自发性高血压大鼠延髓microRNA差异表达分析

目的　探讨自发性高血压大鼠延髓microRNA（miRNA）差异表达谱及其靶基因生物信息学分析。 方法　采用自发性高血压大鼠模型（SHR组）,同周龄SD大鼠为对照组（Control组）。利用miRNA芯片检测大鼠延髓中miRNAs差异表达谱。 结果　与对照组比较,SHR组尾动脉收缩压显著升高（P＜0.0001）;SHR组延髓组织miRNAs有显著差异表达谱,16个miRNAs表达上调和7个miRNAs表达下调（1.5-fold change cutoff, P<0.05）。qRT-PCR验证结果显示,与对照组比较,SHR组延髓miR-153、miR-193及miR-301a表达显著下降,与芯片结果一致。生物信息学分析显示,差异表达miRNAs可能调控2775个靶基因（target score≥83）。这些靶基因主要富集在12个信号通路,包括磷脂酰肌醇3-激酶（Phosphatidylinositide3-kinase,PI3K）通路等。 结论　自发性高血压大鼠延髓组织中miR-153、miR-193及miR-301a明显下调,且生物信息学分析提示PI3K通路介导神经炎症可能作为高血压中枢相关差异表达miRNAs调控靶基因介导的主要致病通路。     

miRNAs可能通过调控MAPK通路参与心室间隔缺损

目的 探讨miRNAs在心室间隔缺损(VSD)中发挥的作用.方法 通过miRNAs表达谱芯片实验分析心室间隔缺损(VSD)组和健康组全血样本中的差异表达miRNAs,通过生物信息数据库miRbase、Targetscan和Miranda进行靶基因测预.通过KEGG和REACTOME通路数据库筛选靶基因信号通路;采用RT...     

人乳腺癌MCF-7细胞在不同HER-2水平miRNAs表达谱检测

目的：通过对比观察人乳腺癌细胞MCF-7和MCF-7/HER-2中微小RNA( miRNAs)的差异性表达,初步筛选出与HER-2表达相关的miRNAs表达谱。方法培养人乳腺癌细胞MCF-7和MCF-7/HER-2,利用miRNA芯片技术检测两株细胞中miR-NAs的表达。结果利用miRNA微阵列,筛选获得217种与HER-2相关的miRNAs,较正常MCF-7细胞上调的miRNAs有123个,下调的有94个。在差异有显著性的miRNAs中,hsa-miR-141和hsa-miR-1299的靶基因有多种生物学功能。结论获得人乳腺癌MCF-7细胞在不同HER-2水平miRNAs的差异表达谱,为进一步分析HER-2在乳腺癌中的作用奠定基础。     

CCl4诱导的小鼠纤维化肝组织微小RNA差异表达谱及初步功能分析

 目的：应用微小RNA(miRNA)芯片技术研究miRNAs在四氯化碳（CCl4）诱导的小鼠纤维化肝脏中的差异表达谱,并基于基因本体论（gene ontology,GO)分析及信号转导通路分析发现差异miRNAs的主要功能。方法：实验分为正常组及模型组,皮下注射 CCl4复制小鼠肝纤维化模型;应用Agilent 小鼠 miRNA 寡核苷酸基因芯片检测各组肝脏miRNA表达谱。用随机方差模型t检验筛选2组间的差异miRNAs,并预测其靶基因。对靶基因进行GO分析及信号转导通路分析发现差异miRNAs发挥的主要功能。结果：正常组与模型组间共筛选出39个差异miRNAs,其中模型组较正常组上调的23个,下调的16个。GO分析及信号转导通路分析结果提示差异miRNAs可能调控的靶基因及其参与的生物学功能包括细胞的增殖与活化、细胞凋亡、细胞周期、细胞黏附、细胞迁移、炎症反应、转化生长因子β（TGF-β）/Smads信号转导通路、Wnt受体信号转导通路、蛋白代谢过程的调控等。GO分析发现关键的上调miRNA包括mmu-miR-322、mmu-miR-15b、mmu-miR-195、mmu-miR-200b、mmu-miR-214等,关键的下调miRNA包括mmu-miR-16、mmu-miR-130a、mmu-miR-101b、mmu-miR-30a和mmu-miR-30e等。对显著性GO与显著性信号通路所属的靶基因取交集,对网络中miRNA在网络中的调控地位进行评价,结果发现关键的上调miRNAs包括mmu-miR-200b、mmu-miR-322、mmu-miR-106b、mmu-miR-23a、mmu-miR-15b等,关键的下调miRNAs包括mmu-miR-16、mmu-miR-30e、mmu-miR-30c、mmu-miR-30a、mmu-miR-130a等。结论：纤维化肝组织miRNAs表达较正常肝组织发生明显变化;肝纤维化形成的各个环节,包括细胞的增殖与活化、细胞黏附、细胞凋亡、细胞迁移与分化、物质代谢、TGF-β信号通路等都可能受miRNAs的调控。     

