miR-146a调控人脐静脉内皮细胞的衰老及机制

目的探究miR-146a在人脐静脉内皮细胞衰老中的调节作用,并初步探究其机制。方法以人脐静脉内皮ECV-304细胞为研究对象,连续传代建立ECV-304细胞复制性衰老模型,衰老相关β-半乳糖苷酶(SA-β-gal)法检测不同代次(P2、P6、P9、P12、P15代)细胞中衰老细胞比例,实时定量PCR法检测不同代次(P2、P6、P9、P12、P15代)细胞中miR-146a表达。靶基因预测软件预测miR-146a靶基因。细胞转染建立miR-146a表达上调的P12代细胞(Pre-miR146a组)和miR-146a表达下调的P12代细胞(Anti-miR146a组),并建立相应对照组,SA-β-gal法检测衰老细胞比例,Western blot检测NADPH氧化酶4(NOX4)蛋白表达。结果 P6、P9、P12和P15代细胞的衰老细胞比例明显高于P2代细胞(P0.05),miR-146a表达则明显低于P2代细胞(P0.05)。靶基因预测miR-146a的靶基因为NOX4;转染后,Pre-miR146a组衰老细胞比例明显低于对照组(P0.05),Anti-miR146a组衰老细胞比例明显高于对照组(P0.05); Pre-miR146a组NOX4蛋白表达明显低于对照组(P0.05),Anti-miR146a组NOX4蛋白表达明显高于对照组(P0.05)。结论 miR-146a在衰老的人脐静脉内皮细胞ECV-304中表达下降,上调miR-146a表达能够抑制ECV-304衰老,其机制与调节NOX表达有关。     

Correlation between pre-miR-146a C/G polymorphism and gastric cancer risk in Chinese population

AIM:To investigate the association between pre-miR- 146a C/G polymorphism and gastric cancer risk. METHODS:We performed a hospital-based,case-control study using polymerase chain reaction-restriction fragment length polymorphism method in 608 individuals(304 gastric cancer patients and 304 age and sex matched cancer-free controls).RESULTS:The frequencies of pre-miR-146a C/G genotypes in the case group were significantly different from those in the control groups(P=0.037).Compared with CC genotype carriers,s...     

Correlation of miR-146a-5p plasma levels and rs2910164 polymorphism with left ventricle outflow tract obstruction in hypertrophic cardiomyopathy

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内皮细胞缺氧后miR-210、miR-92a、miR-126表达及意义

目的进一步探讨缺氧后血管新生的调控机制。方法常规方法培养人微血管内皮细胞,低氧培养箱制备内皮细胞缺氧模型（观察组）,正常细胞作为对照组。采用real time PCR法检测两组内皮特异性microRNA（miR-210、miR-92a、miR-126）表达变化。结果与对照组比较,miR-210、miR-92a、miR-126分别上调了（5.19±0.99）、（3.02±0.88）、（3.87±0.83）倍,P均〈0.05。结论缺氧可促使内皮细胞特异性microRNA表达改变,此可能为缺氧后血管新生的主要调控机制。     

1,25（OH）2D3通过调节miR-146a水平抑制大鼠肝纤维化的体内和体外观察

目的 体内体外观察1,25（OH）₂D₃通过调节miR-146a水平抑制大鼠肝纤维化的作用机制。方法 建立CCl₄诱导的大鼠肝纤维化模型,体外转染肝星状细胞（HSCs）miR-146a 模拟剂/抑制剂,观察1,25（OH）₂D₃处理对动物肝组织变化和HSC增殖和凋亡的影响。采用qPCR法检测肝组织miR-146a水平,采用CCK8法检测细胞增殖,使用流式细胞术检测HSC凋亡。结果 在干预8 w末,1,25（OH）₂D₃干预组大鼠肝纤维化程度明显减轻; 1,25（OH）₂D₃干预组大鼠肝组织miR-146a水平为（0.70± 0.03）,显著高于橄榄油组【（0.33±0.17,P<0.05）】; 1,25（OH）₂D₃组细胞增殖率为58.8%,较DMSO组下降了15.9%,转染miR-146a 模拟剂组大鼠HSC增殖率为46.5%,较对照组下降了53.3%,转染miR-146a 抑制剂组HSC增殖率为132.8%,较对照物组升高了32.8%（P<0.05）,1,25（OH）₂D₃干预组细胞凋亡率为12.6%,较DMSO组增加了5.2%,转染miR-146a 模拟剂组细胞凋亡率为16.8%,较对照组细胞凋亡率增加了8.2%,转染miR-146a 抑制剂组细胞凋亡率为6.3%,较对照组细胞凋亡率减少了2.2%（P<0.05）,提示1,25（OH）₂D₃具有抑制HSC增殖、促进凋亡作用。结论 1,25（OH）₂D₃可能通过调节miR-146a水平抑制HSC活化和抑制大鼠肝纤维化。     

