miR-27a在猿猴病毒小T抗原诱导支气管上皮细胞恶性转化中的作用

目的:检测猿猴病毒40(simian virus 40,SV40)小T抗原(small T antigen,ST)诱导人支气管上皮细胞(human bronchialepithelial cell,HBE)恶性转化中miRNAs的表达谱,寻找与细胞转化相关的miRNAs。方法:选择HBE、HBER和HBERST细胞株,提取总RNA,利用miRNA芯片和实时荧光定量PCR技术检测和验证永生化HBE、HBER和HBERST细胞中差异表达的miRNAs。通过细胞生长曲线检测、细胞周期分析、细胞克隆形成试验等确证与SV40 ST诱导HBE细胞恶性转化相关的miRNAs。结果:在HBE、HBER和HBERST细胞856个miRNA的表达谱中筛选出6个与SV40 ST诱导细胞转化相关的miRNA,2个表达上调(miR-20a和miR-27a).4个表达下调(let-7d,let-7f,miR-1246和miR-3746)。抑制miR-27a能减缓HBERST细胞的增殖速度(P0.01),延长细胞在G0～G1期的停留时间(P0.01)和降低HBERST细胞在软琼脂上形成克隆的数目(P0.01)。结论:miR-27a的异常表达参与了SV40ST诱导的HBE细胞恶性转化。     

Function of miR-27a in malignantly transformed human bronchial epithelial cells induced by SV40 small T antigen

目的:检测猿猴病毒40 (simian virus 40,SV40)小T抗原(small T antigen,ST)诱导人支气管上皮细胞(human bronchial epithelial cell,HBE)恶性转化中miRNAs的表达谱,寻找与细胞转化相关的miRNAs.方法:选择HBE、HBER和HBERST细胞株,提取总RNA,利用miRNA芯片和实时荧光定量PCR技术检测和验证永生化HBE、HBER和HBERST细胞中差异表达的miRNAs.通过细胞生长曲线检测、细胞周期分析、细胞克隆形成试验等确证与SV40 ST诱导HBE细胞恶性转化相关的miRNAs.结果:在HBE、HBER和HBERST细胞856个miRNA的表达谱中筛选出6个与SV40 ST诱导细胞转化相关的miRNA,2个表达上调(miR-20a和miR-27a),4个表达下调(let-7d,let-7f,miR-1246和miR-3746).抑制miR-27a能减缓HBERST细胞的增殖速度(P＜0.01),延长细胞在G0～G1期的停留时间(P＜0.01)和降低HBERST细胞在软琼脂上形成克隆的数目(P＜0.01).结论:miR-27a的异常表达参与了SV40ST秀导的HBE细胞恶性转化.     

α4在化学致癌物诱导细胞转化中的作用

目的：探讨α4在化学致癌物诱导细胞转化中的可能作用。方法：采用免疫印迹检测化学致癌物诱导既往转化细胞模型和肝肿瘤细胞株中α4的表达水平，再利用病毒感染法在肝永生化细胞株L02R上构建α4高表达（L02R-α4）和低表达（L02R-SHα4）的细胞株，检测其细胞生长速度和转化能力。进一步选择已建立的细胞株、化学致癌物AFB1诱导转化的细胞（L02RT-AFB1）及肝肿瘤细胞株HepG2和SMMC等，在有或无mTOR通路抑制剂雷帕霉素处理下，通过免疫印迹检测mTOR下游两个分子p70S6K1和4E-BP1的表达及磷酸化水平。结果：在化学致癌物诱导转化的细胞模型和肝肿瘤细胞株中发现α4的表达比对照细胞上调1.9~5.9倍。蛋白印迹结果证实L02R-α4和L02R-SHα4细胞株构建成功，α4表达上调能够促进L02R细胞增殖并发生转化（P＜0.05）。在α4高表达的转化细胞L02R-α4中，p70S6K1和4E-BP1呈高磷酸化状态。当有雷帕霉素作用时，所有细胞中p70S6K1和4E-BP1的磷酸化水平明显下降，在L02R-SHα4细胞中下降尤为显著。结论：α4具有癌基因功能，α4的异常上调激活mTOR通路，促进细胞增殖并诱导细胞恶性转化。     

