A Nonradioactive Method for Detecting DNA-binding Activity of Nuclear Transcription Factors

To determine the feasibility of a nonradioactive electrophoresis mobility shift assay for detecting nuclear transcription factor, double-stranded oligonucleotides encoding the consensus target sequence of NF-κB were labled with DIG by terminal transferase After nuclear protein stimulated with phorbol 12-myristate 13-acetate (PMA) or PMA and pyrrolidine dithiocarbamate (PDTC) electrophoresed on 8 % nondenaturing poliacrylamide gel together with oligeonucleotide probe, they were electro-blotted nylon membrane positively charged Anti-DIG-AP antibody catalyzed chemiluminescent substrate CSPD to image on X-film The results showed that nuclear proteins binded specifically to the NF-κB consensus sequence in the EMSA by chemiluminescent technique method and the activity of NF-κB in PMA group was more than that in PMA+PDTC group It is suggested that detection of NF-κB by EMSA with chemiluminescent technique is feasible and simple, which can be performed in ordinary laboratories

Isoflavone Attenuates the Nuclear Transcription Factor Kappa B (NF-<i>κ</i>B) Activation on MPP⁺-Induced Apoptosis of PC12 Cells

Objective: To explore the underlying molecular mechanisms of cellular response to the challenge by 1-methyl-4-phenylpyridinium (MPP+)-induced apoptosis of PC12 cells, an in vitro cell model for Parkinson’s disease, and the effect of NF-κB activation on the protection of Parkinson’s disease by Isoflavone (I). Methods: PC12 cells were used to establish the cell model of Parkinson’s disease, and are divided into five groups: control group;MPP+ group;I (Isoflavone) + MPP+ group;I group;SN-50 + MPP+ group. The content of NF-κB in PC12 cells was determined by immunocytochemistry;The viability of PC12 cells after treated with cell-permeable NF-κB inhibitor SN-50 and cell viability were measured by MTT assay;the expression levels of NF-κB p65 in cytoplasm and nuclear fractions were evaluated by western blot analysis;the mRNA expression of NF-κB p65 was analyzed by in situ hybridization (ISH). Results: Compared with the control group, the protein of NF-κB p65 both in cytoplasm and in nuclei was significantly higher than in I + MPP+ and MPP+ groups;similarly, the mRNA expression level of NF-κB p65 gene was also significantly higher;moreover, the protein expression of NF-κB p65 was much lower in I group (P + group, the protein of NF-κB p65 was significantly lower in I + MPP+ group, the mRNA expression level of NF-κB p65 gene was also significantly lower, and the protein expression level of NF-κB p65 was much lower in I + MPP+ group (P + group (P > 0.05). Conclusion: NF-κB activation is essential to MPP+-induced apoptosis in PC12 cells;but Isoflavone can inhibit the cell damage to some extent to execute its protective function, which may be involved in nigral neurodegeneration in patients with Parkinson’s disease.

Transcription factors specificity protein and nuclear receptor 4A1 in pancreatic cancer

Specificity protein(Sp)transcription factors(TFs)Sp1,Sp3 and Sp4,and the orphan nuclear receptor 4A1(NR4A1)are highly expressed in pancreatic tumors and Sp1 is a negative prognostic factor for pancreatic cancer patient survival.Results of knockdown and overexpression of Sp1,Sp3 and Sp4 in pancreatic and other cancer lines show that these TFs are individually pro-oncogenic factors and loss of one Sp TF is not compensated by other members.NR4A1 is also a prooncogenic factor and both NR4A1 and Sp TFs exhibit similar functions in pancreatic cancer cells and regulate cell growth,survival,migration and invasion.There is also evidence that Sp TFs and NR4A1 regulate some of the same genes including survivin,epidermal growth factor receptor,PAX3-FOXO1,α5-andα6-integrins,β1-,β3-andβ4-integrins;this is due to NR4A1 acting as a cofactor and mediating NR4A1/Sp1/4-regulated gene expression through GC-rich gene promoter sites.Several studies show that drugs targeting Sp downregulation or NR4A1 antagonists are highly effective inhibitors of Sp/NR4A1-regulated pathways and genes in pancreatic and other cancer cells,and the triterpenoid celastrol is a novel dual-acting agent that targets both Sp TFs and NR4A1.

