永久性房颤的转录组分析及miRNA调控网络的建立

目的探索永久性房颤(p AF)发病相关的关键miRNAs及其调控的靶基因。方法联合应用表达谱芯片和miRNA芯片分析p AF患者(n=7)和健康成人(n=4)的左房组织,筛选p AF相关差异表达的miRNAs,进行靶基因预测后,与表达谱芯片的筛选结果进行负相关分析后的基因集合进行显著性功能分析(GO-analysis);利用miRNA与靶基因之间的靶向调控关系,构建差异miRNA与交集靶基因的调控网络(miRNA-gene-network),得到网络中起核心调控作用的miRNA和被调控的关键靶基因;采用RT-q PCR方法检验另一组p AF患者(n=5)和健康成人(n=4)的左房组织标本。结果表达谱基因芯片发现610个mRNA有显著性改变(fold change2,P0.05),miRNA-靶基因调节网络发现与p AF显著相关的20个miRNAs和107个靶基因,相关度最高的是miR-144、miR-1284、miR-1827、miR-1、miR-3613-3p和miR-101;其调控的重要靶基因包括CACNB2、EFNB1、PTEN、TAOK1、RUNX1和TPM3等;RT-q PCR验证结果显示这些miRNAs和靶基因密切相关。结论通过表达谱基因芯片与miRNAs芯片联合分析p AF左房组织标本,构建miRNA调控网络,发现p AF重要的miRNA-靶基因调控及功能,结果更加准确。     

慢性神经病理性疼痛对大鼠脊髓背角miRNA表达的影响

目的 筛选慢性神经病理性疼痛大鼠脊髓背角差异表达的miRNA,并预测其调控的靶基因.方法 建立大鼠坐骨神经慢性压迫损伤CCI模型,在术后疼痛高峰期取腰膨大脊髓背角,用miRNA芯片筛选CCI大鼠差异表达的miRNAs,再用荧光实时定量RT-PCR验证差异表达的miRNAs,并利用MIRANDA、TARGETSCAN、PICTAR 3个数据库找出这些miRNA可能调控的靶基因.结果 CCI大鼠表达上调的有miR-99b,表达下调的有miR-674-3p、miR-879与miR-325-5p.RT-qPCR验证结果与芯片基本相符.预测这些miRNA可能的靶基因约26个,这些基因功能广泛.结论 慢性神经病理性疼痛可导致miRNA的表达发生变化,这些miRNA及其调控的靶基因为进一步研究奠定了基础.     

严重烫伤大鼠早期胫骨前肌组织中微小RNA表达谱数据的靶基因分析

目的初步确定严重烫伤大鼠早期胫骨前肌组织中差异表达微小RNA(miRNA)的靶基因,从而为进一步机制研究指明方向。 方法采用高通量芯片技术检测严重烫伤大鼠早期胫骨前肌miRNA表达谱数据,筛选部分差异明显且相关性较好的miNRA,包括miR-1-3p、miR-206-3p、miR-133a-3p、miR-22-3p、miR-30a-5p、miR-30d-3p、miR-190b-5p、miR-628和miR-678,利用互联网络检索数据库Mirbase、Miranda和Mirdb,对这些差异miRNA进行靶基因预测。 结果3大数据库预测结果显示差异miRNA主要调控79个靶基因,网络关系图显示了miR-1-3p、miR-206-3p、miR-133a-3p、miR-22-3p、miR-30a-5p、miR-30d-3p、miR-190b-5p、miR-628和miR-678等miRNA及其预测靶基因之间复杂的调控网络关系。 结论根据初步的预测结果,miR-1-3p和miR-206-3p共同调控的靶基因较多,可作为进一步机制研究的理论依据。     

