BACKGROUND The function of prohibitin 1(Phb1)during liver regeneration(LR)remains relatively unexplored.Our previous research identified downregulation of Phb1 in rat liver mitochondria 24 h after 70%partial hepatecto...BACKGROUND The function of prohibitin 1(Phb1)during liver regeneration(LR)remains relatively unexplored.Our previous research identified downregulation of Phb1 in rat liver mitochondria 24 h after 70%partial hepatectomy(PHx),as determined by subcellular proteomic analysis.AIM To investigate the potential role of Phb1 during LR.METHODS We examined changes in Phb1 mRNA and protein levels,subcellular distribution,and abundance in rat liver during LR following 70%PHx.We also evaluated mitochondrial changes and apoptosis using electron microscopy and flow cytometry.RNA-interference-mediated knockdown of Phb1(PHBi)was performed in BRL-3A cells.RESULTS Compared with sham-operation control groups,Phb1 mRNA and protein levels in 70%PHx test groups were downregulated at 24 h,then upregulated at 72 and 168 h.Phb1 was mainly located in mitochondria,showed a reduced abundance at 24 h,significantly increased at 72 h,and almost recovered to normal at 168 h.Phb1 was also present in nuclei,with continuous increase in abundance observed 72 and 168 h after 70%PHx.The altered ultrastructure and reduced mass of mitochondria during LR had almost completely recovered to normal at 168 h.PHBi in BRL-3A cells resulted in increased S-phase entry,a higher number of apoptotic cells,and disruption of mitochondrial membrane potential.CONCLUSION Phb1 may contribute to maintaining mitochondrial stability and could play a role in regulating cell proliferation and apoptosis of rat liver cells during LR.展开更多
BACKGROUND Prohibitin 1(PHB1)has been identified as an antiproliferative protein that is highly conserved and ubiquitously expressed,and it participates in a variety of essential cellular functions,including apoptosis...BACKGROUND Prohibitin 1(PHB1)has been identified as an antiproliferative protein that is highly conserved and ubiquitously expressed,and it participates in a variety of essential cellular functions,including apoptosis,cell cycle regulation,prolifera-tion,and survival.Emerging evidence indicates that PHB1 may play an important role in the progression of hepatocellular carcinoma(HCC).However,the role of PHB1 in HCC is controversial.AIM To investigate the effects of PHB1 on the proliferation and apoptosis of human HCC cells and the relevant mechanisms in vitro.METHODS HCC patients and healthy individuals were enrolled in this study according to the inclusion and exclusion criteria;then,PHB1 levels in the sera and liver tissues of these participates were determined using ELISA,RT-PCR,and immunohistoche-mistry.Human HepG2 and SMMC-7721 cells were transfected with the pEGFP-PHB1 plasmid and PHB1-specific shRNA(shRNA-PHB1)for 24-72 h.Cell prolif-eration was analysed with an MTT assay.Cell cycle progression and apoptosis were analysed using flow cytometry(FACS).The mRNA and protein expression levels of the cell cycle-related molecules p21,Cyclin A2,Cyclin E1,and CDK2 and the cell apoptosis-related molecules cytochrome C(Cyt C),p53,Bcl-2,Bax,caspase 3,and caspase 9 were measured by real-time PCR and Western blot,respectively.RESULTS Decreased levels of PHB1 were found in the sera and liver