结直肠癌组织中lncRNA CCAT2和NDRG1的表达及意义

目的探讨结直肠癌(CRC)患者组织中长链非编码RNA(lncRNA)结肠癌相关转录物2(CCAT2)、N-myc下游调节基因(NDRG)1的表达及与临床病理特征及预后的关系。方法选取2018年2月至2020年2月在南通市中医院行CRC根治性手术治疗的96例CRC患者作为研究对象。采用实时荧光定量PCR检测组织中lncRNA CCAT2、NDRG1 mRNA表达,采用免疫组织化学检测组织中NDRG1蛋白表达,采用Kaplan-Meier曲线分析不同lncRNA CCAT2、NDRG1 mRNA表达组患者的预后差异,采用多因素Cox回归分析CRC预后影响因素。结果与癌旁组织比较,癌组织中lncRNA CCAT2表达较高,NDRG1 mRNA表达及蛋白阳性率较低,差异有统计学意义(P<0.05)。与TNM分期Ⅰ~Ⅱ期、无淋巴结转移比较,TNM分期Ⅲ期、有淋巴结转移的CRC患者癌组织中lncRNA CCAT2表达较高,NDRG1 mRNA表达较低(P<0.05)。lncRNA CCAT2高表达组3年累积生存率低于lncRNA CCAT2低表达组,而NDRG1 mRNA高表达组3年累积生存率高于NDRG1 mRNA低表达组,差异有统计学意义(P<0.05)。TNM分期、淋巴结转移,lncRNA CCAT2、NDRG1 mRNA是CRC预后影响因素(P<0.05)。结论CRC组织中lncRNA CCAT2表达升高,NDRG1表达降低,二者均参与CRC肿瘤的进展,可作为评估CRC患者生存预后的新指标。

血清SIRT1、Fibulin-5、Bcl-2/Bax与颈动脉粥样硬化斑块破裂所致脑梗死的关系及联合检测价值

目的 探讨血清沉默信息调节蛋白1 (SIRT1)、衰老关键蛋白抗原-5 (Fibulin-5)、B淋巴细胞瘤基因-2(Bcl-2)/B淋巴细胞瘤基因-2相关X蛋白(Bax)与颈动脉粥样硬化(CAS)斑块破裂所致脑梗死(ACI)的关系及联合检测价值。方法 选取新疆维吾尔自治区人民医院2021年1月至2023年2月CAS斑块破裂所致ACI患者98例作为研究组,另选取同期CAS斑块未破裂患者98例作为对照组,比较两组血清SIRT1、Fibulin-5、Bcl-2、Bax水平,分析各血清指标对CAS斑块破裂所致ACI风险的影响及与病情的关系,并评价各血清学指标单独及联合预测CAS斑块破裂所致ACI的价值。结果 研究组血清SIRT1、Bcl-2水平低于对照组,Fibulin-5、Bax水平高于对照组(P<0.05);大面积梗死(MCI)患者血清SIRT1、Bcl-2水平<小面积梗死患者<腔隙性梗死(LI)患者,Fibulin-5、Bax水平>小面积梗死患者> LI患者(P<0.05);重度神经功能缺损患者血清SIRT1、Bcl-2水平<中度神经功能缺损患者<轻度神经功能缺损患者,Fibulin-5、Bax水平>中度神经功能缺损患者>轻度神经功能缺损患者(P<0.05);血清SIRT1、Bcl-2低水平患者CAS斑块破裂所致ACI风险是高水平患者的2.311倍、2.921倍,Fibulin-5、Bax高水平患者CAS斑块破裂所致ACI风险是低水平患者的3.470倍、3.184倍(P<0.05);血清SIRT1、Bcl-2与梗死面积、神经功能缺损程度呈负相关,Fibulin-5、Bax与梗死面积、神经功能缺损程度呈正相关(P<0.05);血清SIRT1、Fibulin-5、Bcl-2、Bax预测CAS斑块破裂所致ACI的AUC分别为0.716 (95%CI:0.648~0.778)、0.796 (95%CI:0.733~0.850)、0.728 (95%CI:0.660~0.789)、0.763 (95%CI:0.698~0.821),联合预测CAS斑块破裂所致ACI的AUC为0.909 (95%CI:0.860~0.945),优于各血清指标单独预测。结论 血清SIRT1、Fibulin-5、Bcl-2/Bax与CAS斑块破裂所致ACI及其病情程度密切相关,联合预测价值可靠,对临床开展防治工作具有指导意义。