miR-146a对原发性胆汁性肝硬化辅助性T细胞增殖调控作用的研究

目的 研究miR-146a对原发性胆汁性肝硬化(PBC)患者T细胞增殖以及白细胞介素(IL)-2分泌的调控作用.方法 荧光定量聚合酶链反应(RT-PCR)检测20例PBC患者和20名健康个体外周血单个核细胞( PBMC)、辅助性T细胞、细胞毒性T细胞、B细胞和单核细胞内miR-146a的表达.将miR-146a表达载体和表达抑制体转染入PBC患者辅助性T细胞内,以抗CD3抗体和抗CD28抗体刺激辅助性T细胞,以CCK-8法检测辅助性T细胞增殖能力,酶联免疫吸附试验(ELISA)法检测培养基内IL-2水平,以RT-PCR法监测辅助性T细胞内miR-146a的表达.2组间的比较采用t检验.结果 PBC患者PBMC内miR-146a的表达(0.46±0.20)较健康个体(1.00±0.26)明显降低(t=7.47,P＜0.01).PBC患者辅助性T细胞和单核细胞内miR-146a的表达(分别为(0.33±0.13,0.56±0.11)较健康个体(分别为1.00±0.14,1.00±0.11))明显降低(t=6.15,4.97;P＜0.01或P＜0.05),但PBC患者与健康个体细胞毒性T细胞(0.98±0.15,1.00±0.12,t=0.22,P＞0.05)和B细胞(0.91±0.06,1.00±0.14;t=0.97,P＞0.05)内miR-146a的表达量之间的差异无统计学意义.抗CD3和抗CD28可以诱导辅助性T细胞表达miR-146a(刺激前:1.00±0.18;刺激后:9.12±2.05;t=8.81,P＜0.01).PBC患者辅助性T细胞较健康个体具有较强的增殖能力{PBC:0.35±0.06[吸光度(A值)];健康个体:0.26±0.04(A值);t=2.83,P＜0.05}以及释放IL-2[ PBC (686±109)pg/ml;健康个体(512±72) pg/ml;t=2.96,P＜0.05]的能力.miR-146a可以抑制PBC患者T细胞的增殖以及分泌IL-2的能力.结论 miR-146a可以负向调控活化的PBC患者辅助性T细胞增殖以及分泌IL-2的能力.PBC的发病机制可能与患者辅助性T细胞内miR-146a表达降低、对细胞增殖的抑制作用减弱有关.     

HIV-1潜伏感染人Jurkat T细胞模型的建立

目的 建立Ⅰ型艾滋病病毒(HIV-1)潜伏感染人Jurkat T细胞的模型,为进一步研究HIV-1潜伏感染机制奠定基础.方法 利用一种表达增强型绿色荧光蛋白( EGFP)的HIV-1假病毒感染人Jurkat T细胞,采用流式细胞术分选出GFP -细胞群,之后经激活剂曲古抑菌素A(Trichostatin A,TSA...     