tissues of HCC patients compared to those of healthy individuals,and decreased PHB1 was positively correlated with low differentiation,TNM stage III-IV,and alpha-fetoprotein≥400μg/L.Overexpression of PHB1 significantly inhibited human HCC cell proliferation in a time-dependent manner.FACS revealed that the overexpression of PHB1 arrested HCC cells in the G0/G1 phase of the cell cycle and induced apoptosis.The proportion of cells in the G0/G1 phase was significantly increased and the proportion of cells in the S phase was decreased in HepG2 cells that were transfected with pEGFP-PHB1 compared with untreated control and empty vector-tr展开更多
Prohibitin is named due to the negative regulatory role of its gene products in cell proliferation. Prohibitin gene is located at q21 of chromosome 17 in human beings and the protein is found at mitochondria, nucleus...Prohibitin is named due to the negative regulatory role of its gene products in cell proliferation. Prohibitin gene is located at q21 of chromosome 17 in human beings and the protein is found at mitochondria, nucleus and cytoplasm. Due to its size and ring-shaped structure, prohibitin protein defines functional subcompartments in mitochondria. Its subunits, PHB1 and PHB2, suppress cell proliferation as in the protein itself Nevertheless, recent investigation suggests that prohibitin protein enhances cell proliferation as well. It has also been found to suppress cell apoptosis by reducing cytochrome C release via the avoidance of mitochondrial crista remodeling which is facilitated through type 1 optic atrophy protein （OPAl）. Acting as a binding site for ubiquitin, prohibitin protein regulates protein degradation by proteasome. Examples are the degradations of sperm mitochondria in a fertilized ovum or those of an abnormal sperm.展开更多
目的 探讨抗增殖蛋白1(prohibitin1,PHB1)对人肝癌细胞增殖的影响及其作用机制。方法 构建PHB1真核表达重组质粒(pEGFP-PHB1)和PHB1靶向siRNA质粒(shRNA-PHB1)转染人肝癌细胞HepG2和SMMC-7721,诱导PHB1在人肝癌细胞中表达上调和下调,采...目的 探讨抗增殖蛋白1(prohibitin1,PHB1)对人肝癌细胞增殖的影响及其作用机制。方法 构建PHB1真核表达重组质粒(pEGFP-PHB1)和PHB1靶向siRNA质粒(shRNA-PHB1)转染人肝癌细胞HepG2和SMMC-7721,诱导PHB1在人肝癌细胞中表达上调和下调,采用MTT、流式细胞学、荧光定量PCR和免疫印迹等技术检测人肝癌细胞增殖、细胞周期,以及细胞周期关键调控分子的表达情况。结果 高表达PHB1可阻滞HepG2和SMMC-7721细胞于 G0/G1 期[(67.28±2.94)%比(56.71±2.56)%, t =6.64, P =0.00;(69.48±3.82)%比(60.43±2.59)%, t =4.80, P =0.00],使S期比例下降[(14.74±1.45)%比(24.13±1.92)%, t =9.54, P =0.00;(13.73±1.26)%比(25.50±2.30)%, t =10.99, P = 0.00 ],抑制细胞增殖;周期调控分子p53和p21CIP1mRNA和蛋白质水平显著升高,而Cyclin A2、Cyclin E1和CDK2 mRNA和蛋白质水平显著降低( P <0.01)。低表达PHB1可促使HepG2和SMMC-7721细胞趋于S期[(31.65±2.71)%比(24.68±1.28)%, t =5.69, P =0.00;(31.02±2.49)%比(25.88±2.40)%, t =3.64, P =0.005],促进细胞增殖;p53、p21CIP1、Cyclin A2、Cyclin E1、CDK2 mRNA和蛋白质水平与PHB1高表达时相反( P <0.01)。结论 高表达PHB1可以阻滞人肝癌细胞周期于G0/G1期,进而抑制细胞增殖,其作用机制可能与p53介导的G0/G1期相关细胞周期蛋白有关。展开更多
The ability of tumor cells to sustain continuous proliferation is one of the major characteristics of cancer.The activation of oncogenes and the mutation or inactivation of tumor suppressor genes ensure the rapid prol...The ability of tumor cells to sustain continuous proliferation is one of the major characteristics of cancer.The activation of oncogenes and the mutation or inactivation of tumor suppressor genes ensure the rapid proliferation of tumor cells.The PI3K-Akt-mTOR axis is one of the most frequently modified signaling pathways whose activation sustains