Combined characterization of microRNA and mRNA profiles delineates early differentiation pathways of CD133+ and CD34+ hematopoietic stem and progenitor cells

MicroRNAs (miRNAs) have been shown to play an important role in hematopoiesis. To elucidate the role of miRNAs in the early steps of hematopoiesis, we directly compared donor-matched CD133(+) cells with the more differentiated CD34(+) CD133(-) and CD34(-) CD133(-) cells from bone marrow on the miRNA and mRNA level. Using quantitative whole genome miRNA microarray and sequencing-based profiling, we found that between 109 (CD133(+) ) and 216 (CD34(-) CD133(-) ) miRNAs were expressed. Quantification revealed that the 25 highest expressed miRNAs accounted for 73% of the total miRNA pool. miR-142-3p was the highest expressed miRNA with up to 2,000 copies per cell in CD34(+) CD133(-) cells. Eighteen miRNAs were significantly differentially expressed between CD133(+) and CD34(+) CD133(-) cells. We analyzed their biological role by examining the coexpression of miRNAs and its bioinformatically predicted mRNA targets and luciferase-based reporter assays. We provide the first evidence for a direct regulation of CD133 by miR-142-3p as well as tropomyosin 1 and frizzled homolog 5 by miR-29a. Overexpression of miRNAs in CD133(+) cells demonstrated that miR-142-3p has a negative influence on the overall colony-forming ability. In conclusion, the miRNAs expressed differentially between the CD133(+) and CD34(+) CD133(-) cells are involved in inhibition of differentiation, prevention of apoptosis, and cytoskeletal remodeling. These results are highly relevant for stem cell-based therapies with CD133(+) cells and delineate for the first time how the stem cell character of CD133(+) cells is defined by the expression of specific miRNAs.     

Aspectos inmunogenéticos de la psoriasis con énfasis en micro-ARN

Psoriasis is a chronic inflammatory disease of the skin, of autoimmune origin, with different cells implicated in the aetiopathology, such as T helper lymphocytes (Th1 and Th17), keratinocytes, and cytokines produced by these cells. The epigenetic regulatory mechanisms are the junction between environmental exposure and genetic factors. It is known that microRNAs (miRNAs), single chain RNAs, are actively involved in epigenetic regulation. Alterations in the miR-125b, miR-424, miR- 21 and miR-203 expression, and others, have been involved in different aspects of the disease. Global studies of miRNA expression performed using microarrays and by direct RNA sequencing revealed important differences in miRNA expression in normal skin and psoriatic individuals. These miRNAs can be considered as potential therapeutic targets or biomarkers of disease.     

Systematic identification of microRNA and messenger RNA profiles in hepatitis C virus-infected human hepatoma cells

In order to investigate the global and dynamic host microRNAs (miRNAs)/messenger RNAs (mRNAs) expression alteration during in vitro acute HCV infection, a comprehensive microarray analysis was performed using human hepatoma cells. Totally, 108 human miRNAs and 1247 mRNAs were identified whose expression levels changed for more than 2.0-fold in response to HCV infection. Upon HCV infection, signature from the unique miRNA expression pattern reflected the involvement of miRNA-regulated host cellular physiology and antiviral mechanism, whereas a preponderance of differentially regulated genes associated with metabolism, cell growth, apoptosis and cytokine/chemokine pathways. Furthermore, a reverse regulatory association of differentially expressed miRNAs and their predicted targets was constructed. Finally, the differentially expressed miRNAs such as miR-24, miR-149?, miR-638 and miR-1181 were identified to be involved in HCV entry, replication and propagation. These results suggest that combined miRNA and mRNA profiling may have superior potential as a diagnostic and mechanistic feature in HCV infection.     

