基础。
基金supported by the National Natural Science Foundation of China,Nos.82071397 (to XCS),82071332 (to ZDG)the Youth Fund of the National Natural Science Foundation of China,No.81801230 (to JJZ)the Excellent Scientific Research Talents Fund of the First Affiliated Hospital of Chongqing Medical University,China (to JJZ)。
文摘Micro RNA-491-5 p(miR-491-5 p) plays an important role in regulating cell proliferation and migration;however,the effect of miR-491-5 p on neovascularization after traumatic brain injury remains poorly understood.In this study,a controlled cortical injury model in C57 BL/6 mice and an oxygen-glucose deprivation model in microvascular endothelial cells derived from mouse brain were established to simulate traumatic brain injury in vivo and in vitro,respectively.In the in vivo model,quantitative real-time-polymerase chain reaction results showed that the expression of miR-491-5 p increased or decreased following the intracerebroventricular injection of an miR-491-5 p agomir or antagomir,respectively,and the expression of miR-491-5 p decreased slightly after traumatic brain injury.To detect the neuroprotective effects of miR-491-p,neurological severity scores,Morris water maze test,laser speckle techniques,and immunofluorescence staining were assessed,and the results revealed that miR-491-5 p downregulation alleviated neurological dysfunction,promoted the recovery of regional cerebral blood flow,increased the number of lectin-stained microvessels,and increased the survival of neurons after traumatic brain injury.During the in vitro experiments,the potential mechanism of miR-491-5 p on neovascularization was explored through quantitative real-time-polymerase chain reaction,which showed that miR-491-5 p expression increased or decreased in brain microvascular endothelial cells after transfection with an miR-491-5 p mimic or inhibitor,respectively.Dual-luciferase reporter and western blot assays verified that metallothionein-2 was a target gene for miR-491-5 p.Cell counting kit 8(CCK-8) assay,flow cytometry,and 2′,7′-dichlorofluorescein diacetate(DCFH-DA) assay results confirmed that the downregulation of miR-491-5 p increased brain microvascular endothelial cell viability,reduced cell apoptosis,and alleviated oxidative stress under oxygen-glucose deprivation conditions.Cell scratch assay,Transwell assay,tube formation assay,and western blot assay results demonstrated that miR-491-5 p downregulation promoted the migration,proliferation,and tube formation of brain microvascular endothelial cells through a metallothionein-2-dependent hypoxia-inducible factor-1α/vascular endothelial growth factor pathway.These findings confirmed that miR-491-5 p downregulation promotes neovascularization,restores cerebral blood flow,and improves the recovery of neurological function after traumatic brain injury.The mechanism may be mediated through a metallothionein-2-dependent hypoxia-inducible factor-1α/vascular endothelial growth factor signaling pathway and the alleviation of oxidative stress.All procedures were approved by Ethics Committee of the First Affiliated Hospital of Chongqing Medical University,China(approval No.2020-304) on June 22,2020.
文摘目的探讨金属硫蛋白2A(MT-2A)、E钙黏蛋白(E-cadherin)、白细胞介素6(IL-6)、细胞周期蛋白E(cyclin E)、增殖细胞核抗原(PCNA)和抗凋亡蛋白bcl-2在前列腺癌组织中的表达及其与前列腺癌生化复发之间的相关性。方法收集2012年10月至2017年10月于山西大医院接受前列腺癌根治性手术的128例患者的前列腺癌组织标本,记录患者的临床数据。制作组织芯片,采用免疫组织化学生物素-亲和素复合物(ABC)染色法检测芯片组织MT-2A、E-cadherin、IL-6、cyclin E、PCNA和bcl-2的表达水平,并分析不同分子标志物与前列腺癌生化复发之间的相关性。结果 128例前列腺癌患者生化复发率为30.5%(39/128),低危、中危和高危前列腺癌患者生化复发率分别是14.8%(8/54)、38.7%(24/62)和58.3%(7/12)。前列腺癌患者危险分级和病理学T分期与MT-2A、cyclin E、IL-6、E-cadherin表达均有关(均P<0.05)。多因素Cox风险模型结果显示,高危险分级(HR=1.81,95% CI 1.56~2.19,P=0.042)、MT-2A阳性表达(HR=2.01,95% CI 1.08~3.15,P=0.005)、cyclin E阳性表达(HR=1.79,95% CI 1.08~2.21,P=0.042)和E-cadherin阴性表达(HR=1.92,95% CI 1.22~2.45,P=0.020)是前列腺癌生化复发的独立危险因素。结论前列腺癌组织中MT-2A、cyclin E和E-cadherin是预测前列腺癌生化复发的重要分子标志物。