Nuclear reprogramming: the zygotic transcription program is established through an“erase-and-rebuild” strategy

Oocytes display a maternal-specific gene expression profile, which is switched to a zygotic profile when a haploid set of chromatin is passed on to the fertilized egg that develops into an embryo. The mechanism underlying this transcription reprogramming is currently unknown. Here we demonstrate that by the time when transcription is shut down in germinal vesicle oocytes, a range of general transcription factors and transcriptional regulators are dissociated from the chromatin. The global dissociation of chromatin factors （CFs） disrupts physical contacts between the chromatin and CFs and leads to erasure of the maternal transcription program at the functional level. Critical transcription factors and regulators remain separated from chromatin for a prolonged period, and become re-associated with chromatin shortly after pronuclear formation. This is followed temporally by the re-establishment of nuclear functions such as DNA replication and transcription. We propose that the maternal transcription program is erased during oogenesis to generate a relatively naive chromatin and the zygotic transcription program is rebuilt de novo after fertilization. This process is termed as the ＂erase-and-rebuild＂ process, which is used to reset the transcription program, and most likely other nuclear processes as well, from a maternal one to that of the embryo. We further show in the accompanying paper （Gao T, et al., Cell Res 2007; 17：135-150.） that the same strategy is also employed to reprogram transcriptional profiles in somatic cell nuclear transfer and parthenogenesis, suggesting that this model is universally applicable to all forms of transcriptional reprogramming during early embryogenesis. Displacement of CFs from chromatin also offers an explanation for the phenomenon of transcription silence during the maternal to zygotic transition.

Hepatitis C Virus non-structural 5A abrogates signal transducer and activator of transcription-1 nuclear translocation induced by IFN-α through dephosphorylation

AIM： To study the effect of Hepatitis C virus nonstructural 5A （HCV NSSA） on IFNα induced signal transducer and activator of transcription-1 （STAT1） phosphorylation and nuclear translocation.METHODS： Expression of STAT1 Tyr701 phosphorylation at different time points was confirmed by Western blot, and the time point when p-STAT1 expressed most, was taken as the IFN induction time for further studies. Immunocytochemistry was used to confirm the successful transient transfection of NS5A expression plasmid. Immunofluorescene was performed to observe if there was any difference in IFNα-induced STAT1 phosphorylation and nuclear translocation between HCV NSSA-expressed and non-HCV NSSA-expressed cells. Western blot was used to compare the phosphorylated STAT1 protein of the cells.RESULTS： Expression of HCV NS5A was found in the cytoplasm of pCNS5A-transfected Huh7 cells, but not in the PRC/ CMV transfected or non-transfected cells, STAT1 Tyr701 phosphorylation was found strongest in 30 min of IFN induction, STAT1 phosphorylation and nuclear import were much less in the presence of HCV NS5A protein in contrast to pRC/CMV-transfected and non-transfected cells under fluorescent microscopy, which was further confirmed by Western blot.CONCLUSION： HCV NSSA expression plasmid is successfully transfected into Huh7 cells and HCV NS5A protein is expressed in the cytoplasm of the cells. IFN-α is able to induce STAT1 phosphrylation and nuclear translocation, and this effect is inhibited by HCV NS5A protein, which might be another possible resistance mechanism to interferon alpha therapy.

慢性心力衰竭患者血清TFAM、PGC-1α、NRF-1水平与心功能的相关性

目的探究慢性心力衰竭(CHF)患者血清线粒体转录因子A(TFAM)、过氧化物酶体增殖物激活受体γ共激活因子1α(PGC-1α)、核呼吸因子1(NRF-1)水平与心功能的相关性。方法选取2022年1月至2023年1月于哈尔滨市第一医院经检查确诊的105例CHF患者作为CHF组,根据纽约心脏病协会(NYHA)心功能分级将CHF组患者分为Ⅰ~Ⅱ级42例、Ⅲ级35例、Ⅳ级28例;选取同期于本院体检的健康检查者105例为对照组,比较两组及CHF组不同心功能分级血清TFAM、PGC-1α、NRF-1水平以及心功能指标。Pearson法分析CHF组患者血清TFAM、PGC-1α、NRF-1水平与心功能指标的关系。结果CHF组患者血清TFAM、PGC-1α、NRF-1水平低于对照组(P<0.05)。CHF组患者左室舒张末期内径(LVEDD)、左室收缩末期内径(LVESD)水平高于对照组,左室射血分数(LVEF)水平低于对照组(P<0.05)。随着心功能分级(NYHAⅠ~Ⅱ级、Ⅲ级和Ⅳ级)逐渐升高,CHF组患者血清TFAM、PGC-1α、NRF-1水平逐渐降低(P<0.05);LVEDD、LVESD水平逐渐升高,LVEF水平逐渐降低(P<0.05)。相关性分析显示,LVEF与血清TFAM、PGC-1α、NRF-1水平呈正相关(r=0.474、r=0.461、r=0.513,P<0.05);LVEDD与血清TFAM、PGC-1α、NRF-1水平呈负相关(r=-0.511、r=-0.502、r=-0.475,P<0.05);LVESD与血清TFAM、PGC-1α、NRF-1水平呈负相关(r=-0.497、r=-0.515、r=-0.462,P<0.05)。结论CHF患者血清TFAM、PGC-1α、NRF-1水平降低,且三者与心功能指标密切相关。