减体积肝移植模型大鼠肝脏组织miRNAs表达谱变化

BACKGROUND: Liver regeneration is the key factor influencing the prognosis of living donor liver transplantation. There has not been the research on special miRNA of liver regeneration after living donor liver transplantation. OBJECTIVE: To analyze the variation of miRNAs expression profile after rat reduced-size liver transplantation at certain time point, select and verify target miRNA which can provide targeting intervention strategies in liver regeneration after rat reduced-size liver transplantation and provide theoretical evidence for liver regeneration after living donor liver transplantation. METHODS: The reduced-size liver transplantation models were established. miRNAs microarray was used to detect miRNA expression. In differentially expressed microRNAs, real-time quantitative PCR was utilized to detect target miRNAs. The credibility of miRNAs microarray results was verified. RESULTS AND CONCLUSION: Compared with rat liver tissue in the sham operation group, 11 miRNAs up-regulated in reduced-size liver transplantation, including let-7b-5p, let-7c-5p, miR-101a-3p, miR-103-3p, miR-130a-3p, miR-142-5p, miR-186-5p, miR-199a-3p, miR-21-5p, 221-3p and miR-34a-5p. Four miRNAs were down-regulated, including miR-26b-5p, miR-150-5p, miR-19a-3p and rno-miR-146-5p. PCR test further verified that miR-221-3p and miR-199a-3p expression changes approximated the chip results at 24, 48 hours and 1 week, indicating that results of miRNA microarray were believable. These results verified that it exists variation of miRNAs expression profile after rat reduced-size liver transplantation, which picked out and verified the target miRNAs.        

应用基因芯片检测喉鳞状细胞癌甲醛固定石蜡包埋组织microRNA表达谱

目的 建立以甲醛固定石蜡包埋组织为材料、基于基因芯片技术的microRNA(miRNA)表达谱的分析方法 ;筛选与喉鳞状细胞癌(简称喉癌)生物学特征密切相关的差异表达miRNA.方法 从喉癌甲醛固定石蜡包埋组织中制备总RNA,经质量鉴定后进行荧光标记.采用Agilent公司的容纳723条人类miRNA探针的基因芯片完成杂交实验,以获得喉癌的miRNA表达谱.以GeneSpring GX和R-Project软件处理分析基因芯片实验数据,筛选与喉癌转移相关的差异表达miRNA.结果 从24例甲醛固定石蜡包埋组织标本中获得了符合基因芯片实验质量标准的RNA样品,并完成了基因芯片杂交及数据分析.从中共鉴定到319个miRNA,有96个miRNA在24例喉癌中均有表达,其中与淋巴结转移密切相关的(检错率<0.05)差异表达miRNA有5个,分别为miR-23a* 、miR-28-5p、miR-15a、miR-16和miR-425.结论 甲醛固定石蜡包埋组织可以提供符合基因芯片分析质量要求的miRNA,是研究miRNA的重要样品资源.从喉癌的miRNA表达谱中筛选出的转移相关差异表达miRNA(miR-23a*、miR-28-5p、miR-15a、miR-16和miR-425)有可能成为评估喉癌转移风险的新型分子标志.     

应用基因芯片检测喉鳞状细胞癌甲醛固定石蜡包埋组织microRNA表达谱

目的 建立以甲醛固定石蜡包埋组织为材料、基于基因芯片技术的microRNA(miRNA)表达谱的分析方法 ;筛选与喉鳞状细胞癌(简称喉癌)生物学特征密切相关的差异表达miRNA.方法 从喉癌甲醛固定石蜡包埋组织中制备总RNA,经质量鉴定后进行荧光标记.采用Agilent公司的容纳723条人类miRNA探针的基因芯片完成杂交实验,以获得喉癌的miRNA表达谱.以GeneSpring GX和R-Project软件处理分析基因芯片实验数据,筛选与喉癌转移相关的差异表达miRNA.结果 从24例甲醛固定石蜡包埋组织标本中获得了符合基因芯片实验质量标准的RNA样品,并完成了基因芯片杂交及数据分析.从中共鉴定到319个miRNA,有96个miRNA在24例喉癌中均有表达,其中与淋巴结转移密切相关的(检错率<0.05)差异表达miRNA有5个,分别为miR-23a* 、miR-28-5p、miR-15a、miR-16和miR-425.结论 甲醛固定石蜡包埋组织可以提供符合基因芯片分析质量要求的miRNA,是研究miRNA的重要样品资源.从喉癌的miRNA表达谱中筛选出的转移相关差异表达miRNA(miR-23a*、miR-28-5p、miR-15a、miR-16和miR-425)有可能成为评估喉癌转移风险的新型分子标志.     