cancer growth.Unsurprisingly,it is also one of the most commonly attempted targets for cancer therapy.FK506 binding protein 8(FKBP8)is an intrinsic inhibitor of mTOR kinase that also exerts an anti-apoptotic function.We aimed to explain these contradictory aspects of FKBP8 in cancer by identifying a“switch”type regulator.We identified through immunoprecipitation--mass spectrometry-based proteomic analysis that the mitochondrial protein prohibitin 1(PHB1)specifically interacts with FKBP8.Furthermore,the downregulation of PHB1 inhibited the proliferation of ovarian cancer cells and the mTOR signaling pathway,whereas the FKBP8 level in the mitochondria was substantially reduced.Moreover,concomitant with these changes,the interaction between FKBP8 and mTOR substantially increased in the absence of PHB1.Collectively,our finding highlights PHB1 as a potential regulator of FKBP8 because of its subcellular localization and mTOR regulating role.展开更多
目的:高迁移率族蛋白B1(high-mobility group protein 1,HMGB1)在肺动脉高压患者血浆中水平显著升高,但其生物学意义尚不明确。抗增殖蛋白1(prohibitin 1,PHB1)是维持血管细胞稳态的重要蛋白质。本研究旨在探讨HMGB1对肺动脉内皮细胞的...目的:高迁移率族蛋白B1(high-mobility group protein 1,HMGB1)在肺动脉高压患者血浆中水平显著升高,但其生物学意义尚不明确。抗增殖蛋白1(prohibitin 1,PHB1)是维持血管细胞稳态的重要蛋白质。本研究旨在探讨HMGB1对肺动脉内皮细胞的影响以及PHB在其中的作用和机制。方法:体内实验:构建野百合碱(monocrotaline,MCT)诱导的肺动脉高压大鼠模型,采用右心室收缩压(right ventricular systolic pressure,RVSP)以及右心室与左心室加室间隔重量和之比评判造模是否成功,ELISA检测动物血浆中HMGB1水平,蛋白质印迹法检测动物肺组织中PHB1水平,CD31免疫荧光检测肺血管内皮的完整性。体外实验:以HMGB1孵育肺动脉内皮细胞观察HMGB1对PAEC损伤的影响,分别过表达和沉默PHB1,通过检测活性氧水平、细胞色素c(cytochrome c,cyto-c)含量和caspase-3的活化研究PHB1在其中的作用。结果:与对照组相比,肺动脉高压大鼠血浆中HMGB1水平显著升高(P<0.05),肺组织中PHB1表达下降同时伴有肺动脉内皮细胞减少(P<0.05);HMGB1干预可损伤肺动脉内皮细胞,并导致PHB1表达降低(P<0.05);而过表达PHB1能减轻HMGB1诱导的肺动脉内皮细胞损伤和氧化应激(P<0.05)。PHB1通过抑制氧化应激减少HMGB1诱导的cyto-c的表达以及caspase-3的剪切(P<0.05)。结论:PHB1表达下调介导了HMGB1诱导的肺动脉内皮细胞损伤,其机制与诱导氧化应激、增加cyto-c释放以及促进caspase-3剪切有关。展开更多
文摘BACKGROUND The function of prohibitin 1(Phb1)during liver regeneration(LR)remains relatively unexplored.Our previous research identified downregulation of Phb1 in rat liver mitochondria 24 h after 70%partial hepatectomy(PHx),as determined by subcellular proteomic analysis.AIM To investigate the potential role of Phb1 during LR.METHODS We examined changes in Phb1 mRNA and protein levels,subcellular distribution,and abundance in rat liver during LR following 70%PHx.We also evaluated mitochondrial changes and apoptosis using electron microscopy and flow cytometry.RNA-interference-mediated knockdown of Phb1(PHBi)was performed in BRL-3A cells.RESULTS Compared with sham-operation control groups,Phb1 mRNA and protein levels in 70%PHx test groups were downregulated at 24 h,then upregulated at 72 and 168 h.Phb1 was mainly located in mitochondria,showed a reduced abundance at 24 h,significantly increased at 72 h,and almost recovered to normal at 168 h.Phb1 was also present in nuclei,with continuous increase in abundance observed 72 and 168 h after 70%PHx.The altered ultrastructure and reduced mass of mitochondria during LR had almost completely recovered to normal at 168 h.PHBi in BRL-3A cells resulted in increased S-phase entry,a higher number of apoptotic cells,and disruption of mitochondrial membrane potential.CONCLUSION Phb1 may contribute to maintaining mitochondrial stability and could play a role in regulating cell proliferation and apoptosis of rat liver cells during LR.
