乙型脑炎减毒活疫苗SA14-14-2在BHK21细胞上传代后的基因序列研究

目的：研究乙型脑炎减毒活疫苗SA14-14-2在BHK₂₁细胞上传代后的病毒基因序列,以深入控制乙型脑炎减毒活疫苗的安全性。方法：将乙型脑炎减毒活疫苗SA14-14-2在BHK₂₁细胞上连续传代,选取第二代病毒（P2）、第五代病毒（P5）、第十五代病毒（P15）提取RNA,反转录为cDNA后,进行各代病毒全基因序列分析,分析与乙型脑炎病毒毒力密切相关的关键位点基因是否发生改变。结论：乙型脑炎减毒活疫苗SA14-14-2在BHK₂₁细胞上传5代后,病毒E蛋白上关键基因第279位（E279）氨基酸由甲硫氨酸（M）突变为赖氨酸（K）。     

建立以HIF-1α为靶标的高通量筛选防治动脉粥样硬化先导化合物的细胞模型

目的 建立一个高通量筛选防治动脉粥样硬化先导化合物的体外细胞模型。方法 克隆HIF-1α低氧反应元件（hypoxia response element,HRE）至荧光素酶报告基因表达载体pGL3-Enhancer,构建荧光素酶表达载体pGL3-HIF-1α-HRE,转染人单核细胞THP-1并筛选稳定表达细胞株THP-1-HIF-1α-HRE。结果 Real Time-PCR检测表明低氧培养可以有效上调THP-1-HIF-1α-HRE细胞HIF-1α蛋白表达和荧光素酶活性,而洛伐他汀和姜黄素预处理可以有效抑制低氧引起的THP-1-HIF-1α-HRE细胞内HIF-1α蛋白表达及荧光素酶活性。结论 成功建立了筛选抗动脉粥样硬化的先导化合物体外高通量细胞模型THP1-HIF-1α-HRE。     

小儿宝泰康颗粒对呼吸道病毒感染的抗病毒作用研究

目的 观察小儿宝泰康颗粒对呼吸道合胞病毒和腺病毒感染细胞的抑制作用,以便为呼吸道病毒感染的治疗提供新的药物。方法 通过细胞培养技术观察不同浓度的小儿宝泰康颗粒对HEp-2细胞的细胞毒作用,采用MTT法并结合CPE法检测病毒感染细胞存活率和病毒抑制率,以利巴韦林作为阳性药物对照,评价该药物对呼吸道合胞病毒和腺病毒感染细胞的抗病毒活性。结果 小儿宝泰康颗粒对HEp-2细胞的半数细胞毒性浓度（TC₅₀）为4.4 mg·mL^-1,对呼吸道合胞病毒的半数有效浓度（EC₅₀）为2.03 mg·mL^-1,而腺病毒的EC₅₀为0.65 mg·mL^-1,抗病毒指数分别为2.17和6.75。在对两种病毒的直接灭活作用中,EC₅₀分别为4.13和8.99 mg·mL^-1。阳性对照药物病毒唑在128 g·mL^-1时,对呼吸道合胞病毒和腺病毒的抑制率分别为92.7%和80.16%。结论 小儿宝泰康颗粒是一种对呼吸道合胞病毒和腺病毒感染有抗病毒作用的药物,有临床应用前景和进一步研究的价值。     

人白介素-15基因系列启动子克隆及其荧光素酶报告基因载体的构建

目的克隆人白介素-15(IL-15)基因系列启动子,构建IL-15基因启动子荧光素酶报告基因载体。方法通过PCR方法从HaCaT细胞基因组DNA中获得IL-15基因编码序列5′上游七段逐步缺失的IL-15启动子序列;双酶切后,分别重组到含萤火虫荧光素酶的报告基因pGL3-Basic载体上,构建IL-15基因系列启动子报告基因载体;用脂质体转染法将含最长启动子序列的报告基因载体转染至HaCaT细胞,并设置空白对照组和LPS组分别处理;24 h后采用双荧光素酶报告基因系统检测其启动子活性。结果经PCR方法扩增出七段逐步缺失的IL-15基因启动子片段,PCR及双酶切鉴定各重组体构建正确;瞬时转染HaCaT细胞后经报告基因检测得知所克隆最长序列具有启动子活性。结论成功构建了IL-15系列启动子报告基因载体,并在HaCaT细胞中初步活性分析得知所克隆IL-15基因调控系列内包含启动子核心区域,为进一步研究IL-15表达调控机制奠定了实验基础。     

