基于数据挖掘分析microRNAs在冠状动脉支架内再狭窄中的调控机制

目的:通过寻找支架内再狭窄相关的循环microRNAs(miRNAs)及其作用靶基因,阐释miRNAs在经皮冠状动脉介入治疗的同时带来的支架内再狭窄(ISR)中的网络调控作用及机制。方法:由美国NCBI的GEO公共数据平台下载GSE60959中的miRNAs芯片数据,通过GeneSpringGX芯片分析软件分析得到ISR患者和对照组之间差异表达的循环miRNAs;采用miRNAs靶基因预测算法TargetScan和miRanda预测差异表达miRNAs的靶基因,通过DAVID平台和KEGG数据库分析得到靶基因所参与的信号通路;用GSE46560的mRNA芯片数据分析得到ISR患者较非ISR患者循环mRNAs差异表达谱,用来验证miRNAs调控ISR所作用的靶基因;采用Cytoscape软件构建基于miRNAs-信号通路、miRNAs-靶基因的共表达网络。结果:与对照组相比,ISR组存在131个差异表达循环miRNAs,其中上调41个。前5个上调miRNAs分别是miR-1183、miR-512-5p、miR-187-5p、miR-144-3p和miR-1225-5p。5个miRNAs的预测靶基因共6587个,信号通路富集分析显示这些基因主要参与的信号通路包括MAPK signaling pathway、ErbB signaling pathway、Wnt signaling pathway、Focal adhesion、Neurotrophin signaling pathway和Regulation of actin cytoskeleton。进一步对mRNAs芯片进行差异分析显示,与对照组相比,ISR患者差异循环mRNAs共171个,其中最可能受上述5个miRNAs调控的基因共43个。构建的共表达网络显示,miR-512-5p是作用靶基因和参与信号通路最多的miRNAs。结论:ISR患者循环miRNAs和mRNAs表达谱均存在改变,多个差异表达miRNAs通过作用于多个靶mRNAs,进而影响多个信号通路的活性,最终对ISR的发生、发展形成网络调控作用。     

miR-133b可能通过调控foxO1的表达影响小鼠B细胞发育

microRNAs(miRNAs)是一类具有转录后调控作用的非编码RNA,在发育、细胞增殖、凋亡及肿瘤发生等多种生理和病理过程中发挥重要作用.为全面了解小鼠B细胞中miRNAs的表达模式,利用流式细胞仪(FACS)分选处于不同发育时期的B细胞,采用TaqMan誖低密度芯片对其进行检测,筛选到pre-B阶段9个miRNAs表达量显著上调.将筛选出的miRNAs进行靶基因预测,并对预测靶基因进行功能聚类和通路分析,发现约4%的基因参与免疫系统过程,包括Bcl2、Kit等.选取foxO1与miR-19b、miR-142-3p、miR-106b、miR-182及miR-133b进行初步功能验证,双荧光素酶报告系统及Westernblot检测结果均显示,miR-133b可直接作用于foxO1 3′UTR从而降低foxO1的表达.结合人类和小鼠B细胞中foxO1的表达情况分析,其表达模式同miR-133b表达模式呈负相关,说明miR-133b可能参与了B细胞发育过程中foxO1的表达调控过程.     

hsa_circ_0076931可能通过hsa-miR-181a-5p影响胶质瘤发生发展

摘要 目的：探讨hsa_circ_0076931在胶质瘤中的表达及其潜在分子机制。方法：通过生物信息学分析,筛选出目的基因hsa_circ_0076931,在H4细胞系中过表达hsa_circ_0076931后进行转录组测序、生物信息学分析和验证。结果：基因本体（GO）和基因组百科全书（KEGG）结果显示：差异环状RNA（circRNAs）母基因及差异信使核糖核酸（mRNA）主要参与细胞周期、细胞分裂等生物学功能以及代谢、癌症相关和MAPK等信号通路。此外,与hsa_circ_0076931互相作用的基因主要参与细胞增殖、细胞凋亡和细胞迁移等生物功能以及MAPK、PI3K-Akt、Rap1等信号通路。hsa_circ_0076931可以下调靶基因hsa-miR-26a-5p、hsa-miR-181a-5p和hsa-miR-34a-5p表达,上调双特异性磷酸酶 5（DUSP5）、血小板衍生生长因子受体（PDGFRB）和钙通道β3亚基（CACNB3）的表达,并抑制磷酸化ERK（p-ERK）蛋白的表达。结论：hsa_circ_0076931可能通过吸附hsa-miR-181a-5p结合上调DUSP5的表达,从而抑制MAPK信号通路参与胶质瘤的发生发展过程。     

