【目的】对武夷黑猪热休克蛋白A5(heat shock protein family A member 5,HSPA5)进行生物信息学预测与分析,为探究其生物学功能提供参考。【方法】对武夷黑猪HSPA5基因编码区进行克隆并测序,再与不同物种进行相似性比对并构建系统进化树...【目的】对武夷黑猪热休克蛋白A5(heat shock protein family A member 5,HSPA5)进行生物信息学预测与分析,为探究其生物学功能提供参考。【方法】对武夷黑猪HSPA5基因编码区进行克隆并测序,再与不同物种进行相似性比对并构建系统进化树,利用生物信息学方法预测其蛋白的理化特性、结构及功能,最后测定其在武夷黑猪不同组织中的表达量。【结果】武夷黑猪HSPA5基因编码区长1965 bp,编码654个氨基酸。相似性比对和进化树分析结果表明,武夷黑猪HSPA5氨基酸序列与杜洛克猪转录本变体2和鼠的HSPA5氨基酸序列相似性最高(100%),且与鼠的亲源关系最近,而与大黄花鱼的亲源关系最远。武夷黑猪HSPA5蛋白为酸性亲水蛋白,含有信号肽序列,在内质网中含量最高。主要由α-螺旋构成,且保守基序位于2个重叠的功能结构域内;含有56个磷酸化位点,76个O-糖基化位点,24个化学因子作用位点。同时具有26个抗原决定簇、11个T细胞抗原表位,13个B细胞抗原表位。与热休克蛋白90(heat shock protein member 90,HSP90)、激活转录因子6(activating transcription factor 6,ATF6)等互作关系密切。实时荧光定量PCR结果表明,HSPA5基因在武夷黑猪肝脏和子宫中的表达量显著高于其他组织(P<0.05)。【结论】本研究成功克隆了武夷黑猪HSPA5基因,且与鼠的亲源关系最近。武夷黑猪HSPA5蛋白主要定位于细胞质中,包含磷酸化、O-糖基化修饰位点,含有与T或B细胞抗原表位重叠的抗原决定簇序列,与未折叠蛋白反应通路中相关蛋白互作,在肝脏表达量最高。这些特征可为进一步研究HSPA5蛋白生物学功能提供参考。展开更多
Objective: To investigate the effects of regulating heat shock protein 70protein 5(HSPA5) on ferroptosis, proliferation and apoptosis of cervical cancer cells in a rat model of cervical cancer. Methods: A total of 48 ...Objective: To investigate the effects of regulating heat shock protein 70protein 5(HSPA5) on ferroptosis, proliferation and apoptosis of cervical cancer cells in a rat model of cervical cancer. Methods: A total of 48 SPF-grade healthy adult female Wistar rats were selected as the research objects. After adaptive feeding, U14 cells were intraperitoneally injected to construct a cervical cancer model. After successful modeling,the rats were randomly divided into four groups: the blank group was given normal saline by gavage for 7 d. The HM03+1-day group was given 10 mg/kg HM03 by gavage for 1 d followed by normal saline for 6 d, the HM03+3-day group was given 10 mg/kg HM03 by gavage for 3 d followed by normal saline for 4 d, and the HM03+7-day group was given10 mg/kg HM03 by gavage for 7 d. Cervical lesion tissues of the rats were taken to prepare cell suspensions, which were then cultured to assess the expressions of HSPA5,glutathione peroxidase 4(GPX4), overoxidized peroxidase 3(SO2/3-PRDX3), and iron(Fe^(2+))in the cervical cancer cells, as well as the proliferation and apoptosis of the cervical cancer cells, were detected. Subsequently, 6 cell suspensions from each group were treated with 10 μmol/L Ferrostatin-1(Fer-1) for 48 h, after which reactive oxygen species(ROS) and lactate dehydrogenase(LDH) expression were measured. Results: HSPA5 mRNA and protein expression in HM03-treated rat cervical cancer cells were significantly lower than those in the blank group(P<0.05), and with the extension of HM03 treatment time, HSPA5 mRNA and protein expression in rat cervical cancer cells were consequently lower(P<0.05). Relative expression of GPX4 protein in HM03-treated rat cervical cancer cells was significantly lower than that in the blank group(The relative expression of GPX4 protein in HM03-treated rat cervical cancer cells was significantly lower than that in the blank group(P<0.05), and the relative expression of SO_(2/3)-PRDX3protein and Fe^(2+)expression level were significantly higher