DDX46基因慢病毒载体的构建及其在人膀胱癌细胞中的表达

目的 构建DDX46基因低表达慢病毒载体,并检测其在人膀胱癌细胞中的表达效果,为DDX46基因在人膀胱癌细胞中的研究奠定基础。方法 应用实时荧光定量检测5637细胞和T24细胞中DDX46 mRNA表达水平,以寻找合适的细胞系进行实验。分别以DDX46基因序列和普适型阴性序列为模板,设计合成靶点序列,并合成寡核苷酸双链,定向克隆到慢病毒质粒GV115,合成重组质粒shDDX46和shRNA,分别称之为实验组和对照组。将不同重组质粒与pHelper 1.0和pHelper 2.0分别转染293T细胞以包装成慢病毒颗粒后感染5637细胞和T24细胞。应用实时荧光PCR定量检测重组慢病毒感染5637细胞和T24细胞后DDX46 mRNA表达水平,判断其干扰效率。结果 5637细胞和T24细胞均高丰度表达DDX46mRNA,其结果（用ΔCt值表示）分别为（6.53±0.08）和（8.48±0.11）;重组慢病毒感染膀胱癌细胞后,在5637细胞中,实验组DDX46 mRNA的表达水平（用ΔCt值表示）为（0.32±0.01）,低于对照组（1.00±0.10）,差异有统计学意义（P<0.05）,其敲减效率为67.70%;在T24细胞中,实验组DDX46 mRNA的表达水平（用ΔCt值表示）为（0.11±0.01）,低于对照组（1.00±0.03）,差异有统计学意义（P<0.05）,其敲减效率为89.00%。结论 成功构建DDX46基因低表达慢病毒载体,为研究DDX46基因在膀胱癌细胞中的作用奠定了基础。     

姜黄素通过调控miR-7641/PTPN14分子轴抑制乳腺癌发展进程的分子机制研究

目的 探讨姜黄素通过调控miR-7641/PTPN14分子轴抑制乳腺癌发展进程的分子机制。方法 采用实时荧光定量PCR（qRT-PCR）检测乳腺癌患者癌组织及细胞系中miR-7641表达情况;使用Kaplan-Meier方法作乳腺癌患者生存曲线;采用不同浓度的姜黄素处理细胞,或转染miR-7641 mimic、Anti-miR-7641及pcDNA-PTPN14载体,采用qRT-PCR检测miR-7641表达情况,MTT实验及克隆形成实验检测细胞增殖能力,Transwell小室法检测细胞迁移及侵袭,western blotting检测Ki67、pcDNA、CyclinD1、Bax、Bcl-2、caspase-3、caspase-8蛋白表达水平,采用双荧光素酶报告基因系统检测miR-7641与PTPN14靶向调控关系。结果 与癌旁组织或乳腺正常上皮细胞比较,miR-7641在乳腺癌患者癌组织及乳腺癌细胞系中高表达（P<0.01、0.001）,且miR-7641能够明显促进乳腺癌细胞的增殖、迁移及侵袭（P<0.05、0.01、0.001）,并促进Ki67、pcDNA、CyclinD1、Bcl-2蛋白表达,抑制Bax、caspase-3、caspase-8蛋白表达;miR-7641与PTPN14 3''-UTR靶向结合,姜黄素通过miR-7641/PTPN14分子轴抑制乳腺癌细胞的增殖、迁移及侵袭（P<0.01、0.001）,并抑制Ki67、pcDNA、CyclinD1、Bax蛋白表达,促进Bcl-2、caspase-3、caspase-8蛋白表达。结论 姜黄素可通过下调miR-7641促进PTPN14表达,进而抑制乳腺癌细胞的增殖、迁移及侵袭。     