Effects of hypoxia,hyperoxia on the regulation of expression and activity of matrix metalloproteinase-2 in hepatic stellate cells

AIM: To study the effects of hypoxia, hyperoxia on the regulation of expression and activity of matrix metalloproteinase-2 (MMP-2) in hepatic stellate cells (HSC). METHODS: The expressions of MMP-2, tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) and membrane type matrix metalloproteinase-1 (MT1-MMP) in cultured rat HSC were detected by immunocytochemistry (ICC) and in situ hybridization (ISH). The contents of MMP-2 and TIMP-2 in culture supernatant were detected with ELISA and the activity of MMP-2 in supernatant was revealed by zymography. RESULTS: In the situation of hypoxia for 12h, the expression of MMP-2 protein was enhanced (hypoxia group positive indexes: 5.7 +/- 2.0, n=10; control: 3.2 +/- 1.0, n = 7; P【0.05), while TIMP-2 protein was decreased in HSC (hypoxia group positive indexes: 2.5 +/- 0.7, n = 10; control: 3.6 +/- 1.0, n = 7; P 【 0.05), and the activity (total A) of MMP-2 in supernatant declined obviously (hypoxia group: 7.334 +/- 1.922, n = 9; control: 17.277 +/- 7.424, n = 11; P 【 0.01). Compared the varied duration of hypoxia, the changes of expressions including mRNA and protein level as well as activity of MMP-2 were most notable in 6h group. The highest value(A(hypoxia)-A(control)) of the protein and the most intense signal of mRNA were in the period of hypoxia for 6h, along with the lowest activity of MMP-2. In the situation of hyperoxia for 12h, the contents (A(450)) of MMP-2 and TIMP-2 in supernatant were both higher than those in the control, especially the TIMP-2 (hyperoxia group: 0.0499 +/- 0.0144, n = 16; control: 0.0219 +/- 0.0098, n = 14; P 【 0.01), and so was the activity of MMP-2 (hyperoxia group: 5.252 +/- 0.771, n = 14; control: 4.304 +/- 1.083, n = 12; P 【 0.05), and the expression of MT1-MMP was increased. CONCLUSION: HSC is sensitive to the oxygen, hypoxia enhances the expression of MMP-2 and the effect is more marked at the early stage; hyperoxia mainly raises the activity of MMP-2.