肠道病毒71型感染人神经母细胞瘤细胞(SH-SY5Y)的miRNA表达谱

目的 研究肠道病毒71型(EV71)感染神经细胞的miRNA表达谱,探讨miRNA在病毒感染神经细胞中的可能作用.方法 建立EV71感染人神经母细胞瘤细胞(SH-SY5Y)模型,收集感染后48 h细胞.以Taqman低密度芯片检测miRNA表达谱,使用实时RT-PCR对芯片结果进行验证并在TargetScan和miRanda网站预测靶基因,采用GO和KEGG分析靶基因功能.结果 成功建立EV71感染SH-SY5Y细胞模型,通过低密度芯片筛选出215种显著升高的miRNA和25种显著下调的miRNA.经过RT-PCR验证,3种miRNA(MiR-10a*、miR-15b*和miR-195)显著下调,7种miRNA(miR-10a、miR-342-5p、miR-483-5p、Let-7b、miR-99a、miR-140-5p和miR-21)显著上调,与芯片结果相符.GO分析显示发展进程和信号调节条目最富集靶基因.KEGG路径分析显示靶基因在肿瘤路径、蛋白水解、Wnt信号传导、黑素形成、粘附连接、MAPK信号通道最富集.结论 EV71感染神经细胞48 h后miRNA表达谱发生改变,10种变化的miRNA靶基因预测在发展进程、信号传导及凋亡中起着重要的作用,可为后期机制研究提供参考.     

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。     

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。     

以IGF-1、NOS、FGF23和硬骨素为靶基因的骨细胞力学响应微小RNA的筛选

目的 筛选骨细胞分泌因子相关力学响应微小RNA(microRNA, miRNA)。方法 分别向体外培养骨细胞和成骨细胞施加周期性张应变（ε=2.5,f=0.5 Hz）,采用miRNA芯片筛选出仅在骨细胞中差异表达的miRNA。通过生物信息学技术,从这些miRNA中进一步筛选出靶基因为胰岛素样生长因子1（insulin-like growth factor-1）、NO合成酶（nitric oxide synthesase, NOS）、成纤维细胞生长因子23(fibroblast growth factor 23, FGF23)和硬骨素(sclerostin, SOST)等分泌因子的miRNA,与小鼠跑台锻炼后芯片检测出的小鼠股骨组织差异表达miRNA进行比较,然后随机选4个miRNA进行定量PCR验证。结果 仅在体外经力学刺激的骨细胞中差异表达的77个miRNA中,筛选出22个靶基因为4种分泌因子（IGF-1、NOS、FGF23、SOST）的miRNA,并进一步筛选出11个在跑台锻炼后的小鼠骨组织中以同样趋势差异表达的miRNA,其中随机选出的miR-361-3p、miR-3082-5p、miR-6348和miR-706,也在骨细胞和骨组织中以同样趋势同样差异表达。结论 诸如miR-361-3p、miR-3082-5p、miR-6348和miR-706仅在骨细胞中差异表达的力学响应miRNA,很可能通过调节分泌因子影响成骨分化或骨代谢。     

miR-224-5p靶向GABRE对人结肠癌细胞系SW480增殖的影响

目的 探讨miR-224-5p通过靶点GABRE对结直肠癌细胞系增殖的抑制作用和可能的调控机制。方法 从基因表达综合数据库（GEO）中检索并筛选GPL18058的microRNA(miRNA)表达阵列,筛选出结直肠癌与正常组织差异表达的miR-224-5p后,通过Target Scan Human数据库预测其下游靶点GABRE,并利用UALCAN分析GABRE在结直肠癌组织中的表达情况和结直肠癌预后关系。利用实时定量PCR(qRT-PCR)检测结直肠癌细胞系中的miR-224-5p和GABRE的表达水平。在结直肠癌细胞系SW480中,沉默miR-224-5p后,CCK-8法与克隆形成实验分析其增殖情况。RT-PCR和Western blot检测miR-224-5p对GABRE表达的影响。在SW480细胞中低表达GABRE,评估其对结直肠癌调节中的miR-224-5p的选择性作用。结果 在结直肠癌组织和细胞系中,miR-224-5p的表达异常上调,抑制其表达后,SW480细胞的增殖能力降低（P<0.05）。在结直肠癌组织和细胞系的基因和蛋白质水平上,miR-224-5p正调控GAB...     