miR-186-5p靶向CXCL13基因通过Wnt/β-catenin信号调节胃癌HGC-27细胞增殖、凋亡、细胞周期、迁移和侵袭

目的 探讨miR-186-5p对胃癌HGC-27细胞增殖、凋亡、细胞周期、迁移和侵袭的影响及作用机制。方法 在胃癌HGC-27细胞中通过脂质体转染miR-186-5p模拟物（mimics）,采用实时荧光定量PCR检测转染效果, MTT实验分析HGC-27细胞增殖水平,流式细胞术分析细胞周期变化和细胞凋亡情况,Transwell实验分析HGC-27细胞迁移和侵袭能力,生物信息学、双荧光素酶报告基因实验以及Western blotting分析miR-186-5p和CXCL13的靶向关系,Western blotting分析Wnt/β-catenin信号通路活化情况。结果 体外转染miR-186-5p mimics可上调HGC-27细胞中miR-186-5p的表达。上调miR-186-5p表达能够抑制HGC-27细胞体外增殖、细胞周期进程、迁移和侵袭能力,并促进细胞凋亡。miR-186-5p可靶向抑制CXCL13的表达,上调miR-186-5p表达能够抑制β-catenin和c-Myc的表达。结论 miR-186-5p可靶向抑制CXCL13的表达,阻断Wnt/β-catenin信号通路,并抑制胃癌HGC-27细胞增殖、细胞周期、迁移和侵袭,促进细胞凋亡。     

基于戊肝病毒的嵌合病毒样颗粒对人乳头瘤病毒16型肿瘤免疫治疗作用的研究

目的 研究基于戊肝病毒(hepatitis E virus,HEV)的嵌合病毒样颗粒(virus-like particles,VLPs)对人乳头瘤病毒16型(human papillomavirus type 16,HPV 16)肿瘤免疫治疗作用。方法 将HPV 16 E7插入HEV的p239蛋白形成重组嵌合蛋白p239-HPV16 E7。所构建的重组蛋白经大肠杆菌表达、纯化、复性后,通过电镜和动态光散射对所得蛋白颗粒大小形态进行表征。将蛋白颗粒免疫C57B/L小鼠,通过流式细胞技术与酶联免疫斑点免疫试验检测脾淋巴细胞特异性免疫细胞分化情况;并且利用TC-1肿瘤细胞在C57B/L小鼠中构建肿瘤模型,以此评价蛋白颗粒在小鼠体内的抗肿瘤免疫效果。结果 体外复性后的嵌合蛋白在电镜下观察到颗粒结构,粒径大小为22.80 nm。所得蛋白颗粒在C57B/L小鼠体内诱导产生良好的特异性细胞免疫反应。与对照组相比,试验组脾淋巴细胞的CD3⁺/CD4⁺、CD3⁺/CD8⁺比例均有明显差异(P＜0.05),且分泌IFN-γ干扰素的效应T细胞显著增加。同时,所得蛋白颗粒能有效抑制TC-1荷瘤小鼠体内肿瘤细胞的生长,在实验周期内小鼠未出现死亡,而对照组小鼠体内肿瘤快速生长,且6周后全部死亡。结论 原核表达的嵌合蛋白p239-HPV16 E7形成病毒样颗粒并有效诱导针对HPV 16的抗肿瘤免疫。     

CYP1A1体外诱导活性评价模型的建立及其在人参皂苷筛选中的应用

目的 建立hAhR介导的CYP1A1体外诱导活性评价模型,应用于体外快速筛选通过hAhR途径介导的对CYP1A1具有激活能力的药物。方法 利用双荧光素酶报告基因系统,将CYP1A1启动子序列插入报告基因质粒上游,构建pGL4.17-CYP1A1质粒,并与含有hAhR编码区序列的pcDNA3.1-hAhR表达质粒瞬时共转染HepG2细胞, pGL4.17-control、pcDNA3.1（+）分别为pGL4.17-CYP1A1、pcDNA3.1-hAhR的空载体,建立hAhR-CYP1A1报告基因模型;应用AhR的完全激动剂TCDD（2 nmol/L）验证该报告基因模型的可靠性,应用该模型考察人参皂苷Rf、Rc、Re、Rg₁、Rb、Rd、Rh₂和Rg₃（20 μmol/L）通过hAhR途径对CYP1A1的诱导作用。结果 经验证,报告基因模型构建成功;人参皂苷Rf、Re、Rg₁和Rc对AhR具有激活作用,其中与对照组比较,人参皂苷Rg₁、Rc对AhR激活效应显著（P<0.05、0.01）,人参皂苷Rb、Rd、Rh₂和Rg₃对AhR无激活作用。结论 本研究建立基于hAhR的CYP1A1体外诱导活性评价模型,可为具有潜在CYP1A1诱导作用的目标化合物的筛选提供有效、快速的体外筛选手段,人参皂苷Rg₁、Rc对AhR-CYP1A1激活效应显著。     