滋阴明目方通过Akt/FoxO1/FasL通路抑制感光细胞凋亡治疗视网膜色素变性的机制研究

目的观察滋阴明目方对视网膜色素变性(retinitis pigmentosa,RP)小鼠视网膜组织中磷酸化蛋白激酶B(phosphorylated protein kinase B,p-Akt)、叉头框蛋白1(forkhead box transcription factor O1,FoxO1)以及凋亡相关因子配体(Fas ligand,FasL)表达的影响,探讨滋阴明目方抑制感光细胞凋亡的机制。方法将60只rd10小鼠随机分为模型组、滋阴明目方低剂量组[10 g/(kg·d)]、滋阴明目方中剂量组[20 g/(kg·d)]、滋阴明目方高剂量组[40 g/(kg·d)]、维生素A组[5 g/(kg·d)],每组12只;选取12只C57小鼠作为空白对照组(等量生理盐水),每组连续干预28 d。通过眼底照相观察小鼠眼底形态改变;进行视网膜电图检查并记录A波和B波振幅;HE染色观察病理形态学变化并测定外核层厚度;Western blot法检测小鼠视网膜组织p-Akt、FoxO1、FasL、半胱氨酸天冬氨酸特异性蛋白(cysteine aspartate-specific protease,Caspase)-3和Caspase-8蛋白表达。结果与空白对照组比较,模型组小鼠视盘苍白、变形,血管萎缩,视网膜电图的A波与B波振幅均降低(P<0.01),视网膜结构模糊,各层界限不清,感光细胞大量丧失,外核层明显变薄(P<0.01),视网膜组织中p-Akt蛋白表达水平明显降低(P<0.01),FoxO1、FasL、Caspase-3和Caspase-8蛋白表达水平明显升高(P<0.01)。与模型组比较,滋阴明目方中、高剂量组及维生素A组小鼠眼底血管较清晰,无视盘苍白表现;视网膜各层结构清晰,细胞排列相对整齐。与模型组、滋阴明目方低剂量组比较,滋阴明目方中、高剂量组的A波与B波振幅、视网膜外核层厚度均明显升高(P<0.01),维生素A组的B波振幅、视网膜外核层厚度明显升高(P<0.01);与滋阴明目方中剂量组比较,滋阴明目方高剂量组的A波与B波振幅、视网膜外核层厚度均明显升高(P<0.01),维生素A组的A波与B波振幅均明显降低(P<0.01);与滋阴明目方高剂量组比较,维生素A组的A波与B波振幅、视网膜外核层厚度均明显降低(P<0.01)。与模型组比较,滋阴明目方低、中、高剂量组和维生素A组p-Akt蛋白表达水平明显升高(P<0.01),FoxO1、FasL和Caspase-3蛋白表达水平降低(P<0.05,P<0.01),滋阴明目方中、高剂量组Caspase-8蛋白表达水平明显降低(P<0.01)。与滋阴明目方低剂量组比较,滋阴明目方高剂量组和维生素A组p-Akt蛋白表达水平明显升高(P<0.01),滋阴明目方中、高剂量组FoxO1、FasL、Caspase-3和Caspase-8蛋白表达水平明显降低(P<0.01)。与滋阴明目方中剂量组比较,滋阴明目方高剂量组和维生素A组p-Akt蛋白表达水平明显升高(P<0.01),滋阴明目方高剂量组FoxO1、Caspase-3和Caspase-8蛋白表达水平明显降低(P<0.01),维生素A组FoxO1、FasL、Caspase-3和Caspase-8蛋白表达水平明显升高(P<0.01)。与滋阴明目方高剂量组比较,维生素A组p-Akt蛋白表达水平明显降低(P<0.01),FoxO1、FasL、Caspase-3和Caspase-8蛋白表达水平明显升高(P<0.01)。结论滋阴明目方可能通过调控Akt/FoxO1/FasL通路,增强p-Akt表达,抑制FoxO1及下游基因FasL、Caspase-3和Caspase-8的蛋白表达,从而减少rd10小鼠视网膜细胞的凋亡,保护视网膜结构和功能,延缓RP的进展。

The heterodimeric structure of heterogeneous nuclear ribonucleoprotein C1/C2 dictates 1,25-dihydroxyvitamin D-directed transcriptional events in osteoblasts