血管肉瘤相关miRNAs的生物信息学预测及分析

目的探讨血管肉瘤相关miRNAs及其靶基因在其恶性生物学行为中的作用。方法从GEO数据库中下载血管肉瘤miRNAs表达谱芯片数据,应用GEO2R筛选出差异表达的miRNAs并进行靶基因预测,对靶基因进行GO及KEGG生物功能富集分析及蛋白互作分析。结果筛选出53个差异表达的miRNAs (P0.05,|log fold-change|≥4),其中上调者51个,下调者2个;GO功能富集分析发现差异表达miRNAs的靶基因主要参与细胞通讯、信号转导等生物学过程;KEGG信号通路富集分析发现靶基因主要参与肿瘤信号通路、代谢通路、环腺苷酸(cAMP)信号通路、丝裂原活化蛋白激酶(MAPK)信号通路、PI3K/AKT信号通路、Ras信号通路等重要通路;String蛋白互作分析发现,STAT3、TP53、MAPK1、SRC等在蛋白互作网络中处于核心地位。结论异常表达的miRNAs在血管肉瘤的恶性生物学行为中发挥着关键作用,为进一步探讨血管肉瘤发生、发展的具体分子机制、分子标志物的筛选及靶向药物的开发奠定基础。     

采用非标记microRNA芯片筛选早产胎盘差异表达microRNA

目的应用非标记microRNA芯片筛选在早产胎盘组织中差异表达的miRNA,并分析其相关的靶基因。方法收集早产儿16例为病例组,同期健康足月产儿18例为对照组,取胎盘组织。通过非标记miRNA芯片技术构建早产胎盘组织miRNA表达谱,采用荧光实时定量PCR进行验证,并分析差异表达miRNAs的靶基因。结果 miRNA芯片结果显示44种miRNAs在早产胎盘组织和对照组胎盘组织中差异表达,其中下调表达的11种,上调表达的33种,荧光实时定量PCR验证出miR-493-3p显著下调,mi R-4632-5p显著上调,与芯片结果一致。经软件分析,miRNA靶基因有IL16、SH2D2A、CCNG2、PHB、WNT1、CCDC92、GIT1和ST7L等相关靶基因。结论早产胎盘组织中存在差异表达的miRNAs,提示这些差异表达的miRNAs在早产的发生中有重要的调控作用。     

早发型重度子痫前期血浆外泌体miRNAs表达谱分析

目的 通过比较早发型重度子痫前期（EOPE）患者与正常妊娠妇女血浆外泌体源性miRNAs表达谱的差异,初步探讨EOPE的发病机制。方法 收集于我院产检并最终诊断为EOPE的孕妇血浆（EOPE组,n=4）及正常妊娠妇女血浆（对照组,n=4）;对外泌体进行鉴定,提取血浆外泌体中的miRNAs进行测序,对差异表达的miRNAs进行生物信息学分析。分别利用3个靶基因预测网站进行靶基因预测,利用Venn图绘制数据库所预测的靶基因交集。使用在线分析网站DAVID分析2组差异表达蛋白中的GO功能注释结果,然后进行KEGG通路富集分析。用RT-PCR检测胎盘及血浆外泌体miRNAs水平。结果 检测到的颗粒直径为30～180 nm,峰值为112.8 nm;外形为茶状或椭圆形;2组均显著表达TSG101和CD63。测序结果显示：与对照组相比,EOPE组中共筛查出27个有显著表达差异的miRNAs,其中15个表达上调,12个表达下调;表达差异最显著的上调miRNA为miR-152。在线数据库网站预测所有差异表达miRNAs靶基因共8 414个,经生物信息学分析显示其靶基因在补体、凝血级联、MMPs信号通路及...     