NDRG2调控IRE1α-XBP1介导内质网应激逆转ER+乳腺癌他莫昔芬耐药

他莫昔芬(tamoxifen,TAM)作为雌激素受体阳性(estrogen receptor,ER+)乳腺癌的一线化疗药物使大多数患者受益,但原发性和继发性耐药问题严重影响临床治疗效果。深入研究ER+乳腺癌TAM耐药机制,改善治疗效果是当前亟待解决的问题。抑癌因子NDRG2(N-myc downstream regulated gene 2,NDRG2)在肿瘤发生发展中发挥重要作用,但是否参与ER+乳腺癌TAM耐药尚不清楚。本研究旨在探明NDRG2在ER+乳腺癌TAM耐药中发挥的作用和机制。通过RT-PCR与免疫印迹分析对比TAM敏感型和耐药型ER+乳腺癌细胞发现,NDRG 2的mRNA转录水平和蛋白质翻译水平在TAM耐药细胞中表达显著下调,且与耐药能力负相关(P<0.001);CCK-8细胞毒性实验和软琼脂克隆形成实验证实,在耐药细胞中过表达NDRG2可显著降低TAM药物半抑制浓度IC 50和软琼脂克隆形成率(P<0.001),逆转耐药表型。分子机制上,X-box结合蛋白1(X-box binding protein 1,XBP1)mRNA剪切实验与内质网相关降解(endoplasmic-reticulum associated degradation,ERAD)报告蛋白的结果显示,过表达NDRG2可增强耐药细胞中剪切型XBP1s mRNA转录与ERAD报告蛋白CD3ε-YFP表达(P<0.001),引发耐药细胞内质网强应激反应;免疫印迹检测结果显示,过表达NDRG2可显著提高耐药细胞中内质网应激感受器肌醇需要激酶1α(inositol requiring enzyme 1,IRE1α)的磷酸化水平及其下游因子,例如内质网EIP辅助因子(endoplasmic reticulum-localized DnaJ 4,ERdj4)、PKR蛋白激酶的细胞抑制剂(cellular Inhibitor of the PKR protein kinase,P58 IPK)、α甘露糖苷酶样应激蛋白(er degradation enhancingαmannosidase likeprotein,EDEM)和蛋白质二硫键异构酶家族A成员5(protein disulfide isomerase family a member 5,PDIA5)的表达水平(P<0.001)。小鼠异种移植瘤研究进一步证实,在耐药细胞中过表达NDRG2可增强TAM治疗效果,显著抑制耐药移植瘤生长(P<0.001)。以上研究结果表明,通过提高耐药细胞中NDRG2表达,增强TAM治疗引发的内质网强烈应激,可逆转ER+乳腺癌细胞耐药性,改善TAM治疗效果。研究结果为解决ER+乳腺癌TAM耐药问题提供了新的思路和有价值的潜在药物靶点。

Robo2基因对黑色素形成影响的研究进展

黑色素是一类生物色素,具有抗氧化、调控体温和防辐射等重要作用,虽不是生物生长发育所必需物质,但它的存在可提高生物的生存竞争能力。动物黑色素是由黑素细胞内的细胞器黑素小体合成储存,动物黑素细胞主要是神经嵴细胞沿背侧路径迁移分化形成。环形交叉路口引导受体2(roundabout guidance receptor 2,Robo2)基因属于轴突导向分子家族Robo的一员,是指导背部轴突导向的主要受体。笔者通过介绍动物黑色素形成和合成途径,简述Robo2基因的来源及功能,分述Robo2基因在黑色素形成中对神经元生长锥方向移动、小眼畸形相关转录因子(microphthalmia-associated transcription factor, MITF)表达和轴突发育的作用,并对黑色素合成机制进行补充,为后期寻找影响黑色素合成的主效基因和揭示黑色素合成机制提供理论依据。但目前对于Robo2基因在黑色素形成中的具体作用机制和调控途径还存在许多未解之谜,需要进一步的研究来揭示。

N-myc downstream regulated gene 1 inhibition of tumor progression in Caco2 cells

BACKGROUND Invasion and migration are the irreversible stages of colorectal cancer(CRC).The key is to find a sensitive,reliable molecular marker that can predict the migration of CRC at an early stage.N-myc downstream regulated gene 1(NDRG1)is a multifunctional gene that has been tentatively reported to have a strong relationship with tumor invasion and migration,however the current molecular role of NDRG1 in CRC remains unknown.AIM To explore the role of NDRG1 in the development of CRC.METHODS NDRG1 stably over-expressed Caco2 cell line was established by lentiviral infection and NDRG1 knock-out Caco2 cell line was established by CRISPR/Cas9.Furthermore,the mRNA and protein levels of NDRG1 in Caco2 cells after NDRG1 over-expression and knockout were detected by real-time polymerase chain reaction and western blot.The cell proliferation rate was measured by the cell counting kit-8 method;cell cycle and apoptosis were detected by flow cytometry;invasion and migration ability were detected by the 24-transwell method.RESULTS NDRG1 over-expression inhibited Caco2 proliferation and the cell cycle could be arrested at the G1/S phase when NDRG1 was over-expressed,while the number of cells in the G2 phase was significantly increased when NDRG1 was knocked out.This suggests that NDRG1 inhibited the proliferation of Caco2 cells by arresting the cell cycle in the G1/S phase.Our data also demonstrated that NDRG1 promotes early cell apoptosis.Invasion and migration of cells were extensively inhibited when NDRG1 was over-expressed.CONCLUSION NDRG1 inhibits tumor progression in Caco2 cells which may represent a potential novel therapeutic strategy for the treatment of CRC.