than that in the blank group,and with the extension of the treatment time of HM03, the relative expression of GPX4protein in rat cervical cancer cells was reduced(P<0.05), and the relative expression of SO2/3-PRDX3 protein and Fe^(2+)expression were increased. The proliferation of cervical cancer cells in HM03-treated rats was significantly reduced, the apoptosis was promoted,and the number of cells decreased, and with the extension of HM03 treatment, the proliferation of cervical cancer cells was reduced, and the rate of apoptosis was increased.After the rat cervical cancer cells were partially treated with Fer-1, the expression levels of both ROS and LDH were significantly reduced compared with those of the same group without Fer-1 treatment(P<0.05). Conclusion: Down-regulation of HSPA5 expression can reduce the proliferation ability of cervical cancer cells and promote apoptosis by activating ferroptosis in rat cervical cancer cells.展开更多
目的探讨热休克蛋白A5基因(HSPA5)与乙型肝炎病毒(HBV)侵袭的关系。方法对210例HBV携带者和194例性别年龄匹配的健康者标本的PCR产物进行基因测序分型。酶联免疫吸附试验(ELISA)或临床化学方法检测HBV感染的血清学标志物。结果病例组的...目的探讨热休克蛋白A5基因(HSPA5)与乙型肝炎病毒(HBV)侵袭的关系。方法对210例HBV携带者和194例性别年龄匹配的健康者标本的PCR产物进行基因测序分型。酶联免疫吸附试验(ELISA)或临床化学方法检测HBV感染的血清学标志物。结果病例组的等位基因和基因型分布与对照组相比差异无统计学意义。此外,HBV感染的个体中,ALT/HBeAg和HBV-DNA均与等位基因或基因型的变异无相关性。结论 HSPA5基因的-86 bp T/A多态性与HBV侵袭的临床风险和急性恶化没有相关性。展开更多
文摘【目的】对武夷黑猪热休克蛋白A5(heat shock protein family A member 5,HSPA5)进行生物信息学预测与分析,为探究其生物学功能提供参考。【方法】对武夷黑猪HSPA5基因编码区进行克隆并测序,再与不同物种进行相似性比对并构建系统进化树,利用生物信息学方法预测其蛋白的理化特性、结构及功能,最后测定其在武夷黑猪不同组织中的表达量。【结果】武夷黑猪HSPA5基因编码区长1965 bp,编码654个氨基酸。相似性比对和进化树分析结果表明,武夷黑猪HSPA5氨基酸序列与杜洛克猪转录本变体2和鼠的HSPA5氨基酸序列相似性最高(100%),且与鼠的亲源关系最近,而与大黄花鱼的亲源关系最远。武夷黑猪HSPA5蛋白为酸性亲水蛋白,含有信号肽序列,在内质网中含量最高。主要由α-螺旋构成,且保守基序位于2个重叠的功能结构域内;含有56个磷酸化位点,76个O-糖基化位点,24个化学因子作用位点。同时具有26个抗原决定簇、11个T细胞抗原表位,13个B细胞抗原表位。与热休克蛋白90(heat shock protein member 90,HSP90)、激活转录因子6(activating transcription factor 6,ATF6)等互作关系密切。实时荧光定量PCR结果表明,HSPA5基因在武夷黑猪肝脏和子宫中的表达量显著高于其他组织(P<0.05)。【结论】本研究成功克隆了武夷黑猪HSPA5基因,且与鼠的亲源关系最近。武夷黑猪HSPA5蛋白主要定位于细胞质中,包含磷酸化、O-糖基化修饰位点,含有与T或B细胞抗原表位重叠的抗原决定簇序列,与未折叠蛋白反应通路中相关蛋白互作,在肝脏表达量最高。这些特征可为进一步研究HSPA5蛋白生物学功能提供参考。
文摘Objective: To investigate the effects of regulating heat shock protein 70protein 5(HSPA5) on ferroptosis, proliferation and apoptosis of cervical cancer cells in a rat model of cervical cancer. Methods: A total of 48 SPF-grade healthy adult female Wistar rats were selected as the research objects. After adaptive feeding, U14 cells were intraperitoneally injected to construct a cervical cancer model. After successful modeling,the rats were randomly divided into four groups: the blank group was given normal saline by gavage for 7 d. The HM03+1-day group was given 10 mg/kg HM03 by gavage for 1 d followed by normal saline for 6 d, the HM03+3-day group was given 10 mg/kg HM03 by gavage for 3 d followed by normal saline for 4 d, and the HM03+7-day group was given10 mg/kg HM03 by gavage for 7 d. Cervical lesion tissues of the rats were taken to prepare cell suspensions, which were then cultured to assess the expressions of HSPA5,glutathione peroxidase 4(GPX4), overoxidized peroxidase 3(SO2/3-PRDX3), and iron(Fe^(2+))in the cervical cancer