闭塞性细支气管炎模型大鼠微小RNA差异表达谱分析

背景：目前对于气管移植后并发闭塞性细支气管炎尚无有效的治疗方法。miRNA分子水平机制的治疗策略在防治器官移植后并发症方面具有重要意义。 目的：分析大鼠原位气管移植模拟肺移植后发生闭塞性细支气管炎的微小RNA 表达谱变化。 方法：通过近交系大鼠原位气管移植,模拟建立肺移植闭塞性细支气管炎的动物模型并经病理学证实;通过微小RNA芯片筛选出供体移植气管闭塞性细支气管炎组织中显著差异表达的微小RNA,并选取差异表达显著的miR-146a、miR-155和miR-451进行相对定量研究,应用实时定量RT-PCR(RT-qPCR)法验证芯片结果的可靠性。 结果与结论：原位气管移植后4周经病理检查证实大鼠闭塞性细支气管炎模型成功建立。microarray检测获得29个与闭塞性细支气管炎相关的微小RNA,包括15个微小RNA表达显著上调,14个微小RNA表达显著下调,其中显著上调miR-146a、miR-155和显著下调miR-451的功能涉及免疫炎症反应等。提示微小RNA在肺移植后闭塞性细支气管炎病理进程中发挥着重要的调控作用。 中国组织工程研究杂志出版内容重点：肾移植;肝移植;移植;心脏移植;组织移植;皮肤移植;皮瓣移植;血管移植;器官移植;组织工程全文链接：     

A real-time PCR approach to evaluate adipogenic potential of amniotic fluid-derived human mesenchymal stem cells

Regulation of adipocyte differentiation is an important process in the control of adipose tissue development. So far, adipogenesis has been investigated through the use of various experimental models. In this work, we used human mesenchymal stem cells (hMSCs) obtained from amniotic fluid (AF) as an alternative model more representative of what naturally happens in vivo. In our opinion, these hMSCs are still not influenced by differentiation stimuli and could act in a way more correspondent to the physiological process of adipogenesis, representing also an ethically acceptable alternative to totipotent human embryonic stem cells (ES). Adipocyte differentiation was monitorated following the expressions of key genes. We measured the expression levels of PPARgamma2, PPARgamma-C1alpha, UCP-1, adipsin, and leptin genes using quantitative real-time PCR. We tested our experimental model with two different media. Understanding in vivo adipogenesis mechanisms will shed light on the pathophysiology of many diseases.     

反义miR-221/222上调p27~(kipl)对胶质母细胞瘤U251的放射增敏作用

目的 探讨敲低微小RNA(micro RNA,miRs)-221/222表达上调p27~(kipl)对U251胶质母细胞瘤细胞系放射敏感性的影响.方法 经生物信息学分析查询miR-221/222成熟体序列和它们与p27~(kipl)的关系.脂质体共转染反义寡聚核苷酸(反义miR-221/222)下调U251胶质母细胞瘤细胞系miR-221与miR-222的表达.使用Northern印迹方法鉴定转染后U251细胞miR-221、miR-222表达水平下调;流式细胞术检测转染后U251联合X线照射的细胞周期分布;克隆形成实验验证各组细胞增殖性死亡;Western 印迹分析p27kipl蛋白的表达变化.结果 经生物信息学分析显示miR-221/222成熟体序列的种子序列几乎一致,p27~(kipl)是miR-221/222的靶基因.Northern印迹分析显示反义miR-221/222共转染组使miR-221/miR-222的表达水平明显下降.转染无义序列组及对照组的miR-221/miR-222表达水平没有改变.流式细胞术检测可见反义miR-221/222共转染组细胞周期阻滞于G0/G1期且明显高于其它各组.经X线照射后,可明显降低S期比例.反义miR-221/222联合X线照射可增加U251细胞增殖性死亡.Western印迹分析显示反义miR-221/222共转染组的p27~(kipl)蛋白表达明显上调.结论 反义miR-221/222通过上调p27kipl蛋白表达可增加U251胶质母细胞瘤细胞系的放射敏感性.     