甲基丙烯酸环氧丙酯致人支气管上皮细胞恶性转化过程中DNMT1及MBD1基因表达的变化

目的：分析甲基丙烯酸环氧丙酯（glycidyl methacrylate,GMA）致人支气管上皮16HBE细胞恶性转化过程中不同时点甲基化转移酶及甲基CpG结合蛋白家族基因的表达差异,初步探讨GMA诱导16HBE细胞发生恶性转化的表观遗传机制。方法：采用人类全基因组表达谱芯片筛选GMA诱导16HBE细胞恶性转化过程中甲基化转移酶及甲基CpG结合蛋白家族的差异表达基因,并采用实时定量PCR（Real time PCR）技术检测筛选所得差异基因DNMT1及MBD1 mRNA的表达情况。结果：芯片结果显示在转化第10代（前期）细胞中DNMT1及MBD1基因较同代龄对照细胞表达上调,实时定量PCR进一步确证DNMT1及MBD1基因表达分别上调80.60%、79.10%,与芯片结果一致。结论：甲基化可能是GMA致16HBE细胞发生恶性转化的一种机制,DNMT1及MBD1基因可能是多个甲基化相关基因转录表达下调的关键调控点,其在GMA致16HBE细胞恶性转化过程中起着重要作用。     

大鼠腹部手术应激后血清miRNA表达谱的差异分析

目的:应用miRNA芯片技术建立大鼠腹部手术应激后血清miRNA差异表达谱,从中发现与早期手术创伤后应激相关的miRNAs。方法:建立大鼠部分肝切除手术模型。检测手术前后大鼠血清中ALT、AST和CRP浓度水平及肝脏病理改变,评估腹部创伤后大鼠应激情况。采用miRNA表达谱芯片筛选手术前后大鼠血清中差异表达的miRNAs,并采用实时荧光定量RT-PCR(real-time RT-PCR)进行验证。采用生物信息学软件(MiRand和TargetScan)预测候选miRNA的靶基因。结果:血清miRNA表达谱出现明显改变,24个miRNAs在2/3部分肝切除术（PH）后24h大鼠血清中表达明显升高,特别是miR-9,其在2/3PH术后24h大鼠血清中的表达水平为术前的50多倍,real-time RT-PCR检测miR-9的结果与芯片结果相符。通过生物信息学预测,CDH1、E-cadherin、MTHFD2、PDYN、MCPIP1、BCL2L11、CMA1、Map3k1等可能是miR-9的靶基因。结论:腹部手术创伤后,大鼠血清表达谱发生明显变化,提示miRNA可能参与调控创伤后应激反应。     

微小RNA-181a、320a及其靶基因TGF-β1在食管鳞癌中的表达

目的：检测人食管鳞癌组织中miR-181a、320a及TGF-β1的表达情况,研究其在食管鳞癌发生中的作用。方法：收集食管鳞癌及癌旁正常组织各3例,抽取总RNA,利用miRNA芯片技术检测miRNA的表达;采用实时定量反转录一聚合酶链反应(qRT-PCR)方法验证miRNA芯片结果的可靠性。采用软件和数据库对差异表达miRNA调控的靶基因进行初步分析。收集食管鳞癌及对照正常组织各22例,采用实时定量反转录-聚合酶链反应(qRT-PCR)方法检测miR-181a、320a和TGF-β1mRNA。结果：miRNA芯片结果显示,共有22种miRNA在食管鳞癌中差异表达（p<0.05）,其中6种显著上调,16种显著下调;qRT-PCR证实miR-320a表达下降,miR-181a表达上升,差异均有统计学意义（P﹤0.05）,与芯片结果一致; TGF-β1mRNA在食管鳞癌中表达较对照正常组织稍升高,但差异无显著性。结论：miR-181a、miR-320a可能参与食管鳞癌的发生发展过程。     