应用基因芯片检测喉鳞状细胞癌甲醛固定石蜡包埋组织microRNA表达谱

目的 建立以甲醛固定石蜡包埋组织为材料、基于基因芯片技术的microRNA(miRNA)表达谱的分析方法 ;筛选与喉鳞状细胞癌(简称喉癌)生物学特征密切相关的差异表达miRNA.方法 从喉癌甲醛固定石蜡包埋组织中制备总RNA,经质量鉴定后进行荧光标记.采用Agilent公司的容纳723条人类miRNA探针的基因芯片完成杂交实验,以获得喉癌的miRNA表达谱.以GeneSpring GX和R-Project软件处理分析基因芯片实验数据,筛选与喉癌转移相关的差异表达miRNA.结果 从24例甲醛固定石蜡包埋组织标本中获得了符合基因芯片实验质量标准的RNA样品,并完成了基因芯片杂交及数据分析.从中共鉴定到319个miRNA,有96个miRNA在24例喉癌中均有表达,其中与淋巴结转移密切相关的(检错率<0.05)差异表达miRNA有5个,分别为miR-23a* 、miR-28-5p、miR-15a、miR-16和miR-425.结论 甲醛固定石蜡包埋组织可以提供符合基因芯片分析质量要求的miRNA,是研究miRNA的重要样品资源.从喉癌的miRNA表达谱中筛选出的转移相关差异表达miRNA(miR-23a*、miR-28-5p、miR-15a、miR-16和miR-425)有可能成为评估喉癌转移风险的新型分子标志.     

Primary biliary cirrhosis is associated with altered hepatic microRNA expression

MicroRNAs (miRNAs) are small RNA molecules that negatively regulate protein coding gene expression and are thought to play a critical role in many biological processes. Aberrant levels of miRNAs have been associated with numerous diseases and cancers, and as such, miRNAs have gain much interests as diagnostic biomarkers, and as therapeutic targets. However, their role in autoimmunity is largely unknown. The aims of this study are to: (1) identify differentially expressed miRNAs in human primary biliary cirrhosis (PBC); (2) validate these independently; and (3) identify potential targets of differentially expressed miRNAs. We compared the expression of 377 miRNAs in explanted livers form subjects with PBC versus controls with normal liver histology. A total of 35 independent miRNAs were found to be differentially expressed in PBC (p < 0.001). Quantitative PCR was employed to validate down-regulation of microRNA-122a (miR-122a) and miR-26a and the increased expression of miR-328 and miR-299-5p. The predicted targets of these miRNAs are known to affect cell proliferation, apoptosis, inflammation, oxidative stress, and metabolism. Our data are the first to demonstrate that PBC is characterized by altered expression of hepatic miRNA; however additional studies are required to demonstrate a causal link between those miRNA and the development of PBC.     

微小RNA活性分析法筛选前列腺癌去势抵抗转化相关微小RNA的实验研究

目的 探讨微小RNA(miRNA)活性分析法筛选前列腺癌去势抵抗转化相关miRNA的效率。方法 应用miRNA活性分析法筛选出在前列腺癌去势抵抗过程中潜在的发挥活性作用的miRNAs。培养人激素敏感型前列腺癌LNCAP细胞(对照组)及人去势抵抗型前列腺癌C4-2细胞(C4-2组)、PC-3细胞(PC-3组)、DU-145细胞(DU-145组),提取各组细胞总RNA,采用实时荧光定量PCR(qPCR)检测miRNAs,比较各组细胞miRNAs的表达情况。结果 应用miRNA活性分析法,依据筛选流程,共选出9个差异表达的miRNAs,分别为miR-1、miR-122、miR-218、miR-145、miR-155、miR-210、miR-197、 miR-346、let-7b。采用qPCR检测这9个miRNAs,结果显示,7个miRNAs在两种不同状态下的前列腺癌细胞中存在差异表达;在不同去势抵抗型前列腺癌细胞中,miR-210、miR-197、miR-346、miR-218均明显高表达,而miR-122、miR-145、let-7b均明显低表达。结论 采用miRNA活性分析法筛选前列腺癌去势抵抗转化相关miRNA,有着较高的准确性与可信度;其具体转化过程还需进一步证实。     

Angiotensin II induced differentially expressed microRNAs in adult rat cardiac fibroblasts

Angiotensin II (Ang II) plays a pivotal role in cardiac fibrosis, and microRNAs (miRNAs) have been shown to participate in diverse pathological processes. Our aim is to identify the Ang II-induced miRNAs in cardiac fibroblasts (CFs). The miRNA array was used to analyze the miRNA expression profile in CFs treated by Ang II and control cells. Stem-loop real-time PCR was performed to re-measure the levels of the differentially expressed miRNAs. Analysis of miRNA arrays showed that 33 miRNAs were differentially expressed (13 up- and 20 downregulated) in response to Ang II (100 nM) for 24 h as compared to control cells. Quantitative PCR revealed that Ang II upregulated the levels of miR-132, -125b-3p and miR-146b but downregulated the levels of miR-300-5p, -204* and miR-181b in CFs. The trend of miRNA change is consistent with microarray and qRT-PCR. Bioinformatic analysis revealed that MMP9 as the target of miR-132, MMP16 as the target of miR-146b and TIMP3 as the target of miR-181b have been listed in the miR database with experimentally validated targets, indicating the potential role of those miRNAs in cardiac fibrosis. Our results demonstrated that we did identify a subset of miRNAs that was differentially expressed in Ang II-treated CFs, which provide a starting point to explore their potential roles in cardiac fibrosis and hypertension.     