MiR‐221 and MiR‐222 alterations in sporadic ovarian carcinoma: Relationship to CDKN1B,CDKNIC and overall survival

MicroRNAs are often aberrantly expressed in human neoplasms and are postulated to play a role in neoplastic initiation and progression. miR‐221 and miR‐222 negatively regulate expression of CDKN1B (p27) and CDKN1C (p57), two cell cycle regulators expressed in ovarian surface epithelium and down‐regulated in ovarian carcinomas. We characterized miR‐221 and miR‐222 expression in 49 sporadic high grade ovarian carcinomas and determined whether somatic mutation or epigenetic alterations explained the differences in expression of these miRNAs. We correlated these findings with protein expression of CDKN1B and CDKN1C as assessed by immunohistochemistry. Expression of miR‐221 and miR‐222 were closely correlated with each other (P = 0.0001). Interestingly, a lower ratio of miR‐221 to miR‐222 expression was significantly correlated with worse overall survival (P = 0.01) and remained a significant predictor of overall survival in multivariate analysis using the covariate adequacy of surgical cytoreduction (P = 0.03). Higher miR‐222 and miR‐221 expression were significantly associated with decreased CDKN1C expression (P = 0.009 and 0.01). In contrast, CDKN1B expression was not associated with miR‐221 or miR‐222 expression. Neither somatic mutations nor methylation of the studied region explained the alterations in miR‐221 and miR‐222 expression in most carcinomas. © 2010 Wiley‐Liss, Inc.     

Differential Expression of miRNAs in Papillary Thyroid Carcinoma Compared to Multinodular Goiter Using Formalin Fixed Paraffin Embedded Tissues

microRNAs (miRNAs) are approximately 22 nt RNAs that negatively regulate target gene expression. Their dysregulation has been implicated in the pathogenesis of a number of human cancers, including papillary thyroid carcinoma (PTC). Whereas previous studies using microarray technologies have largely relied on the ability to procure fresh tissue at the time of surgery to characterize miRNA signatures in PTC, we exploited the ability to procure sufficient miRNA from formalin-fixed paraffin-embedded (FFPE) tissue to describe a series of miRNAs whose expression is dysregulated in PTC compared to benign proliferative multinodular goiter (MNG). We identified 13 miRNAs upregulated and 26 miRNAs downregulated in PTC versus MNG. These include miRNA-21, miRNA-31, miRNA-221, and miRNA-222. Their dysregulation was further validated by real time RT-PCR analysis in an independent set of FFPE tissues. Many of these have previously been described in fresh tissue studies as altered in PTC, confirming the utility of this approach. These results further highlight the applicability of miRNA expression patterns as potential markers of human cancer, and our results suggest that FFPE tissues are suitable resources for such miRNA expression analyses. The ability to utilize FFPE tissue in the molecular characterization of human malignancy will unlock a rich resource for future cancer studies. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Hedgehog signaling alters adipocyte maturation of human mesenchymal stem cells

Human stem cells are powerful tools by which to investigate molecular mechanisms of cell growth and differentiation under normal and pathological conditions. Hedgehog signaling, the dysregulation of which causes several pathologies, such as congenital defects and cancer, is involved in several cell differentiation processes and interferes with adipocyte differentiation of rodent cells. The present study was aimed at investigating the effect of Hedgehog pathway modulation on adipocyte phenotype using different sources of human mesenchymal cells, such as bone marrow stromal cells and human multipotent adipose-derived stem cells. We bring evidence that Hedgehog signaling decreases during human adipocyte differentiation. Inhibition of this pathway is not sufficient to trigger adipogenesis, but activation of Hedgehog pathway alters adipocyte morphology as well as insulin sensitivity. Analysis of glycerol-3-phosphate dehydrogenase activity and expression of adipocyte marker genes indicate that activation of Hedgehog signaling by purmorphamine impairs adipogenesis. In sharp contrast to reports in rodent cells, the maturation process, but not the early steps of human mesenchymal stem cell differentiation, is affected by Hedgehog activation. Hedgehog interferes with adipocyte differentiation by targeting CCAAT enhancer-binding protein alpha and peroxisome proliferator-activated receptor (PPAR) gamma2 expression, whereas PPARgamma1 level remains unaffected. Although Hedgehog pathway stimulation does not modify the total number of adipocytes, adipogenesis appears dramatically impaired, with reduced lipid accumulation, a decrease in adipocyte-specific markers, and acquisition of an insulin-resistant phenotype. This study indicates that a decrease in Hedgehog signaling is necessary but not sufficient to trigger adipocyte differentiation and unveils a striking difference in the adipocyte differentiation process between rodent and human mesenchymal stem cells.     