lncRNA PVT1通过miR-1207-5p靶向调控Wnt6/β-catenin2信号通路对高级别浆液性卵巢癌的影响

目的 探讨长链非编码RNA（lncRNA）PTV1对高级别浆液性卵巢癌增殖和迁移能力的影响。方法 收集61例高级别浆液性卵巢癌患者的癌组织及相应癌旁正常组织,qRT-PCR检测miR-1207-5p、lncRNA PVT1在高级别浆液性卵巢癌组织、癌旁正常组织及不同细胞系中的表达情况。构建lncRNA PVT1沉默细胞系,分为Ovcar3-siPVT1组、Ovcar3-siNC组、NC组。采用MTT和平板克隆实验、Transwell和划痕实验检测细胞增殖、侵袭和迁移;双荧光素酶报告基因检测验证miR-1207-5p与lncRNA PVT1、Wnt6的靶向关系,StarBase和TargetScan网站预测相应miRNA可靶向结合的基因;Western blotting检测Wnt6/β-catenin2信号通路相关蛋白的表达。构建siPVT1+过表达miR-1207-5p、siPVT1+过表达Wnt6细胞系,以siPVT1+siNC为对照,验证lncRNA PVT1的调控作用。结果 qRT-PCR结果显示,高级别浆液性卵巢癌组织中lncRNA PVT1的表达水平显著高于正常癌旁组织（P<0.05）。与NC组和Ovcar3-siNC组相比,Ovcar3-siPVT1组中lncRNA PVT1的表达显著下调,细胞克隆数、侵袭数减少,细胞迁移率降低,Wnt6、β-catenin2蛋白表达水平明显下降,差异均具有统计学意义（P<0.05）。双荧光素酶报告基因检测结果证明miR-1207-5p与lncRNA PVT1、Wnt6具有靶向关系。经StartBase和TargetScan网站预测分析,miR-1207-5p分别与lncRNA PVT1、Wnt6存在靶向结合位点。与siPVT1+NC组相比,siPVT1+过表达miR-1207-5p组和siPVT1+过表达Wnt6组细胞克隆数和迁移数明显增加（P<0.05）。结论 lncRNA PVT1在高级别浆液性卵巢癌中高表达。高表达lncRNA PVT1可能通过上调miR-1207-5p表达增强Wnt6/β-catenin2信号通路的活性,从而促进高级别浆液性卵巢癌细胞的增殖、侵袭和迁移。     

Development and characterization of a stable luciferase dengue virus for high-throughput screening

To facilitate dengue virus (DENV) drug discovery, we developed a stable luciferase reporter DENV-2. A renilla luciferase gene was engineered into the capsid-coding region of an infectious cDNA clone of DENV-2. Transfection of BHK-21 cells with the cDNA clone-derived RNA generated high titers (>10⁶ PFU/ml) of luciferase reporter DENV-2. The reporter virus was infectious to a variety of cells, producing robust luciferase signals. Compared with wild-type virus, the reporter virus replicated slower in both mammalian Vero and mosquito C6/36 cells. To examine the stability of the reporter virus, we continuously passaged the virus on Vero cells for five rounds. All passaged viruses stably maintained the luciferase gene, demonstrating the stability of the reporter virus. Furthermore, we found that the passaged virus accumulated a mutation (T108M) in viral NS4B gene that could enhance viral RNA replication in a cell-type specific manner. Using the reporter virus, we developed a HTS assay in a 384-well format. The HTS assay was validated with known DENV inhibitors and showed a robust Z′ factor of 0.79. The Luc-DENV-2 HTS assay allows screening for inhibitors of all steps of the viral life cycle. The reporter virus will also be a useful tool for studying DENV replication and pathogenesis.     