乳腺癌相关成纤维细胞miRNA表达的检测和分析

目的研究人乳腺癌微环境癌相关成纤维细胞(CAF)与正常成纤维细胞(NF)miRNA表达的差异及其对CAF的生物学功能的影响。方法运用1型胶原酶消化法分别处理临床乳腺癌患者的癌组织和对应癌旁组织标本,原代分离培养CAF和NF细胞;利用免疫荧光法和Western blot法分析CAF和NF细胞成纤维细胞分泌蛋白(FSP)的表达情况,对所分离培养的细胞进行纯度和特性鉴定;利用TranswellTM实验比较CAF与NF细胞的侵袭能力;利用miRNA芯片和芯片结果显著性差异(SAM)分析法分析CAF与NF细胞miRNA表达的差异;对差异miR-205和miR-221,在原代培养的CAF和NF进行实时定量PCR(qRT-PCR)验证;利用多种生物信息学软件预测差异miRNA的靶基因;利用DAVID软件对靶基因进行信号通路富集度分析。利用ELISA检测TGF-β和IL-6信号通路的核心蛋白基质金属蛋白酶1(MMP-1)、MMP-2和MMP-9的表达差异。结果成功分离得到原代培养的CAF与NF细胞,纯度大于95%;与NF细胞相比,CAF细胞的侵袭能力明显增强;miRNA芯片分析结果显示,CAF有10个变化倍数1.5的异常表达miRNA(P0.05),其中3个上调表达(miR-221-5p、miR-31-3p、miR-221-3p),7个下调表达(miR-205、miR-200b、miR-200c、miR-141、miR-101、miR-342-3p、let-7g)。信号通路富集度分析发现,miRNA靶基因与细胞分化、细胞黏附、细胞迁移、细胞增殖、细胞分泌和细胞间相互作用等密切相关,其中,异常表达的miR-200b/c和miR-141等miRNA通过抑制其靶基因,影响TGF-β信号通路、IL-6信号通路,进而影响CAF的侵袭和转移。结论人乳腺癌微环境CAF的miRNA表达谱发生显著变化,CAF细胞中异常表达miRNA可能参与了NF向CAF的转化,并与CAF细胞的增殖、黏附、侵袭、转移有密切关系。     

mi R-22在压应力下软骨终板细胞中的表达及其对靶基因MMP-1的调控作用

目的 应用miRNA芯片技术筛选压应力下人软骨终板细胞中差异表达的miRNAs,研究与压应力相关的miR-22并进行生物信息学分析,探讨其对靶基因MMP-1的调控。方法 将6个月意外流产胎儿软骨终板进行软骨终板细胞的分离、培养。将第2代软骨细胞按实验要求种板培养并随机分为加压组与对照组。加压组施加周期性压力(5 MPa,6 h×5 d),使用TRIzol法提取各组RNA,miRNA芯片检测miRNA表达情况,然后用Realtime PCR(RT-PCR)对差异表达miR-22进行验证。通过靶基因预测软件预测MMP-1与miR-22基因的关系,miR-22 mimics脂质体转染Hela细胞后通过RT-PCR进一步验证。结果 芯片扫描结果显示加压组miR-22下调明显。RT-PCR检测显示压应力下人软骨终板细胞miR-22表达显著降低。miR-22 mimics转染目标细胞48 h后,miR-22的表达显著增高,同时MMP-1基因表达显著降低。结论 压应力下miR-22异常表达,miR-22可靶向负调控与软骨终板细胞退变相关的MMP-1基因。     

Identification of altered MicroRNA expression in canine lymphoid cell lines and cases of B- and T-Cell lymphomas