Coordinated increase of miRNA-155 and miRNA-196b expression correlates with the detection of the antigenomic strand of hepatitis C virus in peripheral blood mononuclear cells

A tight relationship has been revealed between cellular microRNAs (miRNAs) and the course of hepatitis C virus (HCV) replication in human hepatoma cells. Although the detection of the antigenomic HCV RNA strand in peripheral blood mononuclear cells (PBMCs) has provided evidence for viral replication in PBMCs, no reports have shown how miRNAs are affected upon HCV RNA synthesis in PBMCs. The aim of the present study was to assess if and how the expression levels of miRNA-155 and miRNA-196b in PBMCs are related to HCV replication in PBMCs of chronic hepatitis C (CHC) patients. Supporting analyses were performed to evaluate the expression of precursor pri-miR-155 (BIC) and Dicer protein. The genomic and antigenomic HCV RNA strands in PBMCs were detected by strand-specific qRT-PCR. The expression levels of miRNAs, BIC RNA and Dicer protein were assayed on PBMCs by qRT-PCR and Western blotting, respectively. miRNA-155 and miRNA-196b were detected in all studied PBMC samples, but their levels varied according to the presence of the antigenomic HCV RNA strand in PBMCs. Increased expression levels of miRNA-155 and miRNA-196b were associated with the presence of the antigenomic HCV RNA strand in PBMCs. In this group of patients higher frequency of BIC RNA and Dicer protein detection was also found. This study demonstrates that HCV RNA replication in PBMCs of CHC patients is connected with the increased and coordinated expression of miRNA-155 and miRNA-196b.     

微小RNA调节骨骼肌发育与损伤修复的作用及其机制

microRNAs是一类非编码小RNAs分子,新近发现其具有重要的调节基因表达的功能,它能通过抑制翻译和降解靶mRNA来负性调控转录后水平的基因表达,miRNAs已经被证实在肌肉发育和肌细胞增殖和分化的调节中具有重要作用。最近研究发现,肌肉特异性转录因子控制一些microRNAs的表达,通过多种机制调节肌肉发育和功能。结合信息学、生物化学和遗传基因学方法,不仅将阐明骨骼肌microRNAs调控网络,更好地理解肌肉组织的调节,还将通过鉴定候选microRNAs的潜在临床应用靶点,增加肌肉营养不良的治疗干预的新机会。     

Adipogenic human adenovirus Ad-36 induces commitment, differentiation, and lipid accumulation in human adipose-derived stem cells

Human adenovirus Ad-36 is causatively and correlatively linked with animal and human obesity, respectively. Ad-36 enhances differentiation of rodent preadipocytes, but its effect on adipogenesis in humans is unknown. To indirectly assess the role of Ad-36-induced adipogenesis in human obesity, the effect of the virus on commitment, differentiation, and lipid accumulation was investigated in vitro in primary human adipose-derived stem/stromal cells (hASC). Ad-36 infected hASC in a time- and dose-dependent manner. Even in the presence of osteogenic media, Ad-36-infected hASC showed significantly greater lipid accumulation, suggestive of their commitment to the adipocyte lineage. Even in the absence of adipogenic inducers, Ad-36 significantly increased hASC differentiation, as indicated by a time-dependent expression of genes within the adipogenic cascade-CCAAT/Enhancer binding protein-beta, peroxisome proliferator-activated receptor-gamma, and fatty acid-binding protein-and consequentially increased lipid accumulation in a time- and viral dose-dependent manner. Induction of hASC to the adipocyte state by Ad-36 was further supported by increased expression of lipoprotein lipase and the accumulation of its extracellular fraction. hASC from subjects harboring Ad-36 DNA in their adipose tissue due to natural infection had significantly greater ability to differentiate compared with Ad-36 DNA-negative counterparts, which offers a proof of concept. Thus, Ad-36 has the potential to induce adipogenesis in hASC, which may contribute to adiposity induced by the virus.     