干扰NSUN2通过调控细胞周期蛋白表达抑制黑素瘤细胞增殖

为探讨太阳结构域家族蛋白酶2(NOP2/Sun RNA methyltransferase 2, Nsun2)RNA甲基化酶在黑素瘤细胞中的生物学功能,本研究以小鼠黑素瘤B16细胞为对象,构建靶向干扰Nsun2基因的shRNA慢病毒干扰载体,包装病毒后感染B16细胞。与对照组相比,干扰组细胞中Nsun2的敲低效率达到80%。EdU染色结果表明,干扰Nsun2显著抑制B16细胞DNA合成能力。转录物组测序技术系统分析干扰组与对照组细胞基因表达水平,共筛选获得1 062个差异表达基因(DEGs),其中678个表达上调,384个表达下调。DEGs主要富集在染色体、着丝粒区、蛋白质结合等GO条目。KEGG分析表明,DEGs显著富集在细胞周期、DNA复制、细胞衰老等通路。荧光定量PCR和转录物组测序结果均发现,Cdk2、Ccna2、Cdc25b等促进细胞分裂的相关基因显著下调,而Gadd45g和Gadd45a等阻滞细胞增殖的基因显著上调。本研究表明,NSUN2通过调控细胞周期和DNA复制等生物学过程,影响黑素瘤细胞增殖,为黑素瘤发生和发展的分子机制研究提供参考。     

环状RNA作为潜在治疗靶点在子宫内膜异位症中的调控机制

环状RNA (circular RNAs, circRNAs)属结构稳定的非编码RNA,具有miRNA海绵、与蛋白质互作等生物学功能。近年,子宫内膜异位症(endometriosis, EMs;简称内异症)中的circRNAs逐渐得到广泛研究。诸多学者通过多种生物信息学方法预测分析出EMs中差异表达的circRNAs、miRNAs及相关信号通路,并对已知的差异表达的circRNA进行敲除或过表达来探究其在上皮-间质转化(epithelial-mesenchymal transition, EMT)、雌激素介导细胞侵袭、细胞自噬与凋亡、缺氧等生物学过程中的具体调控作用。该文以差异表达的circRNAs为起点,从ceRNA轴出发,在circRNA-miRNA-mRNA网络中对参与EMs发病的相关信号通路进行全面综述,强调了circRNAs作为子宫内膜异位症潜在治疗靶点的作用,以期为内异症临床治疗提供新思路。     