人肺腺癌吉非替尼耐药细胞株中微小RNA的表达谱

目的:分析人肺腺癌吉非替尼耐药细胞株PC9/AB2与亲本细胞株PC9中微小RNA(microRNA,miRNA)表达谱的差异.方法:应用Agilent human miRNA芯片分别检测PC9/AB2细胞与PC9细胞miRNA的表达谱,随后采用Agilent Feature Extraction软件分析并筛选差异表达的miRNA,应用生物信息软件预测筛选出的差异表达miRNA的潜在靶基因.应用实时荧光定量-PCR(real-time fluorogenic quantitative-PCR,RFQ-PCR)验证芯片检测结果.结果:与PC9细胞比较,PC9/AB2细胞中有36个miRNA表达上调＞2倍,有24个miRNA表达下调＞2倍.RFQ-PCR验证了芯片的结果.软件预测发现,16个miRNA有潜在靶基因,其中11个miRNA的靶基因＞100个.结论:筛选获得的差异表达miRNA可能通过调控其靶基因而参与人肺腺癌细胞对吉非替尼的耐药.     

结肠癌组织和癌旁组织miRNA表达谱研究

目的探讨结肠癌癌组织和癌旁组织中miRNA的表达差异,寻找潜在的结肠癌特异表达谱。方法miRNA表达芯片检测手术切除未发生转移的原位结肠癌组织和癌旁组织标本中miRNA表达水平,并采用Real-time PCR对差异表达的miRNA进行验证。结果miRNA表达芯片分析发现肿瘤细胞中24种miRNA表达下调,27种表达上调。miR-145、miR-143在癌组织中表达显著下调,miR-451在癌组织中表达显著上调,并被Real-time PCR方法进一步证实。结论结肠癌癌组织和癌旁组织中miRNA的表达存在差异,miR-145、miR-143在结肠癌组织中表达下调,miR-451表达上调,结肠癌组织具有特异miRNA表达谱,可作为结肠癌潜在诊断芯片进行开发。     

不同转移潜能人大细胞肺癌细胞株转移相关microRNAs的筛选研究

背景与目的微小RNA(microRNAs,miRNAs)参与调节肿瘤发生发展的多个过程,包括细胞的分裂增殖、细胞周期、凋亡、血管形成、侵袭和转移等。本研究应用miRNA芯片检测具有高低不同转移潜能人大细胞肺癌细胞株L9981和NL9980的miRNA表达谱,从中筛选出与大细胞肺癌转移相关的miRNAs。方法收集L9981和NL9980细胞,抽提总RNA进行CY3标记,将标记RNA在miRNA芯片上进行杂交反应。通过数据统计分析,筛选出表达明显差异的miRNAs。应用Real-timePCR验证芯片结果,并应用生物信息学方法预测靶基因。结果在不同转移潜能人大细胞肺癌L9981和NL9980细胞株中共筛选到22个表达明显差异的miRNAs。与NL9980相比,在L9981中有13个miRNAs表达上调,9个表达下调。Real-timePCR验证miR-125a-3p在细胞中的表达水平与芯片结果趋势一致,预测其靶基因可能为胰岛素样生长因子2。结论筛选得到与大细胞肺癌转移相关的miRNA表达谱。     

丝裂霉素对乳腺癌细胞microRNAs表达谱的影响

[目的]探讨丝裂霉素（MMC）对乳腺癌细胞miRNA表达谱的影响,并分析潜在的治疗靶点。[方法]培养人乳腺癌细胞株MCF-7;MTT法检测细胞增殖情况;MMC的IC50浓度干预MCF-7细胞48h后,利用miRNA芯片技术,检测用药前后MCF-7细胞miRNAs的表达;采用实时定量RT-PCR进行验证;对miRNA表达谱进行差异性分析;运用TargetScan软件预测miRNA可能调控的靶基因。[结果]MMC与MCF-7细胞生长抑制率之间有浓度依赖关系,48h的IC50为4.75μmol/L;用4.75μmol/L浓度的MMC干预细胞48h后,与模型组比较,获得82个与MMC干预相关miRNAs（P〈0.01）,其中,显著上调的10个,显著下调的8个;对MMC显著上调的miR-936进行生物信息学分析,共筛选出7个潜在靶基因的生物学功能与乳腺癌相关。[结论]MMC对miRNAs的表达调控可能是其抑制乳腺癌细胞生长的作用途径之一。     