共沉默miR-221-3p/222-3p表达抑制骨髓间充质干细胞增殖及促成软骨分化

背景：基于干细胞的组织工程技术作为治疗骨关节损伤修复的有效手段,具有广泛的应用前景。研究表明miRNA在调控干细胞向软骨分化过程中具有重要的作用。 目的：探讨共感染慢病毒介导的反义miR-221-3p/222-3p(microRNA-221-3p,microRNA-222-3p)对人骨髓间充质干细胞成软骨分化的作用,为临床软骨损伤修复提供新的策略。 方法：利用miRNA基因芯片技术筛选转化生长因子β3诱导人骨髓间充质干细胞成软骨分化过程中不同阶段表达差异的miRNA,并利用荧光实时定量PCR(RT-qPCR)验证;构建慢病毒人反义miR-221-3p/222-3p载体(Lv-miR-221-3p-inhibition,Lv-miR-222-3p-inhibition)并共转染人骨髓间充质干细胞,空载体组(Lv-GFP)以及未转染组作为对照。利用CCK-8法检测沉默miR-221-3p/222-3p 6 d后细胞的增殖情况;通过番红O染色法、免疫组织化学方法以及RT-PCR验证各组软骨诱导21 d后软骨分化相关标志物的表达。 结果与结论：构建的Lv-miRNA-221-3p/222-3p inhibition 共转染人骨髓间充质干细胞,成功沉默了细胞中的miR-221-3p/222-3p表达水平;miRNA基因芯片与RT-qPCR验证结果显示miR-221-3p/222-3p在软骨分化后期表达明显降低;转染组与未转染组以及空载体组相比：①细胞增殖明显受到抑制。②番红Ｏ染色以及免疫组织化学显示软骨分化特征标志物硫酸软骨素以及Ⅱ型胶原表达增强。③RT-qPCR也证实硫酸软骨素以及Ⅱ型胶原的mRNA表达也明显上调。结果显示沉默人骨髓间充质干细胞miRNA-221-3p/222-3p,抑制了细胞增殖并促进了成软骨分化。  中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

MicroRNA‐664a‐5p promotes neuronal differentiation of SH‐SY5Y cells

MicroRNAs (miRNAs) belong to a class of small noncoding RNAs that play important roles in the translational regulation of gene expression. A number of miRNAs are known to act as key regulators of diverse processes such as neuronal differentiation. In this study, we have attempted to identify novel miRNAs related to neuronal differentiation via microarray analysis in the human neuronal differentiation model neuroblastoma SH‐SY5Y cells. We identified 15 up‐regulated and eight down‐regulated miRNAs in SH‐SY5Y cells treated with all‐trans retinoic acid to induce differentiation. We further showed that one of the up‐regulated miRNAs, miR‐664a‐5p, promoted neuronal differentiation of SH‐SY5Y cells. These findings enhance our understanding of the miRNAs involved in the process of neurogenesis and, in particular, highlight an important role of miR‐664a‐5p in SH‐SY5Y cell neuronal differentiation. Further studies will be required to confirm the function of miR‐664‐5p in neuronal development and disease and to identify its relevant target genes.     

miR-21对血管内皮功能及血管生成调节作用的研究进展

<正>血管内皮细胞作为血管的内衬,一方面参与构成血管的通透性屏障,调节液体、气体及生物大分子物质的选择性通透;另一方面可分泌大量血管活性因子,参与对凝血与抗凝功能、纤溶系统和炎症发生发展等的调控,与血管功能和循环系统的稳定密切相关。研究表明,血管内皮细胞功能激活是血管生成的先决条件~([1])。血管内皮细胞在多种细胞因子的诱导下增殖迁移,为血管管腔形成提供内皮细胞数量的积累。血管生成因子与内皮细胞受体结合可激     