乙型脑炎减毒活疫苗病毒群体的异质性分析

 目的  通过观察疫苗病毒群体中病毒个体的遗传和生物学特征,分析SA14-14-2株乙型脑炎减毒活疫苗病毒的均一性,为疫苗病毒的遗传稳定性提供实验证据。 方法   对3批疫苗病毒连续进行3次蚀斑纯化后,各挑选8个蚀斑纯化株,扩大培养一代。比较24个病毒纯化株的包膜（envelope,E）蛋白基因序列、单斑培养病毒滴度以及蚀斑特征。 结果   24个纯化株中共有6个病毒株（25%）的E蛋白核苷酸发生变化,其中2株（8.3%）的核苷酸变异导致其编码氨基酸发生改变,这些变异占总核苷酸变异数的28.6%。动物实验表明,这2个纯化株没有引起小鼠神经毒力变化。在所有24个纯化株中,我国2010年版药典规定的影响疫苗病毒遗传稳定性的8个E蛋白关键位点的氨基酸均未发生改变。24个纯化株病毒的蚀斑大小和形态以及单斑培养滴度均无明显差异。 结论   疫苗病毒具有典型的准种群体多样性特征,但在群体病毒中未检测到E蛋白氨基酸位点为野生型病毒位点的个体病毒,因此,疫苗病毒具有很好的减毒群体特征和遗传稳定性。     

Glucose-Modulated Transgene Expression via Recombinant Adeno-Associated Virus

Purpose. The objective of this study was to examine glucose-modulated reporter gene expression via recombinant adeno-associated viral vectors both in vitro and in vivo. Methods. Huh7 human hepatoma cells were transduced by recombinant adeno-associated virus (rAAV) vectors containing the luciferase gene under control of the rat insulin I gene promoter and a cytomegalovirus immediate-early promoter driving-enhanced green fluorescence protein gene. The reporter gene expression was evaluated by glucose stimulation either in the absence or presence of insulin secretagogues, including phorbol-12-myristate-13-acetate, dibutyryl cyclic AMP, and forskolin. In vivo studies were performed by injecting rAAV into the livers of streptozotocin-induced diabetic C57BL/6J mice followed by measurements of blood glucose concentration and luciferase activity assays 2 weeks after rAAV injection. Results. At a multiplicity of infection of 500, approximately 66-69% of cells expressed enhanced green fluorescence protein at 48 h post-transduction. Luciferase activities, driven by the insulin gene promoter, in the rAAV-transduced hepatoma cells responded to millimolars of glucose. The addition of phorbol-12-myristate-13-acetate, dibutyryl cyclic AMP, and forskolin increased luciferase expression in the presence of either 1 mM or 25 mM glucose. The stimulation of luciferase activities by these substances was inhibited by the presence of 100 nM staurosporine. Exposure to increments of exogenous insulin up to 10^-7 M inhibited luciferase gene expression in rAAV-transduced Huh7 cells. The in vivo experiments demonstrated good correlation between luciferase activities and blood glucose levels in streptozotocin-induced diabetic animals. Conclusion. rAAV is a promising vector for hepatic gene therapy for diabetes. Glucose and insulin secretagogues modulated transgene expression in rAAV-transduced hepatoma cells, suggesting that conditions affecting insulin gene promoter function in pancreatic islet beta cells also affect transgene expression in human hepatoma cells conferred with insulin gene promoter. Results obtained from in vivo experiments demonstrated that glucose modulated transgene expression can be obtained in rAAV-treated diabetic C57BL/6J mice.     

The upregulation of miR-98-5p affects the glycosylation of IgA1 through cytokines in IgA nephropathy