Heterogeneous nuclear ribonucleoprotein （hnRNP） C plays a key role in RNA processing but also exerts a dominant negative effect on responses to 1,25-dihydroxyvitamin D （1,25（OH）2D） by functioning as a vitamin D response element-binding protein （VDRE-BP）. hnRNPC acts a tetramer of hnRNPC1 （huC1） and hnRNPC2 （huC2）, and organization of these subunits is critical to in vivo nucleic acid-binding. Overexpression of either huC1 or huC2 in human osteoblasts is sufficient to confer VDRE-BP suppression of 1,25（OH）2D-mediated transcription. However, huC1 or huC2 alone did not suppress 1,25（OH）2D-induced transcription in mouse osteoblastic cells. By contrast, overexpression of huC1 and huC2 in combination or transfection with a bone-specific polycistronic vector using a ＂self-cleaving＂ 2A peptide to co-express huC1/C2 suppressed 1,25D-mediated induction of osteoblast target gene expression. Structural diversity of hnRNPC between human/NWPs and mouse/rat/rabbit/dog was investigated by analysis of sequence variations within the hnRNP CLZ domain. The predicted loss of distal helical function in hnRNPC from lower species provides an explanation for the altered interaction between huC1/C2 and their mouse counterparts. These data provide new evidence of a role for hnRNPC1/C2 in 1,25（OH）2D-driven gene expression, and further suggest that species-specific tetramerization is a crucial determinant of its actions as a regulator of VDR-directed transactivation.

NMR assignments and characterization of the DNA-binding domain of Arabidopsis transcription factor WRKY11--Dedicated to Professor Xiuwen Han on the occasion of her 80th birthday

The WRKY proteins are a family of plant-specific transcription factors(TFs)that are widely involved in plant development and anti-stress responses.Arabidopsis WRKY11(AtWRKY11)functions in regulating plant defense against abiotic stress and belongs to the Ild subgroup of WRKY TFs.We herein report the expres sion,purification and preliminary structural characterization of AtWRKY11 DNA-binding domain(DBD)using solution NMR Almost complete backbone chemical shift assignments of AtWRKY11-DBD have been ob-tained.Chemical shift-based secondary structure analysis suggests that AtWRKY11-DBD may exhibit local conformational diferences from the X-ray structure of the C-terminal WRKY domain of AtWRKY1,particularly in the β1 and β5 strands.Our current study provides the basis for further structural and interactional studies.

Glycine Attenuates Myocardial Fibrosis in Myocardial Infarction in Rats Partly through Modulating Signal Transducer and Activator of Transcription 3/Nuclear Factor-κB/Transforming Growth Factor-β axis

Objective: Inflammation and fibrosis are strongly associated with each other. Glycine is present in various traditional Chinese medicines and exhibits anti-inflammatory activity. However, the effects of glycine on myocardial fibrosis(MF) in rats with myocardial infarction(MI) have not been reported. The purpose of this study is to investigate the effects of glycine therapy on MF and comprehend its underlying mechanisms. Materials and Methods: Left anterior descending artery ligation-induced MI in Sprague Dawley rats was leveraged to assess the therapeutic effects of Glycine. Rats received either normal saline or glycine(0.5 mg/g bodyweight) for 7 days. Results: Glycine upregulated cardiac ejection fraction and fractional shortening to improve cardiac function, as evaluated by echocardiography. Histological and immunohistochemical analyses demonstrated that glycine could decrease inflammatory cell infiltration and alleviate collagen deposition. Western blotting revealed that nuclear factor-κB(NF-κB)-mediated inflammatory signaling was also downregulated by glycine treatment. The expression of signal transducer and activator of transcription 3(STAT3), tumor necrosis factor-α, and transforming growth factor-β(TGF-β) was decreased significantly in the glycine-treated group compared to the model group. Thus, glycine plays a protective role against myocardial ischemia and subsequent MF. Conclusion: The protective effects of glycine were achieved partly through STAT3/NF-κB/TGF-β signaling pathway.