乳腺癌筛选差异表达的miRNAs-mRNA及其与靶基因的关系

目的 筛选乳腺癌差异表达的miRNAs-mRNA,探讨差异miRNAs的表达及与靶基因的关系。方法 利用肿瘤基因组图谱(TCGA)筛选乳腺癌中差异表达的4种miRNAs;利用qRT-PCR验证4种miRNAs在多种细胞株中的表达;结合GO和KEGG确定4种miRNAs的靶基因。利用CCK-8法检测各组细胞的活力;利用双荧光素酶报告基因验证miRNAs与对应靶基因的作用关系。结果 通过TCGA结合文献筛选差异的miRNAs分别为hsa-miR-122、hsa-miR-133b、hsa-miR-449a和hsa-miR-767。根据GO和KEGG分析hsa-miR-122-5p、hsa-miR-133b-5p、hsa-miR-449a-5p和hsa-miR-767-5p的靶基因分别为ESR1、EGFR、HMGB1和IGF-1。与mimics-NC组(1.52±0.01)比较,mimics hsa-miR-122组(1.75±0.01)、mimics hsa-miR-767组(1.87±0)和mimics hsa-miR-449a组(1.71±0.01)的细胞活力显著升高(P<0.01...     

结肠癌化疗耐药相关miRNA表达谱的初步研究

目的:应用基因芯片技术筛选结肠癌耐药相关微小RNAs (miRNAs),探究miRNAs对化疗耐药的调控机制。方法:采用基因芯片技术分析结肠癌细胞系HCT8及其耐长春新碱细胞系HCT8/v中miRNAs的表达差异,对部分差异表达的miRNAs应用RT-qPCR进行验证,对表达差异显著的miRNAs进行靶基因预测,利用Gene Ontology (GO)和京都基因与基因组百科全书(KEGG)数据库对预测到的靶基因进行生物信息学分析。结果:筛选出342个差异表达miRNAs,其中190个表达上调,152个表达下调。RT-qPCR验证结果示miR-125-5p、miR-181c-5p和miR-153-3的表达情况和芯片检测结果一致;miR-130a-3p和miR-149-3p的表达与芯片检测结果不一致。GO分析结果显示,耐药相关基因主要富集的旁路是RNA聚合酶II调控区序列特异性DNA结合旁路,主要通过正向调节发挥作用,位置主要是在细胞内有界细胞器上。KEGG分析结果显示,耐药相关基因最为富集的是轴突导向通路、胰岛素信号通路及磷脂酶D信号通路。结论:miRNAs与结肠癌化疗耐药密切相关。对这些miRNAs的研究能使我们对结肠癌的化疗耐药机制有更深入的理解,并为逆转化疗耐药提供新的思路。     

高脂饮食诱导胰岛素抵抗小鼠血浆microRNA 表达谱及其与TLR4 的关系

目的:筛选高脂饮食诱导的胰岛素抵抗小鼠,以及应用Toll 样受体4(Toll-like receptor4,TLR4)抑制剂TAK-242 干预的胰岛素抵抗小鼠血浆差异表达的微小RNA(microRNA,miRNA),探讨胰岛素抵抗、TLR4 和差异表达miRNAs 的关系。方法:血浆标本来自前期实验的3 组小鼠,即以普通基础饲料喂养(Low fat diet,LFD)的正常对照组、给予TAK-242 的高脂饮食组(Treated high fat diet,HFD-T)和单纯高脂饮食组HFD-C,应用小鼠miRNA 芯片检测血浆miRNA 表达谱,筛选差异表达的miRNAs;采用实时荧光定量PCR 方法验证芯片结果。应用生物信息学方法,以TLR4 及其信号传导通路为中心,预测差异表达miRNAs 的靶基因,并对靶基因分别进行GO 和KEGG 富集分析,以判定差异miRNAs 主要影响的生物学功能或者通路。结果:miRNA 基因芯片筛查结果显示,单纯高脂饮食组与正常饮食组比对,筛选出差异显著的miRNAs 共计185 种,其中6 种miRNAs 表达上调,179 种miRANs 表达下调;给予TAK-242 的高脂饮食组与正常饮食组比对,筛选出差异显著的miRANs共计171 种,均表达下调;给予TAK-242 的高脂饮食组与单纯高脂饮食组比对,筛选出差异表达的miRNAs 共计13 种,均为下调。生物信息学分析结果显示,以TLR4 为中心挖掘与其互作的蛋白质,共发现有10 种蛋白;在TAK-242 给予的高脂饮食组与单纯高脂饮食组中差异表达倍数均在1 000 以上的4 种miRNAs (mmu-miR-3095-3p、mmu-miR-5113、mmu-miR-709 和mmu-miR-335-3p),可在TLR4 的互作蛋白质或Toll 样受体信号通路中找到其作用的靶基因,发现这些靶基因74% 属于生物过程基因,其中转录调控因子占82%。实时荧光定量PCR 检测mmu-miR-3095-3p、mmu-miR-5113、mmu-miR-709 和mmu-miR鄄335鄄3p水平,变化趋势与芯片结果一致。结论:胰岛素抵抗发生时,血浆miRNAs 表达谱存在改变,此变化与TLR4 及其信号通路相关。该研究结果丰富了胰岛素抵抗发生的机制,为寻找胰岛素抵抗血浆miRNAs 诊断标志物提供了实验依据。     