ObjectiveIncreases in galactose-deficient IgA1 (Gd-IgA1) play a crucial role in the pathogenesis of IgA nephropathy (IgAN), and several recent experiments have shown that microRNAs (miRNAs) are involved in regulating the development and physiological function of the kidney. The aims of this study were to identify miRNAs that can affect the pathogenesis of IgAN and reveal the underlying regulatory mechanism of IgA1 glycosylation in peripheral blood.MethodsThe differentially expressed miRNAs in peripheral blood mononuclear cells (PBMCs) between IgAN patients and healthy controls were screened by high-throughput sequencing, and the targets of these miRNAs were predicted and verified by dual-luciferase reporter assays. We also explored the miRNA regulation of Gd-IgA1 through the transfection of miRNA mimics and related plasmids.ResultsThe high-throughput sequencing results showed that miR-98-5p was more highly expressed in the PBMCs of IgAN patients compared with healthy controls, and the luciferase reporter gene system confirmed that miR-98-5p might target chemokine ligand 3 (CCL3). The transfection of si-CCL3 confirmed that a decrease in CCL3 can affect the expression of interleukin-6 (IL-6) and C1GALT1. The overexpression of miR-98-5p in PBMCs through the transfection of miR-98-5p mimic reduced the CCL3 and C1GALT1 levels and increased the IL-6 levels, and these changes in PBMCs were attenuated by cotransfection with the CCL3 plasmid.ConclusionThe results showed that in PBMCs, miR-98-5p can target CCL3 to decrease its expression and thereby increase the IL-6 levels, and the resulting increase in IL-6 can decrease C1GALT1 expression. Therefore, miR-98-5p might be involved in the development of IgAN.     

miR-98-5p靶向CCR7对乳腺癌细胞MCF-7运动能力的影响及其机制研究

【目的】探究miR-98-5p靶向作用于趋化因子受体(CCR7)对乳腺癌细胞MCF-7运动能力的影响及相关机制。【方法】MCF-7细胞分为Ctrl组、miR-98-5p mimic组、miR-98-5p NC组、pc-CCR7组、miR-98-5p+pc-CCR7组,分别转染相应的miRNA和CCR7过表达载体,RT-PCR检测miR-98-5p和CCR7基因表达水平,荧光素酶报告实验检测miR-98-5p和CCR7靶向关系,Transwell法检测细胞侵袭,划痕法检测细胞迁移,Western blot检测CCR7、基质金属蛋白酶2(MMP-2)、MMP-9、血管内皮生长因子(VEGF)、E-钙粘着蛋白(E-cadherin)、N-cadherin、波形蛋白(Vimentin)蛋白表达水平。【结果】荧光素酶报告实验中,与CCR7 WT+miR-98-5p NC比较,CCR7 WT+miR-98-5p mimic荧光素酶活性显著降低。与Ctrl组比较,miR-98-5p mimic组中miR-98-5p表达水平、E-cadherin蛋白表达水平显著升高,CCR7基因和蛋白表达水平、细胞侵袭和迁移能力、MMP-2、MMP-9、VEGF、N-cadherin、Vimentin蛋白表达水平显著降低,pc-CCR7组细胞侵袭和迁移能力、CCR7、MMP-2、MMP-9、VEGF、N-cadherin、Vimentin蛋白表达水平显著升高,E-cadherin蛋白表达水平显著降低;与pc-CCR7组比较,miR-98-5p+pc-CCR7组细胞侵袭和迁移能力、CCR7、MMP-2、MMP-9、VEGF、N-cadherin、Vimentin蛋白表达水平显著降低,E-cadherin蛋白表达水平显著升高。【结论】miR-98-5p可靶向作用于CCR7,抑制乳腺癌细胞MCF-7运动能力,其作用机制与上皮细胞间充质转化(EMT)有关。     

乙型脑炎病毒C蛋白编码基因重组质粒构建与表达

目的构建流行性乙型脑炎病毒(JEV)C蛋白编码基因重组子并鉴定。方法以JEV SA14-14-2株总RNA为模板,应用反转录-聚合酶链反应(RT-PCR)方法扩增JEV C蛋白编码基因,克隆至pMD19-TSimple载体测序。为便于分析JEV C蛋白编码基因重组子在哺乳动物细胞中的表达,在JEV C蛋白编码基因5′端附加FLAG序列,并亚克隆至pcDNA 3.1(+)载体中,构建重组子pJc并经酶切及DNA测序分析。脂质体法将pJc转染中华仓鼠卵巢(CHO)细胞。免疫荧光检测转染的CHO细胞中JEV C蛋白分布与表达。结果 pJC经BamH I/EcoR I酶切释出的插入子片段(414bp)分别与预期结果相符合。所编码的融合蛋白主要分布于胞质,少量分布于胞膜。结论 pJC成功构建,转染的CHO细胞可表达JEV C蛋白。     