低压低氧对小鼠精子发生及其小RNA表达的影响

作为青藏高原最为关键的环境因子,低压低氧对高原非习服动物繁殖和生殖系统功能有不利影响。已有的研究表明,低氧环境会导致雄性生殖细胞凋亡、精子畸形率升高、精子质量下降,进而影响受精和早期胚胎发育,但目前缺乏低氧损伤精子功能的机理研究。小RNA (small RNA) 是在转录后及翻译水平上调控基因表达的重要功能分子,不同类型的small RNA通过诱导基因沉默或调控翻译等方式参与调节精子发生。本研究通过低压氧舱模拟海拔5 000 m处理4周建立缺氧小鼠模型,发现低氧处理导致小鼠曲精细管中生精细胞排列紊乱,精子数量未发生显著变化但畸形率增加17.5倍 (P < 0.001)。通过small RNA测序发现,低氧组小鼠精子中的small RNA碱基偏好性与对照组一致,第一碱基对尿嘧啶 (U) 有很强的偏好性。低氧组小鼠精子中21 nt长度的small RNA比例显著减少4.4% (P < 0.05)。低氧组小鼠精子中piRNA、tsRNA表达无差异,但miRNA表达上调21个,下调58个。对差异miRNAs靶基因与相同低氧处理小鼠睾丸组织差异基因比对,共比对到831个差异表达基因,其中上调miRNAs的靶基因中有429个差异表达基因;下调miRNAs的靶基因中有813个差异表达基因。此外,上调miRNAs的靶基因富集在FoxO信号通路、甲状腺激素信号通路、类固醇生物合成和HIF?1信号等通路,而下调miRNAs的靶基因富集在脂肪酸代谢等通路。本研究获得了缺氧小鼠精子small RNA变化图谱,对人类和其他动物在缺氧环境下精子表观遗传修饰变化的研究具有参考价值。     

AICAR通过激活Foxc1信号来调节小鼠F9细胞的增殖

为了探索5-氨基咪唑-4-氨甲酰核糖核苷（AICAR）抑制小鼠F9 细胞（F9 embryonal carcinoma cells）增殖的作用机制,本文构建了Foxc1的慢病毒真核表达载体,通过实时定量PCR、免疫荧光染色、双荧光素酶报告基因检测系统以及细胞增殖检测试验,探索AICAR抑制小鼠F9细胞的增殖作用机制. 结果发现,AICAR可以在RNA和蛋白水平促进Foxc1的基因表达,并可以作用于核转录因子κB通路. 另外在培养液中添加AICAR或过表达Foxc1都能抑制F9细胞的增殖. 信号通路报告载体检测发现Foxc1可以激活核转录因子κB通路以及细胞周期相关的通路. 总之,本研究证明,AICAR 通过激活Foxc1通路及其下游多条信号通路来抑制F9细胞增殖.     

Map3k1调控小鼠眼睑闭合的研究进展

小鼠(Mus musculus)眼睑发育需要协调细胞增殖、细胞形态改变、迁移及凋亡.Map3k1是MAPK家族中重要的一员,Map3k1表达的蛋白质MEKK1是MAPK信号通路重要的节点.MEKK1-JNK信号通路活化c-jun,增强AP-1转录因子的转录活性,从而调控肌动蛋白纤维的形成和细胞的迁移.MEKK1还有可能...     

miR-24-3p对宫颈癌细胞增殖与迁移的作用及机制研究

该文探讨了miR-24-3p对宫颈癌细胞增殖和迁移的促进作用及其机制。采用miR-24-3p抑制剂下调宫颈癌细胞中miR-24-3p的表达后,通过MTT、Transwell实验和Western blot检测细胞增殖、迁移和PCNA蛋白水平;采用生物信息学方法预测miR-24-3p的靶基因并进行功能注释和筛选;双荧光素酶报告实验和Western blot验证靶基因类血管动蛋白-2(angiomotin-like 2,AMOTL2),并通过siRNA抑制AMOTL2检测其对宫颈癌细胞迁移的影响。结果显示,下调miR-24-3p能抑制宫颈癌细胞的增殖和迁移能力并减少PCNA蛋白表达;其靶基因主要存在细胞与细胞连接组分中,显著富集于蛋白激酶活性分子功能、蛋白质自身磷酸化生物学过程和癌症中microRNA信号通路;miR-24-3p能靶向负调控最佳靶基因AMOTL2,下调AMOTL2可促进宫颈癌细胞CaSki的迁移。总之,miR-24-3p可调控多靶基因参与多个生物学过程和多条信号通路,在宫颈癌中可促进细胞增殖且通过靶向AMOTL2促进迁移。     

Circular RNA circ-VANGL1 as a competing endogenous RNA contributes to bladder cancer progression by regulating miR-605-3p/VANGL1 pathway