FADD基因对宫颈癌细胞凋亡和侵袭及NF-κB信号通路的影响

目的探讨Fas相关死亡结构域(FADD)基因对宫颈癌细胞凋亡和侵袭及NF-κB信号通路的影响。方法qRT-PCR检测60例宫颈鳞癌患者的癌组织及配对癌旁组织,人宫颈鳞癌细胞系SiHa及人宫颈上皮永生化细胞系H8中FADD mRNA水平。将SiHa细胞分为对照组、NC-sh组、FADD-sh组、NC-OE组和FADD-OE组。对照组SiHa细胞正常培养,不进行转染;NC-sh组、FADD-sh组、NC-OE组和FADD-OE组SiHa细胞分别使用Lipofectamine 2000试剂转染shRNA慢病毒阴性对照(NC-sh)、FADD shRNA慢病毒(FADD-sh)、过表达慢病毒阴性对照(NC-OE)和FADD过表达慢病毒(FADD-OE)。通过MTT法和集落形成实验检测细胞增殖。通过AnnexinⅤ-FITC/PI双染色法检测细胞凋亡。通过Transwell检测细胞侵袭。通过qRT-PCR检测FADD mRNA水平。通过Western blot检测cleaved Caspase-3、cleaved Caspase-8、基质金属蛋白酶(MMP)2、MMP9、核因子(NF)-κB p65和p-NF-κB p65的表达水平。结果与配对癌旁组织相比,宫颈鳞癌组织中FADD mRNA水平降低(t=29.333,P<0.001)。与H8细胞相比,SiHa细胞中FADD mRNA水平降低(t=27.219,P<0.001)。与Ⅰ-Ⅱ期相比,Ⅲ-Ⅳ期患者癌组织中FADD mRNA水平降低(t=7.727,P<0.001)。与淋巴结未转移患者相比,淋巴结转移患者癌组织中FADD mRNA水平降低(t=7.618,P<0.001)。与NC-sh组比较,FADD-sh组细胞的FADD mRNA和蛋白相对表达量降低(P<0.05),相对细胞活力和集落数量升高(P<0.05),细胞凋亡率、cleaved Caspase-3和cleaved Caspase-8蛋白相对表达量降低(P<0.05),侵袭细胞数量、MMP2和MMP9蛋白相对表达量升高(P<0.05),NF-κB p65相对磷酸化水平升高(P<0.05)。与NC-OE组比较,FADD-OE组细胞的FADD mRNA和蛋白相对表达量升高(P<0.05),相对细胞活力和集落数量降低(P<0.05),细胞凋亡率、cleaved Caspase-3和cleaved Caspase-8蛋白相对表达量升高(P<0.05),侵袭细胞数量、MMP2和MMP9蛋白相对表达量降低(P<0.05),NF-κB p65相对磷酸化水平降低(P<0.05)。对照组、NC-sh组和NC-OE组上述指标均差异无统计学意义(P>0.05)。结论FADD在宫颈癌中下调,并且与TNM分期和淋巴结转移有关,宫颈癌中FADD的表达缺失可能通过激活NF-κB信号通路抑制癌细胞的凋亡并促进侵袭。

Revealing the role of CCoAOMT1: fine-tuning bHLH transcription factors for optimal anther development

The tapetum,a crucial innermost layer encompassing male reproductive cells within the anther wall,plays a pivotal role in normal pollen development.The transcription factors (TFs) bHLH010/089/091 redundantly facilitate the rapid nuclear accumulation of DYSFUNCTIONAL TAPETUM 1,a gatekeeper TF in the tapetum.Nevertheless,the regulatory mechanisms governing the activity of bHLH010/089/091 remain unknown.In this study,we reveal that caffeoyl coenzyme A O-methyltransferase 1 (CCoAOMT1) is a negative regulator affecting the nuclear localization and function of bHLH010 and bHLH089,probably through their K259 site.Our findings underscore that CCoAOMT1 promotes the nuclear export and degradation of bHLH010 and bHLH089.Intriguingly,elevated CCoAOMT1 expression resulted in defective pollen development,mirroring the phenotype observed in bhlh010 bhlh089 mutants.Moreover,our investigation revealed that the K259A mutation in the bHLH089 protein disrupted its translocation from the nucleus to the cytosol and impeded its degradation induced by CCoAOMT1.Importantly,transgenic plants with the probHLH089::bHLH089^(K259A)construct failed to rescue proper pollen development or gene expression in bhlh010 bhlh089 mutants.Collectively,these findings emphasize the need to maintain balanced TF homeostasis for male fertility.They firmly establish CCoAOMT1 as a pivotal regulator that is instrumental in achieving equilibrium between the induction of the tapetum transcriptional network and ensuring appropriate anther development.

Role of hepatocyte nuclear factor 4-alpha in gastrointestinal and liver diseases

Hepatocyte nuclear factor 4-alpha(HNF4α)is a highly conserved member of nuclear receptor superfamily of ligand-dependent transcription factors that is expressed in liver and gastrointestinal organs(pancreas,stomach,and intestine).In liver,HNF4αis best known for its role as a master regulator of liver-specific gene expression and essential for adult and fetal liver function.Dysregulation of HNF4αexpression has been associated with many human diseases such as ulcerative colitis,colon cancer,maturity-onset diabetes of the young,liver cirrhosis,and hepatocellular carcinoma.However,the precise role of HNF4αin the etiology of these human pathogenesis is not well understood.Limited information is known about the role of HNF4αisoforms in liver and gastrointestinal disease progression.There is,therefore,a critical need to know how disruption of the expression of these isoforms may impact on disease progression and phenotypes.In this review,we will update our current understanding on the role of HNF4αin human liver and gastrointestinal diseases.We further provide additional information on possible use of HNF4αas a target for potential therapeutic approaches.