基金the Key Research and Development Program of Shaanxi,No.2021SF-227 and No.2020SF-297the Natural Science Basic Research Program of Shaanxi,No.2023-JC-YB-770。
文摘BACKGROUND Prohibitin 1(PHB1)has been identified as an antiproliferative protein that is highly conserved and ubiquitously expressed,and it participates in a variety of essential cellular functions,including apoptosis,cell cycle regulation,prolifera-tion,and survival.Emerging evidence indicates that PHB1 may play an important role in the progression of hepatocellular carcinoma(HCC).However,the role of PHB1 in HCC is controversial.AIM To investigate the effects of PHB1 on the proliferation and apoptosis of human HCC cells and the relevant mechanisms in vitro.METHODS HCC patients and healthy individuals were enrolled in this study according to the inclusion and exclusion criteria;then,PHB1 levels in the sera and liver tissues of these participates were determined using ELISA,RT-PCR,and immunohistoche-mistry.Human HepG2 and SMMC-7721 cells were transfected with the pEGFP-PHB1 plasmid and PHB1-specific shRNA(shRNA-PHB1)for 24-72 h.Cell prolif-eration was analysed with an MTT assay.Cell cycle progression and apoptosis were analysed using flow cytometry(FACS).The mRNA and protein expression levels of the cell cycle-related molecules p21,Cyclin A2,Cyclin E1,and CDK2 and the cell apoptosis-related molecules cytochrome C(Cyt C),p53,Bcl-2,Bax,caspase 3,and caspase 9 were measured by real-time PCR and Western blot,respectively.RESULTS Decreased levels of PHB1 were found in the sera and liver tissues of HCC patients compared to those of healthy individuals,and decreased PHB1 was positively correlated with low differentiation,TNM stage III-IV,and alpha-fetoprotein≥400μg/L.Overexpression of PHB1 significantly inhibited human HCC cell proliferation in a time-dependent manner.FACS revealed that the overexpression of PHB1 arrested HCC cells in the G0/G1 phase of the cell cycle and induced apoptosis.The proportion of cells in the G0/G1 phase was significantly increased and the proportion of cells in the S phase was decreased in HepG2 cells that were transfected with pEGFP-PHB1 compared with untreated control and empty vector-tr
文摘Prohibitin is named due to the negative regulatory role of its gene products in cell proliferation. Prohibitin gene is located at q21 of chromosome 17 in human beings and the protein is found at mitochondria, nucleus and cytoplasm. Due to its size and ring-shaped structure, prohibitin protein defines functional subcompartments in mitochondria. Its subunits, PHB1 and PHB2, suppress cell proliferation as in the protein itself Nevertheless, recent investigation suggests that prohibitin protein enhances cell proliferation as well. It has also been found to suppress cell apoptosis by reducing cytochrome C release via the avoidance of mitochondrial crista remodeling which is facilitated through type 1 optic atrophy protein （OPAl）. Acting as a binding site for ubiquitin, prohibitin protein regulates protein degradation by proteasome. Examples are the degradations of sperm mitochondria in a fertilized ovum or those of an abnormal sperm.