Increasing reports indicate that circular RNAs (circRNAs) are very important regulators in human diseases, including cancers. In bladder cancer (BC), several circRNAs have been reported to be involved in tumor progressions, such as circ-ITCH and circTCF25. However, the functions of most circRNAs in BC still remains largely unknown. In this study, we identified a novel circRNA termed as circ-VANGL1 by bioinformatics analysis. We found that circ-VANGL1 was highly expressed in BC tissues compared with adjacent normal tissues. Furthermore, we showed that circ-VANGL1 could serve as a prognostic marker for patients with BC. Through functional experiments, we found that circ-VANGL1 knockdown significantly suppressed BC cell proliferation, cell cycle, migration, and invasion in vitro. Besides, circ-VANGL1 silence inhibited BC cell propagation in vivo. Mechanistically, we identified circ-VANGL1 as a sponge of miR-605-3p which targeted VANGL1 in BC cells. Through repressing miR-605-3p availability, circ-VANGL1 contributes to VANGL1 expression, consequently leading to BC cell proliferation, migration, and invasion. Taken together, our study demonstrated circ-VANGL1/miR-605-3p/VANGL1 as a novel essential signaling pathway involved in BC progression.     

Differential circular RNAs expression in ovary during oviposition in honey bees

Circular RNAs (circRNAs) are non-coding RNAs newly identified and play important roles in RNA regulation. The mechanism and function of circRNAs have been reported in some species. However, little is known regarding circRNAs in honey bees. In this study, we analyzed circRNAs through bioinformatics, and predicted 12,211 circRNAs in the ovary of honey bee queens. 1340, 175 and 100 circRNAs were differentially expressed in comparisons of egg-laying queens vs virgin queens, egg-laying inhibited queens vs egg-laying queens and egg-laying recovery queens vs egg-laying inhibited queens. Further, functional annotation of differentially expressed circRNAs revealed several pathways that are closely related to ovary activation and oviposition, including insulin secretion and calcium signaling pathways. Moreover, the potential interactions among circRNAs, miRNAs, lncRNAs and mRNAs were investigated. Ame_circ_0005197 and ame_circ_0016640 were observed to sponge several reproductive related miRNAs. These findings demonstrate that circRNAs have potential effects in ovary activation and oviposition of honey bees.     

Roles of stem cell factor/c-Kit and effects of Glivec/STI571 in human uveal melanoma cell tumorigenesis

The B-Raf(V599E)-mediated constitutive activation of ERK1/2 is involved in establishing the transformed phenotype of some uveal melanoma cells (Calipel, A., Lefevre, G., Pouponnot, C., Mouriaux, F., Eychene, A., and Mascarelli, F. (2003) J. Biol. Chem. 278, 42409-42418). We have shown that stem cell factor (SCF) is involved in the proliferation of normal uveal melanocytes and that c-Kit is expressed in 75% of primary uveal melanomas. This suggests that the acquisition of autonomous growth during melanoma progression may involve the SCF/c-Kit axis. We used six human uveal melanoma tumor-derived cell lines and normal uveal melanocytes to characterize the SCF/c-Kit system and to assess its specific role in transformation. We investigated the possible roles of activating mutations in c-KIT, the overexpression of this gene, and ligand-dependent c-Kit overactivation in uveal melanoma cell tumorigenesis. Four cell lines (92.1, SP6.5, Mel270, and TP31) expressed both SCF and c-Kit, and none harbored the c-KIT mutations in exons 9, 11, 13, and 17 that have been shown to induce SCF-independent c-Kit activation. Melanoma cell proliferation was strongly inhibited by small interfering RNA-mediated depletion of c-Kit in these cells, despite the presence of (V599E)B-Raf in SP6.5 and TP31 cells. We characterized the signaling pathways involved in SCF/c-Kit-mediated cell growth and survival in normal and tumoral melanocytes and found that constitutive ERK1/2 activation played a key role in both the SCF/c-Kit autocrine loop and the gain of function of (V599E)B-Raf for melanoma cell proliferation and transformation. We also provide the first evidence that Glivec/STI571, a c-Kit tyrosine kinase inhibitor, could be used to treat uveal melanomas.     