Effect of Sirpα1 on the expression of nuclear factor-kappa B in hepatocellular carcinoma

BACKGROUND:Signal regulatory protein alpha1(Sirpα1) is a member of Sirps families containing four immunoreceptor tyrosine-based inhibitory motifs(ITIMs) domains in the cytoplasm of and an activated substrate of receptor tyrosine kinase(RTK),that negatively regulates the RTK-dependent cell proliferating signal transduction pathway.Previously we found that Sirpα1 was closely associated with the occurrence and development of hepatocellular carcinoma(HCC)as well as liver regeneration.Since it is unclear about the regulatory mechanisms,we established the cell line transfected Sirpα1 gene and preliminarily clarified the mechanisms by which Sirpα1 negatively regulates the carcinogenesis and development of HCC. METHODS:Liver cancer Sk-Hep1 cell was respectively transfected with plasmids of pLXSN,pLXSN-Sirpα1 and pLXSN-Sirpα1Δ4Y 2 ,screened with the drug of G418(1200 μg/ml),and various transfected Sk-Hep1 cell lines were obtained.The protein expressions of P65,P50,IκBα,cyclin D1 and Fas in various Sk-Hep1 cell lines were determined by Western blotting,and P65 and P50 were localized by the immunofluorescence technique. RESULTS:Sirpα1 could significantly upregulate the protein expression of IκBα(vs.other cell lines,P<0.05) in the Sk-Hep1 cell,and downregulate the protein expressions of P65,P50 and cyclin D1(vs.other cell lines, P<0.05)in the Sk-Hep1 cell.P65 protein expression was mainly localized in the cytoplasm in the pLXSN Sk-Hep1 cell,and in the nucleus of the Sk-Hep1 cell with mutantSirpα1Δ4Y 2 ,but in nucleus of the Sk-Hep1 cell with wild Sirpα1.P50 protein expression was localized in the cytoplasm and nucleus of the pLXSN Sk-Hep1 cell,but in the nucleus of the Sk-Hep1 cell with wild Sirpα1 and mutant Sirpα1Δ4Y 2 plasmid. CONCLUSIONS:Sirpα1 might negatively regulate and control the abnormal proliferation of liver cancer cells by influencing the protein content and localization of nuclear factor-kappa B,then influence the expression of cyclins such as cyclin D1 in the signal transduction pathway.It may be one of the important mechanisms by which Sirpα1 negatively regulates the carcinogenesis and development of HCC.

CYLD deletion triggers nuclear factor-κB-signaling and increases cell death resistance in murine hepatocytes

AIM:To analyze the role of CYLD for receptor-mediated cell death of murine hepatocytes in acute liver injury models.METHODS:Hepatocyte cell death in CYLD knockout mice(CYLD-/-)was analyzed by application of liver injury models for CD95-(Jo2)and tumor necrosis factor(TNF)-α-[D-Gal N/lipopolysaccharide(LPS)]induced apoptosis.Liver injury was assessed by measurement of serum transaminases and histological analysis.Apoptosis induction was quantified by cleaved PARP staining and Western blotting of activated caspases.Nuclear factor(NF)-κB,ERK,Akt and jun amino-terminal kinases signaling were assessed.Primary Hepatocytes were isolated by two step-collagenase perfusion and treated with recombinant TNF-αand with the CD95-ligand Jo2.Cell viability was analyzed by MTT-assay.RESULTS:Livers of CYLD-/-mice showed increased anti-apoptotic NF-κB signaling.In both applied liver injury models CYLD-/-mice showed a significantly reduced apoptosis sensitivity.After D-Gal N/LPS treatment CYLD-/-mice exhibited significantly lower levels of alanine aminotransferase(ALT)(295 U/L vs 859 U/L,P<0.05)and aspartate aminotransferase(AST)(560 U/L vs 1025 U/L,P<0.01).After Jo injection CYLD-/-mice showed 2-fold lower ALT(50 U/L vs 110 U/L,P<0.01)and lower AST(250 U/L vs 435 U/L,P<0.01)serumlevels compared to WT mice.In addition,isolated CYLD-/-primary murine hepatocytes(PMH)were less sensitive towards death receptor-mediated apoptosis and showed increased levels of Bcl-2,XIAP,c IAP1/2,survivin and c-FLIP expression upon TNF-and CD95-receptor triggering,respectively.Inhibition of NF-κB activation by the inhibitor of NF-κB phosphorylation inhibitor BAY 11-7085 inhibited the expression of antiapoptotic proteins and re-sensitized CYLD-/-PMH towards TNF-and CD95-receptor mediated cell death.CONCLUSION:CYLD is a central regulator of apoptotic cell death in murine hepatocytes by controlling NF-κB dependent anti-apoptotic signaling.