Siphon-Specific Expression of an Actin Encoding Gene Is Regulated by Six1/2 in Ciona savignyi

Actin is a ubiquitous protein and plays essential roles on cellular structure maintenance and cellular motility in both muscle and non-muscle tissues.Multiple genes encoding muscle actin have been identified from the ascidians,including those expressed in the larval tail muscle,the adult body-wall muscle,and adult heart muscle.In this study,a novel striated non-tail muscle actin gene was identified from the RNA-seq data of Ciona savignyi embryos.Phylogenetic analysis,alignment of the N-terminal amino acid sequences and comparation of diagnostic residues provided evidence that it had high similarity with vertebrate cardiac and skeletal muscle actin.In situ hybridization and promoter-driven GFP reporter assay revealed that it was specifically expressed in the primordia of the oral and atrial siphon.We hereby defined it as siphon-specific muscle actin coding gene(Cs-SMA).A 201 bp(−1350 bp to−1150 bp)sequence containing T-box and Six1/2 binding motif within the upstream region of Cs-SMA confined the expression of GFP in the siphons of electroporated embryos.Six1/2 binding motif was experimentally confirmed to play indispensable role in controlling the siphon-specific expression of Cs-SMA.The tissue-specific expression of Cs-SMA in the siphon primordia indicated its potential crucial roles in Ciona embryogenesis and organogenesis.

Plant Long ncRNAs: A New Frontier for Gene Regulatory Control

Long non-coding RNA (lncRNA) refers to an over 200 nt functional RNA molecule that will not be translated into protein. Previously thought to be dark matters of the genome, lncRNAs have been gradually recognized as crucial gene regulators. Although tremendous progress has been made in animals and human, the study of lncRNAs in plant is still in its infancy. Here, we reviewed the biogenesis and regulation mechanisms of lncRNAs and summarized the achievements that have been made in plant lncRNA identification and functional characterization. Genome-wide identification has uncovered large amount of lncRNAs in Arabidopsis, Rice, Maize and Wheat, and more information from other plant species will be expected with the aid of deep sequencing technologies. Similar to other species, LncRNA-mediated gene regulation also widely exists in plants, even though only a few functionally characterized examples are available. Up to now, at least four divergent lncRNA-mediated regulation mechanisms have been unraveled, including target mimicry, transcription interference, PRC2 associated histone methylation and DNA methylation. lncRNAs may be involved in the regulation of flowering, male sterility, nutrition metabolism, biotic and abiotic stress response in plants.

Measuring Ca^(2+) influxes of TRPC1-dependent Ca^(2+) channels in HL-7702 cells with Non-invasive Micro-test Technique

AIM： To explore the possibility of using the Noninvasive Micro-test Technique （NMT） to investigate the role of Transient Receptor Potential Canonical 1 （TRPC1） in regulating Ca^2＋ influxes in HL-7702 cells, a normal human liver cell line.METHODS： Net Ca^2＋ fluxes were measured with NMT, a technology that can obtain dynamic information of specific/selective ionic/molecular activities on material surfaces, non-invasively. The expression levels of TRPCl were increased by liposomal transfection, whose effectiveness was evaluated by Western-blotting and single cell reverse transcription-polymerase chain reaction.RESULTS： Ca^2＋ influxes could be elicited by adding 1 mmol/L CaCl2 to the test solution of HL-7702 cells. They were enhanced by addition of 20 μmol/L noradrenalin and inhibited by 100 μmol/L LaCl3 （a non-selective Ca^2＋ channel blocker）; 5 μmol/L nifedipine did not induce any change. Overexpression of TRPCl caused increased Ca^2＋ influx. Five micromoles per liter nifedipine did not inhibit this elevation, whereas 100 μmol/L LaCI3 did.CONCLUSION： In HL-7702 cells, there is a type of TRPCl-dependent Ca^2＋ channel, which could be detected v/a NMT and inhibited by La^3＋.