Metabolism of aflatoxin B1 in the upper airways of the rabbit: role of the nonciliated tracheal epithelial cell

Short-term tracheal explant cultures from the rabbit were used to study the metabolism of the carcinogen aflatoxin B1 (AFB1) and to determine the cell types that are susceptible to damage by AFB1 and their relative contents of monooxygenase enzymes. Tracheas were cultured in serum-free medium for 0.5-24 h with 0.7 microM [3H]AFB1, and metabolism was measured by determining the level of binding of the carcinogen to DNA and by the release of metabolites into the medium. The binding of aflatoxin B1 was time dependent and appeared to peak at 12 h in culture. In addition, the metabolites aflatoxicol, aflatoxin M1, and aflatoxin Q1 were produced by the explants. Ultrastructural evaluation of cultured tracheas showed degenerative changes exclusively in nonciliated secretory cells after 4 h in culture. Extensive nonciliated secretory cell necrosis was evident by 12 h. Ciliated cells did not show degenerative changes until 12 h and appeared much more viable after 24-h exposure to AFB1 relative to the nonciliated cells. Tracheal sections stained to demonstrate rabbit lung cytochrome P-450, Forms 2 and 5, and cytochrome P-450 reduced nicotinamide adenine dinucleotide phosphate reductase by an immunoperoxidase technique showed intense staining selectively within nonciliated cells. In total, the data revealed that: (a) rabbit tracheal explants are able to metabolize aflatoxin B1; (b) the nonciliated secretory cell population in this tissue is the target cell for cytotoxicity of this carcinogen; and (c) as is the case in the more distal airways, the nonciliated epithelial cells appear to have a high content of components of the pulmonary cytochrome P-450 monooxygenase system, which may be an important factor in the susceptibility of these cells and this region of the airways to suspected airborne carcinogens.     

小鼠白血病细胞L1210外泌体微RNA表达谱及其功能分析

目的：对白血病细胞外泌体（LCEX）中微 RNA（miRNA）的表达特点及其功能进行分析。方法以小鼠白血病细胞株 L1210为模型,应用密度梯度超速离心法分离其培养上清,获得 LCEX L1210,应用基因芯片技术检测 L1210细胞和 LCEXL1210中 miRNA 的表达,比较二者 miRNA 的表达,对部分差异表达的miRNA 应用实时荧光定量 PCR 进行验证,通过 Gene Ontology 数据库分析。结果在 LCEX L1210中共鉴定出1044种 miRNA,L1210细胞中共鉴定出872种 miRNA,其中732种 miRNA 为两者共有,两者共同表达的 miRNA 占 L1210细胞的83.9%,占 LCEXL1210的70.1%,提示 LCEXL1210中70%以上 miRNA 来自于其母细胞。在 LCEXL1210中有312种 miRNA 在其母体细胞中没有鉴定出,为 LCEXL1210所特有。有些miRNA 在 LCEXL1210中表达明显高于 L1210细胞,如 miR-16-1、miR-210及 miR-195等,提示 LCEX 的miRNA 表达与其母体细胞存在表达差异。对部分表达差异的 miRNA 应用实时荧光定量 PCR 验证显示,这些 LCEX 高表达的 miRNA 参与多种生物学功能及信号转导途径的调控。结论 LCEXL1210所含的miRNA 与其来源细胞 L1210细胞所含的 miRNA 有高度相似性,但有1／3的 miRNA 为其所特有。这些LCEXL1210中高表达的 miRNA 参与多种生物学功能及信号转导途径的调控。     