Proliferating cell nuclear antigen clamp associated factor,a potential proto-oncogene with increased expression in malignant gastrointestinal tumors

Gastrointestinal(GI)cancers,including malignancies in the gastrointestinal tract and accessory organs of digestion,represent the leading cause of death worldwide due to the poor prognosis of most GI cancers.An investigation into the potential molecular targets of prediction,diagnosis,prognosis,and therapy in GI cancers is urgently required.Proliferating cell nuclear antigen(PCNA)clamp associated factor(PCLAF),which plays an essential role in cell proliferation,apoptosis,and cell cycle regulation by binding to PCNA,is a potential molecular target of GI cancers as it contributes to a series of malignant properties,including tumorigenesis,epithelial-mesenchymal transition,migration,and invasion.Furthermore,PCLAF is an underlying plasma prediction target in colorectal cancer and liver cancer.In addition to GI cancers,PCLAF is also involved in other types of cancers and autoimmune diseases.Several pivotal pathways,including the Rb/E2F pathway,NF-κB pathway,and p53-p21 cascade,are implicated in PCLAF-mediated diseases.PCLAF also contributes to some diseases through dysregulation of the p53 pathway,WNT signal pathway,MEK/ERK pathway,and PI3K/AKT/mTOR signal cascade.This review mainly describes in detail the role of PCLAF in physiological status and GI cancers.The signaling pathways involved in PCLAF are also summarized.Suppression of the interaction of PCLAF/PCNA or the expression of PCLAF might be potential biological therapeutic strategies for GI cancers.

Nuclear Factor kappa B p65 Expression in Mouse Cochlea

Nuclear factor kappa B(NF-κB) is one of the best-characterized transcription factors playing important roles in many cellular responses to a large variety of stimuli,including inflammatory cytokines, phorbol esters, growth factors, and bacterial and viral products. The aim of this study is to demonstrate NF-κB expression in the mouse cochlea and its enhancement in response to lipopolysaccharides(LPS) and kanamycin(KA) treatment. Methods KA treatment consisted of subcutaneous KA injections at 700 mg/kg twice a day with an eight-hour interval between the two injections for 3 or 7 days. For animals in the LPS treatment group, a single dose of 0.3 mg LPS dissolved in 0.2 ml sterile saline were injected into both bullae through the tympanic membrane and kept there for 3 hours. Animals in the control group received subcutaneous saline injection for 7 days. Following immmunohistochemichal processing with rabbit polyclonal anti-NF-κB p65 antibodies, cryosections of the cochlea were examined for expression of NF-κB p65 in various structures in the cochlea. Results NF-κB p65 expression, identified by presence of brown reaction products characteristic of DAB immunohistochemistry, was visible in the spiral ligament, spiral prominence, tectorial membrane(TM), spiral ganglion and nerve fibers. Relatively weak NF-κB p65 expression was also visualized in the organ of Corti. Within the organ of Corti, the inner hair cells(IHC), outer hair cells(OHC), inner pillar cells(IP), outer pillar cells (OP), Deiter’s cells(DC), and Boettcher’s cells exhibited stronger staining than the inner sulcus cells, Hensen’s cells(HC) and Claudius’cells. No NF-κB p65 expression was seen in the nucleus of the IHC and OHC. NF-κB p65 expression was increased in animals exposed to LPS or KA, demonstrating significant differences in the staining between control animals and LPS/KA-treated animals. NF-κB p65 expression was not significantly different between LPS treated and KA treated animals or between 3 and 7 days in KA-treated animals. Conclusion LPS and KA exposure increases expression of NF-κB p65 in the mouse cochlea.