Regulation of cancer aggressive features in melanoma cells by microRNAs

MicroRNAs (miRNAs) are small non-coding RNAs with regulatory roles, which are involved in a broad spectrum of physiological and pathological processes, including cancer. A common strategy for identification of miRNAs involved in cell transformation is to compare malignant cells to normal cells. Here we focus on identification of miRNAs that regulate the aggressive phenotype of melanoma cells. To avoid differences due to genetic background, a comparative high-throughput miRNA profiling was performed on two isogenic human melanoma cell lines that display major differences in their net proliferation, invasion and tube formation activities. This screening revealed two major cohorts of differentially expressed miRNAs. We speculated that miRNAs up-regulated in the more-aggressive cell line contribute oncogenic features, while the down-regulated miRNAs are tumor suppressive. This assumption was further tested experimentally on five candidate tumor suppressive miRNAs (miR-31, -34a, -184, -185 and -204) and on one candidate oncogenic miRNA (miR-17-5p), all of which have never been reported before in cutaneous melanoma. Remarkably, all candidate Suppressive-miRNAs inhibited net proliferation, invasion or tube formation, while miR-17-5p enhanced cell proliferation. miR-34a and miR-185 were further shown to inhibit the growth of melanoma xenografts when implanted in SCID-NOD mice. Finally, all six candidate miRNAs were detected in 15 different metastatic melanoma specimens, attesting for the physiological relevance of our findings. Collectively, these findings may prove instrumental for understanding mechanisms of disease and for development of novel therapeutic and staging technologies for melanoma.     

条纹斑竹鲨肝脏环形RNA的筛选、鉴定及功能分析北大核心CSCD

为筛选和验证条纹斑竹鲨肝脏中环形RNA(circRNA)及探究其在肝癌细胞HepG2中过表达对肝癌细胞增殖、迁移能力的影响,本研究主要进行了两项实验:对条纹斑竹鲨肝脏circRNA进行高通量测序和预测,随后设计正、反向引物验证其真实性;构建circRNA过表达载体,将其瞬时转染进肝癌细胞HepG2,进行CCK-8和划痕实验来评价其对肝癌细胞增殖和迁移能力的影响。结果显示:预测到有4558条circRNAs,并确认了14条circRNAs的真实性;qRT-PCR实验表明在肝癌细胞HepG2中能瞬时过表达circRNA 13-566、circRNA 4-475、circRNA 5-402、circRNA 294-177、circRNA 30-219;且CCK-8和划痕实验显示,这5条circRNAs过表达后,均能不同程度地抑制肝癌细胞的增殖和迁移能力,其中circRNA 4-475、circRNA 294-177作用尤为显著。上述结果为深入研究条纹斑竹鲨肝脏中circRNA及其在肝再生、肝癌治疗方面的功能提供了新思路和基础。     

Integrative analysis of circRNAs acting as ceRNAs involved in ethylene pathway in tomato

Circular RNAs (circRNAs) are a large class of non‐coding endogenous RNAs that could act as competing endogenous RNAs (ceRNAs) to terminate the mRNA targets' suppression of miRNAs. To elucidate the intricate regulatory roles of circRNAs in the ethylene pathway in tomato fruit, deep sequencing and bioinformatics methods were performed. After strict screening, a total of 318 circRNAs were identified. Among these circRNAs, 282 were significantly differentially expressed among wild‐type and sense‐/antisense‐LeERF1 transgenic tomato fruits. Besides, 1254 target genes were identified and a large amount of them were found to be involved in ethylene pathway. In addition, a sophisticated regulatory model consisting of circRNAs, target genes and ethylene was set up. Importantly, 61 circRNAs were found to be potential ceRNAs to combine with miRNAs and some of the miRNAs had been revealed to participate in the ethylene signaling pathway. This research further raised the possibility that the ethylene pathway in tomato fruit may be under the regulation of various circRNAs and provided a new perspective of the roles of circRNAs.