A set of miRNAs that involve in the pathways of drug resistance and leukemic stem-cell differentiation is associated with the risk of relapse and glucocorticoid response in childhood ALL

Relapse is a major challenge in the successful treatment of childhood acute lymphoblastic leukemia (ALL). Despite intensive research efforts, the mechanisms of ALL relapse are still not fully understood. An understanding of the molecular mechanisms underlying treatment outcome, therapy response and the biology of relapse is required. In this study, we carried out a genome-wide microRNA (miRNA) microarray analysis to determine the miRNA expression profiles and relapse-associated miRNA patterns in a panel of matched diagnosis-relapse or diagnosis-complete remission (CR) childhood ALL samples. A set of miRNAs differentially expressed either in relapsed patients or at diagnosis compared with CR was further validated by quantitative real-time polymerase chain reaction in an independent sample set. Analysis of the predicted functions of target genes based on gene ontology 'biological process' categories revealed that the abnormally expressed miRNAs are associated with oncogenesis, classical multidrug resistance pathways and leukemic stem cell self-renewal and differentiation pathways. Several targets of the miRNAs associated with ALL relapse were experimentally validated, including FOXO3, BMI1 and E2F1. We further investigated the association of these dysregulated miRNAs with clinical outcome and confirmed significant associations for miR-708, miR-223 and miR-27a with individual relapse-free survival. Notably, miR-708 was also found to be associated with the in vivo glucocorticoid therapy response and with disease risk stratification. These miRNAs and their targets might be used to optimize anti-leukemic therapy, and serve as novel targets for development of new countermeasures of leukemia. This fundamental study may also contribute to establish the mechanisms of relapse in other cancers.     

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.     

Organ-specific testosterone-insensitive response of miRNA expression of C57BL/6 mice to Plasmodium chabaudi malaria

Increasing evidence critically implicates miRNAs in the pathogenesis of diseases, but little is known in context with infectious diseases. This study investigates as to whether the testosterone-induced persistent susceptibility to blood-stage malaria of Plasmodium chabaudi coincides with changes in miRNA expression of the anti-malaria effectors sites spleen and liver. Female C57BL/6 mice were treated with vehicle or testosterone (T) for 3?weeks. Then, T treatment was discontinued for 12?weeks before challenge with 10(6) P. chabaudi-parasitized erythrocytes. The miRNA expression was examined after 12?weeks of T withdrawal and during infections at peak parasitemia on day?8 p.i. using miRXplore? microarray technology. P. chabaudi infections induce an organ-specific response of miRNA expression. We can identify 25 miRNA species to be downregulated by more than 2-fold in the spleen and 169 miRNA species in the liver. Among these 194 miRNA species, there are 12 common miRNA species that are downregulated by 0.48-0.14-fold in both spleen and liver, which are miR-194, miR-192, miR-193A-3P, miR-145, miR-16, miR-99A, miR-99B, miR-15A, miR-152, let-7G, let-7B, and miR-455-3P. Only in the liver, there is an upregulation of the miR-142-5p by 2.5-fold and miR-342-3p by 5.1-fold. After 12?weeks of T withdrawal, the spleen exhibits only the miR-200A that is upregulated by 2.7-fold. In the liver, miR-376B, miR-493*, and miR-188-3P are upregulated by 2.4-fold, 2.2-fold, and 2.1-fold, respectively, and miR-347, miR-200A, and miR-200B are downregulated by approximately 0.4-fold. Upon infection, however, these changes are not sustained, i.e., the miRNA expressions of both spleen and liver of T-pretreated mice exhibit the same response to P. chabaudi malaria as that of vehicle-treated control mice. Our data suggest (1) that the P. chabaudi-induced downregulation of miRNA expression in spleen and liver is required to allow the upregulation of their numerous target genes in response to infection, and (2) that the T-induced persistent susceptibility to P. chabaudi does not affect the responsiveness of miRNA expression in spleen and liver to blood-stage malaria.     

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.