微小RNA miR-199a／a^＊模拟物转染肝癌细胞HepG2中Met原癌基因的表达

目的探讨在肝癌细胞系HepG2中,Met是否为miR-199a／a^＊的靶基因。方法将miR-199a／a^＊的模拟物及阴性对照物分别转染至HepG2细胞中,转染后48h及72h分别提取RNA及蛋白行RT—PCR及Western Blot检测Met的表达。结果与阴性对照相比,转染Up-miR-199a^＊组的MetmRNA（P〈0．05）和蛋白（P〈0．001）表达水平均显著下调;而Up—miR-199a组与阴性对照组相比无统计学差异。结论肝癌细胞HepG2中miR-199a^＊负调控Met的mRNA及蛋白表达。     

Regulatory effects of miR-146a/b on the function of endothelial progenitor cells in acute ischemic stroke in mice

The study aims to explore how microRNA-146a/b (miR-146a/b) regulates the function of endothelial progenitor cells (EPCs) in acute ischemic stroke in mice. Eighty male SPF C57BL/6J mice were evenly divided into the model-6 h, model-12 h, model-24 h (mice suffered from middle cerebral artery occlusion [MCAO] for 6 h, 12 h and model-24 h) and normal groups. EPCs were transfected and assigned into the control, MCAO, MCAO-miR-146a, MCAO-miR-146b and MCAO-miR-146a/b groups. The qRT-PCR was used to detect miR-146a/b expression in EPCs. Expressions of tumor necrosis factor receptor-associated factor 6 (TRAF6) and interleukin-1 receptor-associated kinase 1 (IRAK1) were detected using western blotting. Cell proliferation and migration of EPCs were testified using CCK-8 assay and scratch test, respectively. Angiogenesis ability of EPCs was observed under microscope. MiR-146a and miR-146b expressions were lower in the model groups than the normal group. There were up-regulated TRAF6 and IRAK1 expressions in the model-6 h, model-12 h and model-24 h groups compared with the normal group. And there were down-regulated TRAF6 and IRAK1 expressions in the MCAO-miR-146a, MCAO-miR-146b and MCAO-miR-146a/b groups than in the MCAO group. Compared with the control group, the proliferation, migration and angiogenesis ability of EPCs were significantly lower in the MCAO group, but higher in the MCAO-miR-146a, MCAO-miR-146b and MCAO-miR-146a/b groups. Besides, the miR-146a/b group showed more enhancement than the MCAO-miR-146a and MCAO-miR-146b groups. MiR-146a/b could down-regulate the TRAF6 and IRAK1 expressions and promote proliferation, migration and angiogenesis ability of EPCs, which was important for recovery of patients with hyperacute ischemic stroke.     

急性白血病细胞体外对Jurkat细胞凋亡的影响

为探讨白血病细胞凋亡相关基因 (Fas)的功能性表达情况 ,将 2 7例白血病患者的白血病细胞与 Jurkat细胞体外孵育后 ,用原位末端标记法 (TUNEL )测定单纯 Jurkat细胞 (对照组 )、不加和加入不同浓度抗凋亡相关基因配体 (Fas L)的 Jurkat细胞凋亡率。结果显示 ,不加抗 Fas L 急性非淋巴细胞白血病组 (ANL L 组 )与对照组比较 ,凋亡率明显增高 (P<0 .0 1) ,急性淋巴细胞白血病组 (AL L 组 )与对照组比较仅轻度增高 (P<0 .0 5 ) ,两者均随细胞浓度的增加而凋亡率增高 ;ANL L组加与不加抗 Fas L 相比凋亡率明显减少 (P<0 .0 1) ,随抗 Fas L浓度的增加而凋亡率减少 ,AL L 组加与不加抗 Fas L 相比凋亡率轻度减少 (P<0 .0 5 ) ,也随抗 Fas L 浓度的增高而减少 ,但不如 ANL L组凋亡率变化明显。提示急性白血病细胞存在 Fas L的功能性表达 ,ANL L较 AL L的 Fas L表达高     

砷诱导人T淋巴细胞表达HMG2基因的克隆研究

目的为了解三氧化二砷诱导人T淋巴细胞的差异表达基因,探讨砷对淋巴细胞基因转录的影响机制.方法在体外用三氧化二砷处理人Jurkat T淋巴细胞(5μmol/L,24h)后,应用抑制性消减杂交技术(SSH)和克隆技术构建差异表达的cDNA文库,用PCR技术和测序技术鉴定阳性克隆.结果用SSH技术构建了由三氧化二砷诱导的Jurkat T淋巴细胞的差异表达基因的正向消减cDNA文库.经测序分析结果显示,该文库中含有高泳动类蛋白2(HMG2)基因片段,同源性98%.结论我们的研究结果提示三氧化二砷(5μM,24h)可诱导HMG2的转录,它在砷诱导淋巴细胞凋亡机制中可能起着重要的作用.     