Immunological detection of aflatoxin B1-DNA adducts formed in vivo

Two monoclonal antibodies (6A10 and 12F5) were obtained after fusion of mouse P3X63-AG.8.653 myeloma cells with spleen cells isolated from BALB/c mice immunized with imidazole ring-opened aflatoxin B1 (AFB1)-DNA and characterized by competitive enzyme-linked immunosorbent assays. Both antibodies are highly specific for imidazole ring opened AFB1-DNA and show some cross-reactivity with AFB1-DNA and no cross-reactivity with 8,9-dihydro-8-(7-guanyl)-9-hydroxy-AFB1, AFB1 conjugated with bovine serum albumin, aflatoxin M1 conjugated with bovine serum albumin, AFB1, or aflatoxin G1. Antibody 6A10 was further characterized and showed no cross-reactivity with DNA modified by several other carcinogens. It could detect adducts with 4-fold higher sensitivity in highly modified DNA (2.5 adducts/100 nucleotides) than in low modified DNA (4 adducts/10(5) nucleotides). With low modified DNA the limit of sensitivity is 5 adducts/10(7) nucleotides. Antibody 6A10 reliably detected adducts formed in vivo in rats and mice treated with AFB1. In a pilot study, AFB1 adducts were detected in liver tissues from individuals living in areas with suspected exposure to AFB1. Monitoring adduct levels in human tissue may provide information not only on carcinogen exposure but also on the relationship among infection with hepatitis B virus, dietary exposure to aflatoxin B1, and liver cancer.     

Inhibition of penta-acetyl geniposide on AFB1-induced genotoxicity in C3H10T1/2 cells.

A new compound, penta-acetyl geniposide ((Ac)5-GP), was obtained from modified extract of Gardenia fructus (San-Jee-Chee in Chinese). The structure of the compound was identified as 1-(beta-D-2',3',4',6'-tetraacetyl-glucopyrannosyloxyl)-1,4a, 5,7a-tetrahydro-7-(acetomethyl)-cyclopentapyran-4-carboxylic acid methyl ester, according to the spectral data. The inhibitory effects of (Ac)5-GP on aflatoxin B1 (AFB1)-induced cytotoxicity and DNA damage were studied. In the investigation of the inhibitory effect of (Ac)5-GP on AFB1-cytotoxicity, the plating efficiency of C3H10T1/2 cells in S-9 activation system was increased. In addition, (Ac)5-GP inhibited the DNA damage of AFB1-treated C3H10T1/2 cells, and it interfered with the inhibitory effect of DNA synthesis caused by AFB1. These results suggest that the reduced DNA damage and the increased DNA synthesis from cultured C3H10T1/2 cells are important mechanisms for the inhibition of AFB1-cytotoxicity by (Ac)5-GP.     

Investigation of covalent aflatoxin B1-DNA adducts formed in vivo in rainbow trout (Salmo gairdneri) embryos and liver

The formation of covalent aflatoxin-DNA adducts has been studied in embryo and yearling stages of the rainbow trout (Salmo gairdneri). A linear relationship was found between the amount of aflatoxin B1 (AFB1) absorbed into 21-day-old eggs and the level of covalent modification of embryo DNA after exposure to 0.25 to 0.5 p.p.m. aqueous solutions of AFB1 for 1 h; higher concentrations resulted in absorption of a greater proportion of AFB1. The principal covalent product, identified after acid and enzymatic hydrolysis of isolated embryo DNA, was chromatographically identical to 2,3-dihydro-2-(N7-guanyl)-3-hydroxy aflatoxin B1. Additional AFB1 derivatives which were present are believed to be formed by chemical transformation of this product in DNA producing an aflatoxin-formamidopyrimidine adduct and the metabolic activation of other aflatoxin metabolites, such as aflatoxin M1 and aflatoxin P1. Although the eggs were exposed to AFB1 for a short time, covalent modification of DNA was evident over an extended period. The highest level of covalent products was present at approximately 24 h after exposure to 0.5 p.p.m. AFB1. Ninety-six hours later, this level had decreased slightly; however, the distribution of covalent adducts had changed: formamidopyrimidine adducts now represented up to 50% of the hydrolyzed aflatoxin derivatives. A similar pattern of covalent aflatoxin derivatives was found in the liver DNA of yearling trout 10 h after the administration of 0.3 mg/kg AFB1.     