Transcriptional regulation of human polo-like kinases and early mitotic inhibitors

Human polo-like kinases （PLK1-PLK4） have been implicated in mitotic regulation and carcinogenesis. PLK1 phosphorylates early mitotic inhibitor 1 （Emil） to ensure mitosis entry, whereas Emi2 plays a key role during the meiotic cell cycle. Transcription factor E2F is primarily considered to regulate the G1/S transition of the cell cycle but its involvement in the regulation of mitosis has also been recently suggested. A gap still exists between the molecular basis of E2F and mitotic regulation. The present study was designed to characterize the transcriptional regulation of human PLK and Emi genes. Adenoviral overexpression of E2F1 increased PLK1 and PLK3 mRNA levels in A549 cells. A reporter gene assay revealed that the putative promoter regions of PLK1, PLK3, and PLK4 genes were responsive to activators E2F, E2F1-E2F3. We further characterized the putative promoter regions of Emil and Emi2 genes, and these could be regulated by activators E2F and E2F1-E2F4, respectively. Finally, PLK1-PLK4, Emil, and Emi2 mRNA expression levels in human adult, fetal tissues, and several cell lines indicated that each gene has a unique expression pattern but is uniquely expressed in common tissues and cells such as the testes and thymus. Collectively, these results indicate that E2F can integrate G1/S and G2/M to oscillate the cell cycle by regulating mitotic genes PLK and Emi, leading to determination of the cell fate.

N-myc下游调节基因2对巨噬细胞极化及乳腺癌增殖、侵袭和迁移的影响

目的探究N-myc下游调节基因2(N-myc downstream-regulated gene 2,NDRG2)在乳腺癌中的表达及对巨噬细胞极化和乳腺癌增殖、侵袭及迁移的影响。方法生物信息学分析NDRG2在乳腺癌组织中的表达及与临床相关病理特征和预后的关系。免疫荧光检测乳腺癌组织中NDRG2与巨噬细胞分子标志物的定位与表达。构建巨噬细胞与乳腺癌细胞的共培养体系观察巨噬细胞内NDRG2水平改变对乳腺癌细胞增殖、侵袭和迁移的影响。结果NDRG2在乳腺癌组织中表达下调并与不良预后相关,巨噬细胞内NDRG2表达下调促进巨噬细胞向促瘤表型M2极化,NDRG2表达上调则促进巨噬细胞向抑瘤型M1极化,共培养体系中巨噬细胞内NDRG2表达下调可增加乳腺癌细胞增殖、侵袭和迁移能力,从而促进乳腺癌进展。结论巨噬细胞内NDRG2表达下调不仅可以促进巨噬细胞向促瘤型M2转变,还能增强乳腺癌细胞的增殖、侵袭和迁移能力。

缺血性脑卒中病人外周血中NDRG2蛋白、mTOR含量与预后的相关性分析

目的研究缺血性脑卒中病人外周血中N-Myc下游调节基因2(NDRG2)蛋白、哺乳动物雷帕霉素靶蛋白(mTOR)的含量,并分析其与病人预后的相关性。方法选取缺血性脑卒中病人300例为观察组,根据神经功能缺损评分分为轻度组(n=99)、中度组(n=113)、重度组(n=88),根据出院90 d改良Rankin量表(mRS)分为预后不良组(mRS>3分,n=108)与预后良好组(mRS≤3分,n=192),同期随机选取体检的健康者278名为对照组。收集研究对象的一般资料并检测常规生化指标水平;利用酶联免疫吸附法(ELISA)检测外周血NDRG2蛋白、mTOR含量,Pearson法分析NDRG2蛋白、mTOR与缺血性脑卒中病人常规生化指标的相关性;病人出院后随访3个月,利用ROC曲线分析NDRG2蛋白、mTOR含量预测缺血性脑卒中预后不良的价值。结果观察组病人外周血中NDRG2蛋白与mTOR含量均显著高于对照组(P<0.01);病人外周血中NDRG2蛋白与mTOR含量神经功能缺损重度组高于中度组和轻度组,中度组亦高于轻度组(P<0.05);缺血性脑卒中病人外周血NDRG2蛋白、mTOR含量分别与LDL-C、TG、TC、Hcy、D-D、Lp(a)、Hs-CRP呈正相关关系(P<0.05~P<0.01),与HDL-C呈负相关关系(P<0.01)。预后良好组病人外周血中NDRG2蛋白与mTOR含量均低于预后不良组(P<0.01);外周血NDRG2蛋白、mTOR含量评估缺血性脑卒中预后不良AUC分别为0.733、0.952,敏感度分别为62.04%、89.81%,特异度分别为76.56%、89.58%;二者联合评估缺血性脑卒中AUC为0.958,敏感度为86.11%,特异度为93.23%。结论缺血性脑卒中病人外周血中NDRG2蛋白、mTOR均呈高水平,二者联合检测对缺血性脑卒中预后不良具有较高的预测价值。