cells, as well as the proliferation and apoptosis of the cervical cancer cells, were detected. Subsequently, 6 cell suspensions from each group were treated with 10 μmol/L Ferrostatin-1(Fer-1) for 48 h, after which reactive oxygen species(ROS) and lactate dehydrogenase(LDH) expression were measured. Results: HSPA5 mRNA and protein expression in HM03-treated rat cervical cancer cells were significantly lower than those in the blank group(P<0.05), and with the extension of HM03 treatment time, HSPA5 mRNA and protein expression in rat cervical cancer cells were consequently lower(P<0.05). Relative expression of GPX4 protein in HM03-treated rat cervical cancer cells was significantly lower than that in the blank group(The relative expression of GPX4 protein in HM03-treated rat cervical cancer cells was significantly lower than that in the blank group(P<0.05), and the relative expression of SO_(2/3)-PRDX3protein and Fe^(2+)expression level were significantly higher than that in the blank group,and with the extension of the treatment time of HM03, the relative expression of GPX4protein in rat cervical cancer cells was reduced(P<0.05), and the relative expression of SO2/3-PRDX3 protein and Fe^(2+)expression were increased. The proliferation of cervical cancer cells in HM03-treated rats was significantly reduced, the apoptosis was promoted,and the number of cells decreased, and with the extension of HM03 treatment, the proliferation of cervical cancer cells was reduced, and the rate of apoptosis was increased.After the rat cervical cancer cells were partially treated with Fer-1, the expression levels of both ROS and LDH were significantly reduced compared with those of the same group without Fer-1 treatment(P<0.05). Conclusion: Down-regulation of HSPA5 expression can reduce the proliferation ability of cervical cancer cells and promote apoptosis by activating ferroptosis in rat cervical cancer cells.
基金supported by the National Natural Science Foundation of China(No.81600001BJ6)the Jiangsu Province’s Key Provincial Talents Program(No.ZDRCA2016005)+3 种基金the Natural Science Foundation of Jiangsu ProvinceChina(No.BK20141026 DA14)the Jiangsu Science&Technology ProgramChina(No.BE2016002-4)
文摘本研究旨在探讨S100钙结合蛋白A16 (S100 calcium binding protein A16, S100A16)在肝细胞脂质代谢中的作用及可能的生物学机制。用脂肪酸培养HepG2细胞(人肝癌细胞系)以建立脂肪酸培养模型,对照模型不加脂肪酸培养,每一模型分成3组细胞,分别用S100a16过表达质粒、shRNA质粒、Vector质粒转染。用试剂盒检测细胞甘油三酯浓度,用油红O染色观察脂滴聚集情况,用免疫沉淀和质谱分析寻找和S100A16相互作用的兴趣蛋白,并用免疫共沉淀验证,用Western blot和qRTPCR进行相关机制研究。结果显示,和对照模型相比,脂肪酸培养模型细胞内脂肪和甘油三酯浓度显著增加。S100a16过表达组细胞内脂肪积累显著高于Vector质粒转染组。热休克蛋白A5 (heat shock protein A5, HSPA5)与S100A16之间存在相互作用。S100a16过表达可上调内质网应激的HSPA5/肌醇依赖酶1α-X结合蛋白1 (inositol-requiring enzyme 1α-X binding protein 1,IRE1α-XBP1)通路相关蛋白(HSPA5、IRE1α和pIREα1)表达水平,并上调脂肪合成相关基因Srebp1c、Acc和Fas mRNA表达水平,而转染S100A16 shRNA质粒可使上述蛋白和mRNA水平低于Vector质粒转染组。以上结果提示,S100A16可能通过内质网应激HSPA5/IRE1α-XBP1通路促进HepG2细胞脂质合成。
文摘目的探讨热休克蛋白A5基因(HSPA5)与乙型肝炎病毒(HBV)侵袭的关系。方法对210例HBV携带者和194例性别年龄匹配的健康者标本的PCR产物进行基因测序分型。酶联免疫吸附试验(ELISA)或临床化学方法检测HBV感染的血清学标志物。结果病例组的等位基因和基因型分布与对照组相比差异无统计学意义。此外,HBV感染的个体中,ALT/HBeAg和HBV-DNA均与等位基因或基因型的变异无相关性。结论 HSPA5基因的-86 bp T/A多态性与HBV侵袭的临床风险和急性恶化没有相关性。