肺癌细胞与组织中miR-100、miR-148a的表达变化及临床意义

目的：观察微小RNA 100（miR-100）、微小RNA 148a（miR-148a）在肺癌细胞与组织中的表达,并探讨其临床意义。方法采用RT-PCR方法检测人肺癌细胞系NCI-H1299、NCI-H358、H460、A549及人正常支气管上皮细胞系HBE,以及肺癌及其癌旁组织中miR-100、miR-148a表达;分析肺癌组织中miR-100、miR-148a表达与其临床病理参数的关系。结果与HBE细胞相比,miR-100在NCI-H1299、NCI-H358及A549细胞中表达升高,在H460细胞表达降低,P均＜0．05;miR-148a在NCI-H358、H460细胞中表达升高,在A549细胞中表达降低,P均＜0．05。与癌旁组织相比,肺癌组织中miR-100表达降低,但差异无统计学意义（P＝0．400）;miR-148a表达升高（P＝0．036）。肺癌组织中miR-100、miR-148a表达水平与患者性别、年龄、病理类型无关;有淋巴结转移者肺癌组织中miR-100表达水平较无淋巴结转移者低（P＝0．016）。结论 miR-100、miR-148a在肺癌细胞中表达异常;在肺癌组织中,miR-100表达降低,miR-148a表达增加,二者可能参与肺癌的发生、发展。     

体外与非体外循环冠状动脉旁路移植术后血清miR-1、miR-133a、miR-208a水平变化及分析

目的探究行体外循环冠状动脉旁路移植术(ONCABG)与非体外循环冠状动脉旁路移植术(OPCABG)患者术后血清miR-1、miR-133a、miR-208a水平变化。方法选取2016年2月—2019年2月本院多支冠状动脉病变需要行CABG的患者94例。根据手术方式,将94例患者分为ONCABG组47例和OPCABG组47例。利用荧光实时定量PCR法检测所有患者术前、术后不同时间血清miR-1、miR-133a、miR-208a水平。采用化学发光免疫分析仪检测所有患者术前、术后不同时间血清心肌肌钙蛋白I(cTnI)、肌酸激酶同工酶(CK-MB)水平。分析血清miR-1、miR-133a、miR-208a水平与cTnI、CK-MB水平的相关性。结果与术前相比,两组患者术后4 h、24 h血清miR-1、miR-133a、miR-208a、cTnI、CK-MB水平升高(P0.05)。与术后4 h相比,两组患者术后24 h血清miR-1、miR-133a、miR-208a、cTnI、CK-MB水平降低(P0.05)。与ONCABG组相比,OPCABG组术后4 h、术后24 h血清miR-1、miR-133a、miR-208a、cTnI、CK-MB水平降低(P0.05)。Pearson分析显示,两组患者术后4 h、术后24 h血清miR-1、miR-133a、miR-208a水平与cTnI、CK-MB水平均呈正相关(P0.05)。结论 miR-1、miR-133a、miR-208a有望作为判断ONCABG与OPCABG后心肌损伤的重要生物标志物。本研究为ONCABG与OPCABG在临床上的选择提供了一定的参考依据。     

Common SNP in pre-miR-146a decreases mature miR expression and predisposes to papillary thyroid carcinoma