结直肠癌中NDRG2的表达与微卫星不稳定的相关性及其联合检测的意义

目的探讨结直肠癌(CRC)中N-myc下游调节基因2(NDRG2)的表达及其与微卫星不稳定(MSI)的相关性,进一步探索其临床意义。方法收集2017-06—2021-12湖北医药学院附属人民医院病理科存档的结直肠癌组织蜡块标本262例,采用免疫组化SP法检测NDRG2以及4种错配修复(MMR)蛋白(MLH1、MSH2、MSH6、PMS2)的表达情况,统计学分析其相关性及与患者临床病理特征及预后的关系。结果NDRG2表达阴性的肿瘤浸润较深,分化差,淋巴结易转移,TNM多处于Ⅲ~Ⅳ期(P<0.05)。相较于MSI-L/MSS,MSI-H好发生于女性、多位于右半结肠、肿瘤浸润较浅、多为黏液腺癌、淋巴结不常转移、TNM多为Ⅰ~Ⅱ期(P<0.05)。Spearman相关性分析显示NDRG2阳性表达与MSI-H具有正相关性(r=0.145,P=0.027)。Kaplan-Meier生存分析显示,NDRG2表达阴性和MSI-L/MSS的结直肠癌患者总生存率更低(P<0.05)。多因素COX回归分析显示,TNM分期Ⅲ~Ⅳ期及NDRG2阴性表达是结直肠癌预后的独立危险因素(P<0.05)。结论NDRG2阳性表达与MSI-H呈正相关,且可以预测良好的预后,联合检测可以为结直肠癌患者提供更多有益信息。

NDRG2 gene copy number is not altered in colorectal carcinoma

AIM To investigate if the down-regulation of N-myc Downstream Regulated Gene 2(NDRG2) expression in colorectal carcinoma(CRC) is due to loss of the NDRG2 allele(s).METHODS The following were investigated in the human colorectal cancer cell lines DLD-1, Lo Vo and SW-480: NDRG2 mRNA expression levels using quantitative reverse transcriptionpolymerase chain reaction(qRT-PCR); interaction of the MYC gene-regulatory protein with the NDRG2 promoter using chromatin immunoprecipitation; and NDRG2 promoter methylation using bisulfite sequencing.Furthermore, we performed qPCR to analyse the copy numbers of NDRG2 and MYC genes in the above three cell lines, 8 normal colorectal tissue samples and 40 CRC tissue samples.RESULTS As expected, NDRG2 mRNA levels were low in the three colorectal cancer cell lines, compared to normal colon.Endogenous MYC protein interacted with the NDRG2 core promoter in all three cell lines.In addition, the NDRG2 promoter was heavily methylated in these cell lines, suggesting an epigenetic regulatory mechanism.Unaltered gene copy numbers of NDRG2 were observed in the three cell lines.In the colorectal tissues, one normal and three CRC samples showed partial or complete loss of one NDRG2 allele.In contrast, the MYC gene was amplified in one cell line and in more than 40% of the CRC cases.CONCLUSION Our study suggests that the reduction in NDRG2 expression observed in CRC is due to transcriptional repression by MYC and promoter methylation, and is not due to allelic loss.