Canine lymphoma is a common spontaneous tumor with many similarities to human lymphoma, and thus has potential to be an important animal model of lymphomagenesis. This study determined that microRNA (miRNA) expression in canine tumors can be assessed using a commercially available human cancer miRNA qPCR array. miRNA expression in six different canine lymphoid cell lines and in naturally occurring canine B- and T-cell lymphomas was compared using RNA harvested from normal canine peripheral blood mononuclear cells (PBMC) and normal lymph nodes (LN) as controls. We found that false discovery rate (FDR) correction for multiple testing after quantile normalization controlled for variation across arrays and that they were the best methods for normalization and statistical analysis. Increases in miRNAs known to upregulate oncogenes (miR19a+b, miR17-5p) and decreased expression of miRNAs with tumor suppressor functions (miR-203, miR-218, and miR-181a) also seen in human lymphoid malignancies were observed. However, there were few similarities between canine groups. The results of this study indicate that the use of both PBMC and LN cells as controls provides different, but potentially equally important targets for further analysis. Our findings of miRNA dysregulation in canine lymphoid cell lines and clinical cases of lymphoma emphasize the potential of canine lymphoma as an important spontaneous, large animal model of human B- and T-cell lymphomas.     

MicroRNA-338-3p在肿瘤中的研究进展

微小RNA(microRNA,miRNA)是一种广泛分布于身体各个器官的短链非编码RNA.miRNA可广泛调控基因,通过基因调控控制细胞的生长、分化、增殖以及凋亡等,在正常的细胞生命活动中起着重要作用.随着近年来肿瘤发病率的日益增高,miRNA与肿瘤的关系正成为研究热点,其可作为肿瘤诊断、治疗及预后的标志物.多项研究证实miR-338-3p在多种实质瘤中的表达降低.进一步的研究显示,miR-338-3p可抑制肿瘤基因,通过调控肿瘤基因以及相关信号通路等一系列方式影响肿瘤的进展以及诊断预后.越来越多的证据显示miR-338-3p可作为一种新颖的肿瘤生物标志物,并且有可能成为新的治疗靶点.     

MicroRNA 96 is a post-transcriptional suppressor of anaplastic lymphoma kinase expression

Anaplastic lymphoma kinase (ALK) constitutes a part of the oncogenic fusion proteins nucleophosmin-ALK and echinoderm microtubule-associated protein like 4-ALK, which are aberrantly expressed in a subset of T-cell anaplastic large-cell lymphoma and non-small-cell lung cancer, respectively. The expression of mutated, constitutively active ALK also occurs in a subset of neuroblastoma tumors. ALK is believed to play an important role in promoting tumor survival. Nevertheless, the mechanisms underlying the expression of ALK in cancer cells are not completely known. MicroRNA (miR) has been implicated in the regulation of the expression of both oncogenes and tumor suppressor genes. We tested the hypothesis that the expression of ALK could be regulated by miR. Three Internet-based algorithms identified miR-96 to potentially bind with the ALK 3'-untranslated region. Notably, miR-96 levels were markedly decreased in ALK-expressing cancer cell lines and primary human tumors compared with their normal cellular and tissue counterparts. Transfection of the cell lines with miR-96 decreased levels of the different forms of ALK protein, without significant effects on ALK mRNA. Furthermore, miR-96 decreased the phosphorylation of ALK target proteins, including Akt, STAT3, JNK, and type I insulin-like growth factor receptor, and it down-regulated JunB. These effects were associated with reduced proliferation, colony formation, and migration of ALK-expressing cancer cells. These data provide novel evidence that decreases in miR-96 could represent a mechanism underlying the aberrant expression of ALK in cancer cells.     