MicroRNAs in metabolism

MicroRNAs (miRNAs) have within the past decade emerged as key regulators of metabolic homoeostasis. Major tissues in intermediary metabolism important during development of the metabolic syndrome, such as β‐cells, liver, skeletal and heart muscle as well as adipose tissue, have all been shown to be affected by miRNAs. In the pancreatic β‐cell, a number of miRNAs are important in maintaining the balance between differentiation and proliferation (miR‐200 and miR‐29 families) and insulin exocytosis in the differentiated state is controlled by miR‐7, miR‐375 and miR‐335. MiR‐33a and MiR‐33b play crucial roles in cholesterol and lipid metabolism, whereas miR‐103 and miR‐107 regulates hepatic insulin sensitivity. In muscle tissue, a defined number of miRNAs (miR‐1, miR‐133, miR‐206) control myofibre type switch and induce myogenic differentiation programmes. Similarly, in adipose tissue, a defined number of miRNAs control white to brown adipocyte conversion or differentiation (miR‐365, miR‐133, miR‐455). The discovery of circulating miRNAs in exosomes emphasizes their importance as both endocrine signalling molecules and potentially disease markers. Their dysregulation in metabolic diseases, such as obesity, type 2 diabetes and atherosclerosis stresses their potential as therapeutic targets. This review emphasizes current ideas and controversies within miRNA research in metabolism.     

Duration and magnitude of extracellular signal-regulated protein kinase phosphorylation determine adipogenesis or osteogenesis in human bone marrow-derived stem cells

Purpose

Imbalances between osteogenic and adipogenic differentiation leads to diseases such as osteoporosis. The aim of our study was to demonstrate the differences in extracellular signal-regulated kinase (ERK) phosphorylation during both adipogenesis and osteogenesis of human bone marrow-derived stem cells (BMSCs).

Materials and Methods

Using troglitazone, GW9662 and U0126, we investigated their role in hBMSC differentiation to adipogenic and osteogenic fates.

Results

ERK1/2 inhibition by U0126 suppressed proliferator-activated receptor (PPAR)γ expression and lipid accumulation, while it decreased the mRNA expression of adipogenic genes (lipoprotein lipase, PPARγ, and adipocyte protein) and osteogenic genes (type I collagen and osteopontin). ERK phosphorylation was transient and decreased during adipogenesis, whereas it occurred steadily during osteogenesis. Troglitazone, a PPARγ agonist, induced adipogenesis by inhibiting ERK phosphorylation even in an osteogenic medium, suggesting that ERK signaling needs to be shut off in order to proceed with adipose cell commitment. Cell proliferation was greatly increased in osteogenesis but was not changed during adipogenesis, indicating that ERK might play different roles in cellular proliferation and differentiation between the two committed cell types.

Conclusion

The duration and magnitude of ERK activation might be a crucial factor for the balance between adipogenesis and osteogenesis in human bone marrow-derived stem cells.     

Overexpression of miR-221 inhibits proliferation and promotes apoptosis of human astrocytoma cells

microRNAs (miRNAs) play tumor-promoting roles in a variety of tumors. This study investigated the expression of miRNA-211 (miR-221) in human astrocytoma, and its effect on proliferation and apoptosis of human astrocytoma cells in vitro. miR-221 expression was detected in 10 astrocytoma tissues and 4 adjacent tissues by real-time quantitative PCR (qRT-PCR). miR-221 expression in situ was significantly higher in astrocytoma tissues than in adjacent tissues (P<0.05). To determine whether the upregulation of miR-221 could be associated with tumor development or progression, a synthetic miR-221 mimic was transiently transfected into U251 astrocytoma cells in vitro. qRT-PCR confirmed that the mimic significantly increased the expression of miR-221 in these cells. An MTT colorimetric assay indicated that proliferation was significantly higher in U251 cells transfected with miR-221 mimic than in scramble-transfected control cells (P<0.05). Further analysis of miR-221 transfected cells by flow cytometry revealed an altered cell cycle progression, with more cells in S and G1 phase, as well as an inhibition of apoptosis (P<0.05). These findings indicate that the upregulation of miR-221 in astrocytoma tissues may be associated with development or progression of these tumors. Thus, miR-221 should be explored as a potential molecular marker for the diagnosis and treatment of astrocytoma.