文摘目的 探讨抗增殖蛋白1(prohibitin1,PHB1)对人肝癌细胞增殖的影响及其作用机制。方法 构建PHB1真核表达重组质粒(pEGFP-PHB1)和PHB1靶向siRNA质粒(shRNA-PHB1)转染人肝癌细胞HepG2和SMMC-7721,诱导PHB1在人肝癌细胞中表达上调和下调,采用MTT、流式细胞学、荧光定量PCR和免疫印迹等技术检测人肝癌细胞增殖、细胞周期,以及细胞周期关键调控分子的表达情况。结果 高表达PHB1可阻滞HepG2和SMMC-7721细胞于 G0/G1 期[(67.28±2.94)%比(56.71±2.56)%, t =6.64, P =0.00;(69.48±3.82)%比(60.43±2.59)%, t =4.80, P =0.00],使S期比例下降[(14.74±1.45)%比(24.13±1.92)%, t =9.54, P =0.00;(13.73±1.26)%比(25.50±2.30)%, t =10.99, P = 0.00 ],抑制细胞增殖;周期调控分子p53和p21CIP1mRNA和蛋白质水平显著升高,而Cyclin A2、Cyclin E1和CDK2 mRNA和蛋白质水平显著降低( P <0.01)。低表达PHB1可促使HepG2和SMMC-7721细胞趋于S期[(31.65±2.71)%比(24.68±1.28)%, t =5.69, P =0.00;(31.02±2.49)%比(25.88±2.40)%, t =3.64, P =0.005],促进细胞增殖;p53、p21CIP1、Cyclin A2、Cyclin E1、CDK2 mRNA和蛋白质水平与PHB1高表达时相反( P <0.01)。结论 高表达PHB1可以阻滞人肝癌细胞周期于G0/G1期,进而抑制细胞增殖,其作用机制可能与p53介导的G0/G1期相关细胞周期蛋白有关。
基金This work was funded by Shanghai Pujiang Program(No.18PJ140-6700).
文摘The ability of tumor cells to sustain continuous proliferation is one of the major characteristics of cancer.The activation of oncogenes and the mutation or inactivation of tumor suppressor genes ensure the rapid proliferation of tumor cells.The PI3K-Akt-mTOR axis is one of the most frequently modified signaling pathways whose activation sustains cancer growth.Unsurprisingly,it is also one of the most commonly attempted targets for cancer therapy.FK506 binding protein 8(FKBP8)is an intrinsic inhibitor of mTOR kinase that also exerts an anti-apoptotic function.We aimed to explain these contradictory aspects of FKBP8 in cancer by identifying a“switch”type regulator.We identified through immunoprecipitation--mass spectrometry-based proteomic analysis that the mitochondrial protein prohibitin 1(PHB1)specifically interacts with FKBP8.Furthermore,the downregulation of PHB1 inhibited the proliferation of ovarian cancer cells and the mTOR signaling pathway,whereas the FKBP8 level in the mitochondria was substantially reduced.Moreover,concomitant with these changes,the interaction between FKBP8 and mTOR substantially increased in the absence of PHB1.Collectively,our finding highlights PHB1 as a potential regulator of FKBP8 because of its subcellular localization and mTOR regulating role.
文摘目的:高迁移率族蛋白B1(high-mobility group protein 1,HMGB1)在肺动脉高压患者血浆中水平显著升高,但其生物学意义尚不明确。抗增殖蛋白1(prohibitin 1,PHB1)是维持血管细胞稳态的重要蛋白质。本研究旨在探讨HMGB1对肺动脉内皮细胞的影响以及PHB在其中的作用和机制。方法:体内实验:构建野百合碱(monocrotaline,MCT)诱导的肺动脉高压大鼠模型,采用右心室收缩压(right ventricular systolic pressure,RVSP)以及右心室与左心室加室间隔重量和之比评判造模是否成功,ELISA检测动物血浆中HMGB1水平,蛋白质印迹法检测动物肺组织中PHB1水平,CD31免疫荧光检测肺血管内皮的完整性。体外实验:以HMGB1孵育肺动脉内皮细胞观察HMGB1对PAEC损伤的影响,分别过表达和沉默PHB1,通过检测活性氧水平、细胞色素c(cytochrome c,cyto-c)含量和caspase-3的活化研究PHB1在其中的作用。结果:与对照组相比,肺动脉高压大鼠血浆中HMGB1水平显著升高(P<0.05),肺组织中PHB1表达下降同时伴有肺动脉内皮细胞减少(P<0.05);HMGB1干预可损伤肺动脉内皮细胞,并导致PHB1表达降低(P<0.05);而过表达PHB1能减轻HMGB1诱导的肺动脉内皮细胞损伤和氧化应激(P<0.05)。PHB1通过抑制氧化应激减少HMGB1诱导的cyto-c的表达以及caspase-3的剪切(P<0.05)。结论:PHB1表达下调介导了HMGB1诱导的肺动脉内皮细胞损伤,其机制与诱导氧化应激、增加cyto-c释放以及促进caspase-3剪切有关。