微小 RNA-490-3p靶向 FK506相关蛋白 14表达抑制骨肉瘤 MG63细胞侵袭和迁移的实验研究

目的探讨微小 RNA-490-3p(miR-490-3p)对骨肉瘤 MG63细胞侵袭、迁移的影响及其机制。方法 2019年 9月至 2020年 4月,从中科院上海细胞库购买人骨肉瘤细胞株 MG63进行体外培养,分为对照组(未转染)miR-NC组(转染 miR-NC)、 miR-490-3p组(转染 miR-490-3p mimics),miR-490-3p+pcDNA组(共转染 miR-490-3p mimics与空载体、)和 miR-490-3p+FKBP14组(共转染 miR-490-3p mimics与 FKBP14过表达载体),采用 RT-PCR检测 miR-490-3p表达, Transwell小室法检测细胞侵袭、迁移,蛋白质印迹法检测钙黏蛋白 E(E-cadherin)、基质金属蛋白酶 -2(MMP-2)、上皮间质转化因子 Twist转录因子( Twist)和 FK506相关蛋白 14(FKBP14)蛋白表达,双荧光素酶报告基因实验检测 miR-490-3p和 FKBP14的靶向关系。结果与对照组比较, miR-490-3p组细胞中 miR-490-3p表达水平明显升高( 3.68±0.37比 1.02±0.10,P<0.05),而侵袭细胞数( 33.25±2.02比     

The development of GADD45α luciferase reporter assays in human cells for assessing the genotoxicity of environmental pollutants

Abstract

Objectives: In order to assess the potential carcinogenic and genotoxic responses induced by environmental pollutants, genotoxicity test systems based on a GADD45α promoter-driven luciferase reporter in human A549 and HepG2 cells were established.

Materials and methods: Four different types of environmental toxicants including DNA alkylating agents, precarcinogenic agents, DNA cross-linking agents and non-carcinogenic agents, and three environmental samples collected from a coke oven plant were used to evaluate the test systems. After treated with the tested agents and environmental samples for 12?h, the cell viabilities and luciferase activities of the luciferase reporter cells were determined, respectively.

Results: Methyl methanesulfonate, benzo[a]pyrene, formaldehyde and the extractable organic matter (EOM) from coke oven emissions in ambient air generally produced significant induction of relative luciferase activity in a similar dose-dependent manner in A549- and HepG2-luciferase cells. No significant increases in relative luciferase activity were observed in pyrene-treated A549- or HepG2-luciferase cells. Significant increase in relative luciferase activity was already evident after 2.5?µM benzo[a]pyrene, 5?µM formaldehyde, 0.006?µg/L bottom-EOM, 0.10?µg/L side-EOM or 0.06?µg/L top-EOM, where no cytotoxic damage was observed. Compared with the A549-luciferase cells, the tested pollutants produced higher induction of relative luciferase activity in HepG2-luciferase cells.

Discussion and conclusion: Therefore, the new genotoxicity test systems can detect different types of genotoxic agents and low concentrations of environmental samples. The luciferase reporter cells, especially the HepG2-luciferase cells, could provide a valuable tool for rapid screening of the genotoxic damage of environmental pollutants and their complex mixtures.     

14-3-3ζ as a prognostic marker and therapeutic target for cancer

Importance of the field: The ubiquitously expressed 14-3-3ζ protein is involved in numerous important cellular pathways involved in cancer. Recent research suggests 14-3-3ζ may play a central role regulating multiple pathways responsible for cancer initiation and progression. This review will provide an overview of 14-3-3 proteins and address the role of 14-3-3ζ overexpression in cancer.

Areas covered in this review: The review covers the basic role of 14-3-3 in regulation of multiple pathways with a focus on 14-3-3ζ as a clinically relevant biomarker for cancer recurrence.

What the reader will gain: 14-3-3ζ overexpression has been found in multiple cancers; however, the clinical implications were unclear. Recently, 14-3-3ζ has been identified as a biomarker for poor prognosis and chemoresistance in multiple tumor types, indicating a potential clinical application for using 14-3-3ζ in selecting treatment options and predicting cancer patients' outcome.

Take home message: 14-3-3ζ is a potential prognostic marker of cancer recurrence and predictive marker for therapeutic resistance. The overexpression of 14-3-3ζ in multiple cancers suggests that it may be a common target to intervene tumor progression; therefore, more efforts are needed for the development of 14-3-3 inhibitors.