LMP1对鼻咽癌细胞系CNE1癌基因微小RNA表达谱的影响

目的:比较鼻咽癌细胞系CNE1与其EB病毒潜伏膜蛋白1(LMP1)稳定转染细胞系CNE1-LMP1的癌基因微小RNA(oncomiRs)表达谱的差异,探讨LMP1对鼻咽癌细胞系CNE1 oncomiRs表达的影响。方法:采用包含132个oncomiRs分子的microRNA芯片分析CNE1与CNE1-LMP1的表达差异,并采用荧光定量PCR验证差异表达明显的oncomiRs分子。结果:二者共检出30个miRNA分子,CNE1中检出miRNA分子19个;其中11个为CNE1-LMP1特异性表达。在共同表达的19个miRNA分子中,在CNE1-LMP1中表达升高2倍以上的miRNA分子有6个:hsa-miR-19b、hsa-miR-17-3p、hsa-miR-22、hsa-miR-149、hsa-miR-150和hsa-miR-188。没有表达降低2倍以上的分子。在CNE1-LMP1特异性表达的11个miRNA中,hsa-miR-122a呈高水平表达,其表达水平超过内对照。通过荧光定量PCR验证6个差异分子的表达,发现改变倍数与芯片结果一致(P0.01)。结论:LMP1能影响oncomiRs表达谱,可能是LMP1作为病毒癌基因发挥作用的另一重要途径。     

Differential microRNA expression in the serum of patients with nephrotic syndrome and clinical correlation analysis

Many different microRNAs existed in nephrotic syndrome patients, and they may be involved in nephrotic syndrome occurrence. In order to further clarify miRNAs expression changes in nephrotic syndrome patients and their correlation with clinical features, this study investigated differential microRNA expression in the peripheral serum of patients with nephrotic syndrome and analyzed the correlation between miRNA with largest overexpression level and clinical features. miRNAs microarray was applied to screen different expressed miRNAs in nephrotic syndrome patients. Real-time PCR was performed to verify miRNA expression level. SPSS software was used to analyze correlation between miRNA expression and clinical features. Compared with healthy subjects, 35 miRNAs overexpressed and 24 miRNAs down-regulated in patients. After real-time PCR verification, 6 miRNAs up-regulated in nephrotic syndrome patients, including hsa-miR-181a, hsa-miR-210, hsa-miR-30a, hsa-miR-942, hsa-miR-192 and hsa-miR-586. miRNA-30a significantly overexpressed in nephrotic syndrome patients and with no difference between genders. miRNA-30a expression level in drug resistant nephrotic syndrome patients was obviously higher than the drug sensitive patients. miRNA-30a up-regulated most significantly in mesangial proliferative glomerulonephritis among different pathology types, while it decreased most obviously in glomerular lesions. miRNA differently expressed in the serum of nephrotic syndrome patients. miRNA-30a could be treated as the molecular marker in predict drug resistance and pathological type of nephrotic syndrome.     

Differential microRNA expression and identification of putative miRNA targets and pathways in head and neck cancers

MicroRNAs (miRNAs) are small noncoding RNAs that involved in various cancer-related cellular processes. Diverse studies on expression profiling of miRNAs have been performed and the data showed that some miRNAs are up-regulated or down-regulated in cancer. Until now, there are no data published on the miRNA expression in head and neck cancers from Malaysia. Hence, this study aimed to investigate potentially crucial miRNAs in head and neck cancer patients from Malaysian populations. A global miRNA profiling was performed on 12 samples of head and neck cancer tissue using microarray analysis followed by validation using real-time RT-PCR. Microarray analysis identified 10 miRNAs that could distinguish malignant head and neck cancer lesions from normal tissues; 7 miRNAs (hsa-miR-181a-2*, hsa-miR-29b-1*, hsa-miR-181a, hsa-miR-181b, hsa-miR-744, hsa-miR-1271 and hsa-miR-221*) were up-regulated while 3 miRNAs (hsa-miR-141, hsa-miR-95 and hsa-miR-101) were down-regulated. These miRNAs may contribute in a simple profiling strategy to identify individuals at higher risk of developing head and neck cancers, thus helping in the elucidation of the molecular mechanisms involved in head and neck cancer pathogenesis.     