Increased expression of P-glycoprotein and doxorubicin chemoresistance of metastatic breast cancer is regulated by miR-298

MicroRNAs (miRNAs) are short, noncoding RNA molecules that regulate the expression of a number of genes involved in cancer; therefore, they offer great diagnostic and therapeutic targets. We have developed doxorubicin-resistant and -sensitive metastatic human breast cancer cell lines (MDA-MB-231) to study the chemoresistant mechanisms regulated by miRNAs. We found that doxorubicin localized exclusively to the cytoplasm and was unable to reach the nuclei of resistant tumor cells because of the increased nuclear expression of MDR1/P-glycoprotein (P-gp). An miRNA array between doxorubicin-sensitive and -resistant breast cancer cells showed that reduced expression of miR-298 in doxorubicin-resistant human breast cancer cells was associated with increased expression of P-gp. In a transient transfection experiment, miR-298 directly bound to the MDR1 3' untranslated region and regulated the expression of firefly luciferase reporter in a dose-dependent manner. Overexpression of miR-298 down-regulated P-gp expression, increasing nuclear accumulation of doxorubicin and cytotoxicity in doxorubicin-resistant breast cancer cells. Furthermore, down-regulation of miR-298 increased P-gp expression and induced doxorubicin resistance in sensitive breast cancer cells. In summary, these results suggest that miR-298 directly modulates P-gp expression and is associated with the chemoresistant mechanisms of metastatic human breast cancer. Therefore, miR-298 has diagnostic and therapeutic potential for predicting doxorubicin chemoresistance in human breast cancer.     

Altered microRNA expression signature in Chikungunya-infected mammalian fibroblast cells

Chikungunya virus (CHIKV) infection can cause severe arthralgia and chronic arthritis in humans. MicroRNAs (miRNA) have demonstrated their potential use as biomarker in variety of human pathologies and infections. This study was conducted to understand the miRNA signature in early CHIKV infection stages. In the current study, we used TaqMan-based quantitative PCR method to identify the miRNA signature of host response upon CHIKV infection in human and mouse fibroblast cells. The GO enrichment analysis suggests that the putative target genes of these differentially expressed miRNAs are to be involved in RIG-I pathway, TGF-beta-signaling pathway, JAK–STAT-signaling pathway, MAPK-signaling pathway, cytokine–cytokine receptor interactions, and Fc gamma R-mediated phagocytosis. The results obtained in the current study and earlier studies indicate the potential use of miR15, miR-16, miR-17, let-7e, miR-125, miR-99, and miR-23a as a biomarker in CHIKV infection. miRNAs such as miR-15a, miR-16, miR-140, miR-146a, miR-155, miR203, miR223, miR-499, and miR-363 which are implicated in rheumatoid arthritis showed differential regulation in CHIKV infection. The data obtained in this study provide valuable information on CHIKV-induced miRNA expression in mammalian fibroblast cells, and suggest that CHIKV may establish infection by regulating miRNA expression profile.     

57miRNA regulation during oncogene-induced senescence

Aberrant oncogene activation induces cellular senescence, an irreversible growth arrest which acts as a barrier against tumor formation. To identify miRNAs involved in oncogene-induced senescence we arrayed miRNA expression in primary human fibroblasts (Tig3) following over-expression of B-RAF. 18 miRNA were significantly regulated (P<0.001) 4 days after B-RAF activation; in particular, miR-34a expression increased ∼8-fold. In addition, miR-34a is up-regulated in mouse fibroblasts undergoing replicative senescence. In agreement with previous reports, we find that miR-34a reduces cellular proliferation and induces cell cycle arrest. Using microarrays, we have identified a number of putative miR-34a targets involved in oncogene-induced senescence and we are now evaluating their ability to mediate the cellular effects of miR-34a. Although members of the miR-34 family are known p53 target genes, our data indicate that miR-34a is regulated independently of p53 during oncogene-induced senescence. Hence, miR-34a responds to several independent cancer-related pathways thereby underlining the importance of miR-34a as an important tumor suppressor. We are currently investigating the regulation of miR-34a in normal and senescent cells using ChIP and promoter reporter assays.