Although papillary thyroid carcinoma (PTC) displays strong heritability, no predisposing germ-line mutations have been found. We show that a common G/C polymorphism (rs2910164) within the pre-miR-146a sequence reduced the amount of pre- and mature miR-146a from the C allele 1.9- and 1.8-fold, respectively, compared with the G allele. This is matched by a similar decrease in the amount of each pre-miR generated from the corresponding pri-miR-146a in an in vitro processing reaction. The C allele also interfered with the binding of a nuclear factor to pre-miR-146a. The reduction in miR-146a led to less efficient inhibition of target genes involved in the Toll-like receptor and cytokine signaling pathway (TRAF6, IRAK1), and PTC1 (also known as CCDC6 or H4), a gene frequently rearranged with RET proto-oncogene in PTC. In an association study of 608 PTC patients and 901 controls, we found marked differences in genotype distribution of rs2910164 (P = 0.000002), the GC heterozygous state being associated with an increased risk of acquiring PTC (odds ratio = 1.62, P = 0.000007), and both homozygous states protective with odds ratio = 0.42 for the CC genotype (P = 0.003) and odds ratio = 0.69 for the GG genotype (P = 0.0006). Moreover, 4.7% of tumors had undergone somatic mutations of the SNP sequence. Thus, our data suggest that a common polymorphism in pre-miR-146a affects the miR expression, contributes to the genetic predisposition to PTC, and plays a role in the tumorigenesis through somatic mutation. Preliminary evidence suggests that these effects are mediated through target genes whose expression is affected by the SNP status.     

HIV-1慢性感染Jurkat细胞系的建立及其特性研究

目的建立艾滋病病毒Ⅰ型（HIV-1）慢性感染Jurkat细胞系,研究其生物学特性。方法参照文献方法改良,建立HIV-1ⅢB慢性感染Jurkat细胞系。其细胞系特性采用以下方法检测：光学显微镜观察细胞形态;流式细胞仪检测HIV-1ⅢB感染细胞的阳性率;噻唑蓝（MTT）法检测抗病毒药物对慢性感染细胞的毒性作用;酶联免疫吸附试验（ELISA）检测药物对慢性感染细胞中病毒复制的影响;合胞体形成方法检测药物对HIV-1ⅢB慢性感染细胞与正常细胞融合的阻断作用。结果成功建立了HIV-1慢性感染Jurkat细胞系（Jurkat/HIV-1ⅢB）,感染细胞阳性率〉90%。检测影响病毒复制各周期的抗病毒药物对Jurkat/HIV-1ⅢB细胞的作用,发现Jurkat/HIV-1ⅢB细胞具有HIV-1慢性感染细胞的生物学特征。结论成功建立Jurkat/HIV-1ⅢB细胞系,为抗HIV-1药物的研发和病毒感染机制研究提供了新的工具。     

Effects of metformin on changes of miR-19a and miR-221 expression associated with myocardial infarction in patients with type 2 diabetes

BackgroundThe presence of hyperglycemia is a risk factor for cardiovascular diseases, as it increases the risk of myocardial infarction (MI). Metformin is considered an effective anti-hyperglycemic drug for patients with type 2 diabetes. Prediction of microRNAs is valuable in determining the risk of MI.AimThis study aimed to measure the expression of two microRNAs, which are involved in the risk of MI and vascular stenosis among metformin users and non-users with MI.MethodsIn this study, we analyzed the expression of two microRNAs, collected from the blood samples of 180 subjects with MI, using the quantitative polymerase chain reaction (qPCR) assay. The subjects were categorized into three groups: non-diabetic patients with MI (MIND), diabetic patients with MI not using metformin (MIDMet?), and diabetic patients with MI using metformin (MIDMet+). To assess the sensitivity and specificity of miR-19a and miR-221 expression as potential biomarkers for MI, the receiver operating characteristic curve (ROC) analysis was conducted for both diabetic groups.ResultsThe diabetic MIDMet + group exhibited a significant decrease in the expression levels of miR-221 (7.2 folds) and miR-19a (5.3 folds) as compared to the MIDMet? and MIND groups (p < 0.05). The ROC analysis revealed that the areas under the ROC curve (AUC) for circulating miR-19a and miR-221 were 0.931 and 0.965 in patients with type 2 diabetes, respectively (p < 0.001).ConclusionBased on the present findings, metformin therapy can influence cardiovascular disorders and their outcomes through down-regulation of microRNAs. Also, exploration of microRNAs and the effects of metformin on their reduction can provide a potential therapeutic strategy for patients with type 2 diabetes by reducing the MI risk.