Differential Expression Profiling and Functional Analysis of microRNAs through Stage I-III Papillary Thyroid Carcinoma

Objective: To elucidate the mechanisms undergoing the pathogenesis of PTC, this study try to find stage specific microRNAs (miRNAs) using microarray chip in stage I, II and III papillary thyroid carcinoma (PTC) tissues as well predict miRNAs binding target genes and their molecular functions.Methods: PTC specimens of stage I, II, and III and their paired adjacent non-tumor tissue (one patient for each stage) were collected. The expressions of miRNAs were examined using miRNA microarray chip. The most significant changed miRNAs from microarray were verified by using quantitative RT-PCR. The Potential miRNAs regulating target genes and their preliminary biological functions were forecasted with variety function prediction software.Results: Ten miRNAs exhibited sequential up regulation expression profiles and five miRNAs performed sequential down regulation throughout stage I to III (p<0.05). After normalization, Fifteen miRNAs showed significant different compared to adjacent non-tumor tissues (p<0.05). Among of them, the most significant up regulation and down regulation miRNAs were miR-146b-5p and miR-335, respectively. Both of them were verified with qRT-PCR. 34 target genes for miR-146-5p and 36 target genes for miR-335 was predicted.Conclusion: MicroRNA profile assay successfully detected a branch of differential expression miRNAs between PTC and normal tissue. Some of them also showed stage specific. Biological function analysis showed that target genes were involved in five aspects including cell proliferation, differentiation, apoptosis, cycle, and signaling transduction pathway, suggesting the regulatory role of abnormal expression of critical miRNAs in the pathogenesis of PTC.     

子宫内膜癌microRNA表达谱的初步研究

目的 筛选子宫内膜癌与正常子宫内膜组织中的差异mieroRNA.方法 收集3例患者的新鲜子宫内膜癌组织及正常子宫内膜组织,利用miRNA芯片对其进行实验分析,并采用茎环逆转录荧光定量PCR方法验证芯片结果的准确性.结果 相对正常子宫内膜组织,子宫内膜癌组织中表达上调的miRNA基因68个,下调的miRNA基因81个.采用茎环逆转录荧光定量PCR对显著上调的hsa-miR-205,hsa-miR-200b和hsa-miR200c以及显著下调的hsa-miR-495,hsa-miR-216b的验证结果与芯片检测结果一致.结论 筛选获得了子宫内膜癌的miRNA的差异表达谱,可能在子宫内膜癌的发病机制中发挥潜在作用。     

Curcumin inhibits the proliferation and invasion of human osteosarcoma cell line MG-63 by regulating miR-138

Objective: In this study, we screened the different human osteosarcoma cell line MG-63 miRNAs after the treatment of curcumin and explored the effects of curcumin on MG-63 cells and its mechanism. Methods: Affemitrix miRNA chip was used to detect the changes of miRNA expression profile in MG-63 cells before and after curcumin treatment, and screen different expression of miRNAs. The target gene of miRNA was analyzed by bioinformatics. The expression levels of miRNA-138 target genes Smad4, NFκB p65 and cyclin D3 were detected. MTT and Transwell Cell invasion assays were used to observe the effects of curcumin on MG-63 cells. Results: Curcumin could significantly inhibit the proliferation of MG-63 cells and the expression levels of miRNA-138 target genes Smad4, NFκB p65 and cyclin D3 in MG-63 cells (P<0.05); overexpression of hsa-miR-138 down-regulated the expression levels of Smad4, NFκB p65 and cyclin D3 compared with the treatment of curcumin, while inhibition of hsa-miR-138 up-regulated the expression levels of Smad4, NFκB p65 and cyclin D3. Conclusions: Curcumin could increase the expression of hsa-miR-138, hsa-miR-138 inhibited cell proliferation and invasive ability by inhibition of its target genes.