Modulatory effect of crocetin on aflatoxin B1 cytotoxicity and DNA adduct formation in C3H10T1/2 fibroblast cell

Crocetin is a carotenoid isolated from the seeds of Cape jasmine (Gardenia jasminoides). The cytotoxicity and DNA-adduct formation of rat microsome-activated aflatoxin B1 (AFB1) in the C3H10T1/2 cells were significantly inhibited by pretreatment of crocetin. Most significant inhibition was found at the time of 9 h after crocetin pretreatment. Under these experimental conditions, consistent elevation in the cytosolic glutathione (GSH) levels and the activities of GSH S-transferase (GST) and GSH-peroxidase (GSH-Px) were observed. Crocetin treatment also resulted in a decrease in AFB1-DNA adduct formation in vitro, while no effect of crocetin on the formation of AFB1-8,9-oxide in vitro system was detected as measured by the Trisdiol method. From these results, we suggested that the protective effect of crocetin on the AFB1-cytotoxicity in C3H10T1/2 cells might be due to the cellular defense mechanisms that elevated the cytosol GSH and the activities of GST and GSH-Px.     

Aflatoxin B1-2,3-oxide as a probable intermediate in the covalent binding of aflatoxins B1 and B2 to rat liver DNA and ribosomal RNA in vivo.

Administration of[3H]aflatoxin B2 (2,3-dihydroaflatoxin B1)(AFB2) to male rats resulted in levels of hepatic DNA- and ribosomal(r)RNA-aflatoxin adducts that were about 1% of those for rats given [3H]aflatoxin B1(AFB1). The levels of hepatic protein-aflatoxin adducts were 35 to 70% as great for AFB2-treated as compared to AFB1-treated rats...     

miR-32-5p通过靶向Dickkopf相关蛋白3的表达调控乳腺癌MDA-MB-231 细胞的生物学行为

目的：通过生物信息学手段筛选乳腺癌中差异表达的关键miRNA及其靶基因,干预其在乳腺癌细胞中的表达并观察对乳腺癌细胞功能的影响。方法：利用GEO数据库筛选在乳腺癌中差异表达的miRNA,ENCORI数据库验证差异miRNA的表达,以选定最显著的差异表达 miRNA 为研究对象;利用 Starbase、miRDB 和 miRWalk 数据库预测 miR-32-5p 的靶基因,利用DAVID数据库对靶基因进行GO分析和KEGG分析,利用String数据库联合Cytoscape3.6.2软件进行PPI网络分析及核心基因的筛选,从核心基因中选择相互联系紧密“度值”最显著的Dickkopf相关蛋白3（DDK3）基因进行后续实验。qPCR检测miR-32-5p在人正常乳腺细胞 MCF10A和人乳腺癌细胞MCF7、MDA-MB-231、MDA-MB-453细胞中的表达。向MDA-MB-231细胞中转染miR-32-5p mimic、miR-32-5p inhibitor及各自的对照（NC）序列,分别用CCK-8法、流式细胞术和Transwell实验检测过表达或抑制miR-32-5p对细胞增殖、凋亡和侵袭的影响。结果：从GEO数据库中获取的两个数据集共识别出两个差异miRNA,ENCORI数据库验证差异miRNA的表达发现miR-32-5p的表达水平与GEO数据库的结果一致,故选择其进行研究;预测得到198个miR-32-5p 潜在的靶基因并鉴定出 10 个核心基因（DKK3、WNT2B、SFRP5、SFRP2、SFRP1、LRP6、WNT6、KREMEN1、NEDD4L、TRIP12）,其中DKK3的度值最大可能在乳腺癌中较为重要,于是选择miR-32-5p/DKK3轴进行后续研究。miR-32-5p在3种乳腺癌细胞中的表达水平显著高于正常乳腺细胞（均P<0.01）,其中以MDA-MB-231细胞中表达最高。双荧光素酶基因报告实验验证了miR-32-5p与DKK3基因的靶向结合及其对后者表达的负向调控。转染miR-32-5p mimic、miR-32-5p inhibitor后成功提高或抑制了MDA-MB-231细胞中miR-32-5p的表达。与对照组相比,过表达miR-32-5p可抑制MDA-MB-231细胞的凋亡而促进细胞增殖和侵袭（P<0.05或P<0.01）,敲低miR-32-5p则起相反的作用（均P<0.01）。结论：miR-32-5p/DKK3轴可能是影响乳腺癌发生发展的关键通路,过表达miR-32-5p能够抑制乳腺癌MDA-MB-231细胞的凋亡而促进细胞增殖和侵袭。