LncRNA NEAT1调控miR-505-3p/VEGFA对碱烧伤大鼠角膜新生血管的影响

目的 探讨长链非编码RNA(LncRNA)核旁斑组装转录本1(NEAT1)调控微小RNA-505-3p(miR-505-3p)/血管内皮生长因子A(VEGFA)轴对碱烧伤大鼠角膜新生血管生成的影响及其作用机制。方法 144只SD大鼠随机分为对照组、模型组、sh-NC组、sh-NEAT1组、sh-NEAT1+antagomir-NC组和sh-NEAT1+miR-505-3p antagomir组,每组24只。除对照组外,其余组大鼠均建立碱烧伤模型。观察大鼠角膜新生血管情况,并计算角膜新生血管面积;实时荧光定量PCR(qRT-PCR)检测大鼠角膜中LncRNA NEAT1、miR-505-3p、VEGFA mRNA表达;苏木素-伊红(HE)染色观察大鼠角膜病理形态学改变;免疫组织化学染色、Western blot检测大鼠角膜中VEGFA、CD31蛋白表达;双荧光素酶报告基因实验验证miR-505-3p与LncRNA NEAT1、VEGFA的靶向关系。结果 与对照组比较,模型组大鼠角膜新生血管面积增加,LncRNA NEAT1、VEGFA mRNA相对表达水平以及VEGFA、CD31蛋白表达水平均升高,miR-505-3p相对表达水平降低(均为P<0.05),角膜组织存在炎症细胞浸润、上皮细胞缺损等病理学损伤;与sh-NC组比较,sh-NEAT1组大鼠角膜新生血管面积减少,LncRNA NEAT1、VEGFA mRNA相对表达水平以及VEGFA、CD31蛋白表达水平均降低,miR-505-3p相对表达水平升高(均为P<0.05),角膜组织病理学损伤有所改善;与sh-NEAT1+antagomir-NC组比较,sh-NEAT1+miR-505-3p antagomir组大鼠角膜新生血管面积增加,VEGFA mRNA相对表达水平以及VEGFA、CD31蛋白表达水平均升高,miR-505-3p相对表达水平降低(均为P<0.05),角膜组织病理学损伤加重。经验证,大鼠角膜上皮细胞中miR-505-3p与LncRNA NEAT1、VEGFA均存在靶向关系。结论 干扰LncRNA NEAT1可能通过靶向上调miR-505-3p并下调VEGFA表达抑制碱烧伤大鼠角膜新生血管生成。

肺腺癌转移相关转录本1、Fas相关死亡结构域蛋白、雌激素相关受体α在乳腺癌中的表达及与患者临床分期和预后的关系

目的探讨肺腺癌转移相关转录本1(MALAT1)、Fas相关死亡结构域蛋白(FADD)、雌激素相关受体α(ERRα)在乳腺癌中的表达及与患者临床分期和预后的关系。方法选取122例乳腺癌患者,取其乳腺癌组织及相应癌旁组织,逆转录聚合酶链反应(RT-PCR)检测MALAT1的相对表达量,免疫组化法检测FADD、ERRα蛋白的表达情况。比较不同临床分期及预后乳腺癌患者乳腺癌组织中MALAT1、FADD、ERRα的表达情况。绘制受试者工作特征(ROC)曲线,计算曲线下面积(AUC),评估MALAT1、FADD、ERRα单独及联合检测对乳腺癌患者预后的预测价值。结果乳腺癌组织中MALAT1的相对表达量、ERRα阳性表达率均明显高于癌旁组织(P﹤0.01),FADD阳性表达率明显低于癌旁组织(P﹤0.01)。Ⅲ~Ⅳ期乳腺癌患者乳腺癌组织中MALAT1的相对表达量、ERRα阳性表达率均明显高于Ⅰ~Ⅱ期患者(P﹤0.01),FADD阳性表达率明显低于Ⅰ~Ⅱ期患者(P﹤0.01)。随访1年,死亡23例,生存99例,生存率81.15%(99/122),死亡乳腺癌患者乳腺癌组织中MALAT1的相对表达量、ERRα阳性表达率均明显高于生存患者(P﹤0.01),FADD阳性表达率明显低于生存患者(P﹤0.01)。ROC曲线显示,MALAT1、FADD、ERRα联合检测预测乳腺癌患者预后的AUC为0.872(95%CI:0.813~0.941),高于各指标单独检测。结论MALAT1、FADD及ERRα在乳腺癌组织中异常表达,可能与患者临床分期、预后有关,上述因子联合检测对乳腺癌患者预后的评估价值较高。

大豆GmNF-YA13互作蛋白的筛选及鉴定

大豆GmNF-YA13蛋白是一个核转录因子Y(NF-Y),在干旱和高盐响应过程中均发挥重要作用。为研究其抗旱和耐盐的作用机理,寻找GmNF-YA13的互作蛋白,构建pGBKT7-GmNF-YA13诱饵载体,采用酵母双杂交筛选大豆酵母文库,并进行X-α-gal染色验证。结果显示:酵母双杂交获得85个阳性克隆,测序分析后得到36个候选的互作蛋白。功能预测显示互作蛋白主要参与生长发育、胁迫响应、能量代谢、转录调控和信号转导等生物过程。选择GmUVR8、GmCML41、GmFbox13和GmFBA与诱饵pGBKT7-GmNF-YA13进行一对一验证,只有GmFBA能与GmNF-YA13发生相互作用,预示GmNF-YA13功能的发挥需要GmFBA的参与。该结果可为NF-YA抗逆分子网络的研究提供基础。