自噬蛋白Beclin-1与N-myc下游调控基因2在食管鳞状细胞癌中的表达及其临床意义

目的探索自噬蛋白Beclin-1和N-myc下游调控基因2(NDRG2)在食管鳞状细胞癌(ESCC)中的表达,并探讨两种蛋白作为ESCC预后分子标志物的临床价值。方法采用免疫组织化学染色检测Beclin-1与NDRG2在121例ESCC组织中的表达,通过Pearson相关分析两者表达与临床病理参数的关系。根据Beclin-1与NDRG2的表达情况将所有患者分为2组,高、低表达组的生存概率通过Kaplan-Meier曲线计算,Log-rank检验用于生存分析的比较。采用Cox比例风险模型进行单因素和多因素分析,确定ESCC患者的独立预后因子。结果免疫组织化学染色分析显示Beclin-1蛋白在121例ESCC及癌旁组织中的阳性表达率分别为40.5%(49/121)和59.5%(68/121),差异有统计学意义(χ^(2)=5.973,P=0.015)。NDRG2蛋白在ESCC组织中的阳性表达率亦明显低于癌旁组织,差异有统计学意义(46.3%vs.61.2%;χ^(2)=5.385,P=0.020)。Beclin-1低表达者有更为频繁的淋巴结转移(χ^(2)=6.158,P=0.013)和更晚的TNM分期(χ^(2)=4.538,P=0.033),而NDRG2低表达与更晚的T分期(χ^(2)=6.607,P=0.010)和TNM分期(χ^(2)=5.744,P=0.017)相关。Kaplan-Meier曲线显示,Beclin-1低表达与高表达患者的3年总体生存(OS)率分别为44.3%和60.9%,差异有统计学意义(χ^(2)=5.696,P=0.017)。NDRG2低表达者的预后亦明显低于NDRG2高表达者,3年OS率分别为43.9%和59.2%(χ^(2)=9.004,P=0.003)。单、多因素Cox比例风险模型分析表明,Beclin-1低表达(HR=1.727,95%CI:1.017~2.934,P=0.043)、NDRG2低表达(HR=1.671,95%CI:1.023~2.730,P=0.040)与TNMⅡ~Ⅲ期(HR=3.823,95%CI:2.133~6.852,P<0.001)是ESCC患者预后不良的独立预测因素。结论Beclin-1和NDRG2表达在ESCC中明显下调,且与患者不良预后相关,其可能是预测ESCC患者生存的预后标志物。

Molecular cloning of promoter in human fibrinogenlike protein 2 (hfgl2) gene and functional analysis of its sequence

The aim of this study is to investigate the important regulative elements region which plays an important role on the activation of transcription exerted by the 5' noncoding region of hfgl2 gene in response to HBc and HBx. A series of promoter luciferase report plasmids, in which the hfgl2 gene has been deleted of the 5' and retained the common 3', were constructed. All the plasmids constructed were subjected to electrophoretic analysis and DNA sequencing. A eukaryotic construct expressing HBc or HBx, a luciferase reporter construct containing hfgl2 promoter and aβ-galactosidase (β-gal) plasmid were co-transfected into Chinese hamster ovary (CHO) cells and hepG2 cells, respectively. Luciferase report plasmids containing hfgl2 promoter were successfully constructed, and a serial assays of deletion of hfgl2 gene promoter showed that a strong regulatory region from -817 to -467 (relative to the transcription start site) was responsible for transcription and expression regulation of hfgl2 gene. The important regulative elements region in the promoter of hfgl2 gene was in response to HBc and HBx. which contributes to further pursuit of cis-acting elements and transcriptional factors involved in the transcription of hfgl2 gene.

NDRG2在人胚胎组织中的表达分布特点

本文旨在研究NDRG2在不同胎龄人胚胎组织中的表达水平及细胞定位。利用RT-PCR和Western blot研究NDRG2 mRNA和蛋白在胎心、肺、肝和肾中的表达水平,免疫组织化学分析NDRG2蛋白在多种胚胎组织中的分布特点。结果表明,NDRG2在胚胎组织中的表达随胚龄的延长而增加。NDRG2 mRNA和蛋白在胎心和肺中的变化一致；在胎肝中mRNA表达低而蛋白表达高,在胎肾中则相反。NDRG2蛋白阳性反应产物存在于细胞胞浆,见于小肠绒毛上皮细胞、结肠上皮细胞、皮肤表层细胞及毛囊、肺内小气道内衬上皮细胞、肝细胞、心肌细胞、胸腺小体、肾小管上皮细胞。结果提示,NDRG2蛋白可能不是一个组织特异性蛋白,并在组织和器官的形成中起作用。

Ndrg2基因表达对胃癌细胞增殖调控及其机理的研究

为研究Ndrg2基因在人类肿瘤发生发展中的作用 ,以不表达Ndrg2基因的胃癌细胞系HGC 2 7和表达Ndrg2基因的胃癌细胞系SGC 790 1作为对比材料 ,以Ndrg2基因转染HGC 2 7胃癌细胞系 ,以及用Ndrg2的反义寡核苷酸封闭SGC 790 1胃癌细胞系中Ndrg2基因的表达 .发现Ndrg2可以抑制HGC 2 7胃癌细胞的软琼脂集落形成 ,有一定诱导细胞凋亡的作用 ,对细胞周期蛋白E的表达有明显下调作用 .当封闭了SGC 790 1胃癌细胞中Ndrg2基因表达的软琼脂集落形成受到抑制 ,流式细胞仪检测发现此时的SGC 790 1细胞周期被阻滞在G1期 ,细胞周期蛋白D1和E表达下调 .Ndrg2基因对两种肿瘤细胞中的细胞外信号调节激酶 (ERK)和P38的表达也有不同的影响 .

兔抗人NDR2高效价抗血清的制备、纯化及鉴定

目的 制备高效价的 NDR2抗体 .方法 构建p Rset- A- ndr2 ,p GEX- 4 T- 1- ndr2 - a和 p GEX- 4 T- 1- ndr2 - b三种重组表达质粒 ,并分别在大肠杆菌中诱导表达相应的融合蛋白 ;用全长 GST- NDR2蛋白免疫兔 ,然后用 GST- NDR2 - A和 GST- NDR2 - B片段加强免疫 ;对包涵体形式表达的 6 His-NDR2进行初步的纯化 ;利用固定于硝酸纤维素膜上的 NDR2抗原亲和吸附纯化抗血清 .结果 经免疫得到了高效价的兔抗人 NDR2多克隆抗血清 ,亲和纯化提高了 NDR2抗体的特异性 ;免疫组化表明 NDR2是一种胞浆蛋白 .结论 用全长NDR2蛋白免疫兔 ,再用片段加强免疫 ,可以提高蛋白的抗原性 ,制备得到高效价的抗体 .纯化后的特异性 NDR2抗体 ,为进一步研究

索拉非尼联合热疗对肝癌HepG-2细胞中Bax、Bcl-2表达的影响

目的探讨索拉非尼联合热疗对人肝癌HepG-2细胞株中Bax及Bcl-2蛋白表达的影响。方法将实验细胞分为4组:对照组(正常培养的HepG-2细胞)、索拉非尼组(索拉非尼药物浓度为12μmol/L)、热疗组(加热温度为43℃,加热时间为2 h)及索拉非尼联合热疗组。用免疫组织化学染色法观察各组人肝癌HepG-2细胞株中Bax及Bcl-2的表达情况。结果肝癌HepG-2细胞Bax表达于胞浆和(或)胞膜,Bcl-2主要表达于胞膜,均呈棕黄色弥漫状分布;索拉非尼及热疗均使人肝癌HepG-2细胞中Bax蛋白的表达有所增加(P<0.01),且均使Bcl-2蛋白的表达有所降低(P<0.01),与索拉非尼组及热疗组化较,索拉非尼联合热疗组Bax蛋白的表达明显增加(P<0.01)、Bcl-2蛋白的表达明显降低(P<0.01)。结论索拉非尼联合热疗通过增加人肝癌HepG-2细胞中Bax蛋白的表达和降低Bcl-2蛋白的表达协同诱导肝癌细胞凋亡,可能成为治疗肝癌的新方法。