Systematic identification of mRNAs recruited to argonaute 2 by specific microRNAs and corresponding changes in transcript abundance

microRNAs (miRNAs) are small non-coding RNAs that regulate mRNA stability and translation through the action of the RNAi-induced silencing complex (RISC). Our current understanding of miRNA function is inferred largely from studies of the effects of miRNAs on steady-state mRNA levels and from seed match conservation and context in putative targets. Here we have taken a more direct approach to these issues by comprehensively assessing the miRNAs and mRNAs that are physically associated with Argonaute 2 (Ago2), which is a core RISC component. We transfected HEK293T cells with epitope-tagged Ago2, immunopurified Ago2 together with any associated miRNAs and mRNAs, and quantitatively determined the levels of these RNAs by microarray analyses. We found that Ago2 immunopurified samples contained a representative repertoire of the cell's miRNAs and a select subset of the cell's total mRNAs. Transfection of the miRNAs miR-1 and miR-124 caused significant changes in the association of scores of mRNAs with Ago2. The mRNAs whose association with Ago2 increased upon miRNA expression were much more likely to contain specific miRNA seed matches and to have their overall mRNA levels decrease in response to the miRNA transfection than expected by chance. Hundreds of mRNAs were recruited to Ago2 by each miRNA via seed sequences in 3'-untranslated regions and coding sequences and a few mRNAs appear to be targeted via seed sequences in 5'-untranslated regions. Microarray analysis of Ago2 immunopurified samples provides a simple, direct method for experimentally identifying the targets of miRNAs and for elucidating roles of miRNAs in cellular regulation.     

miR-202-5p在牙鲆性腺中的表达分析及其与cbx2靶向关系验证

为研究miR-202-5p在鱼类中作用的靶基因及功能,采用荧光定量PCR和原位杂交技术构建了miR-202-5p在牙鲆(Paralichthys olivaceus)不同组织和雌雄性腺中的表达谱。定量结果发现, miR-202-5p在牙鲆性腺中具有特异性的高表达,且在精巢中的表达水平高于卵巢。原位杂交结果显示, miR-202-5p主要在精巢的精原细胞和精母细胞中表达,而仅在卵巢的Ⅳ和Ⅴ期卵母细胞中有较强烈表达。研究发现色素框同源蛋白2(Chromobox homolog 2, CBX2)在性腺发育中具有重要调节作用,为进一步研究miR-202-5p在牙鲆性腺发育中的功能,采用生物信息学方法预测和双荧光素酶报告基因技术鉴定了二者的靶向关系,结果证实, cbx2为miR-202-5p直接调节的靶基因,为深入阐述miR-202-5p在牙鲆性腺发育中的作用机制提供了基础。     

microRNAs associated with the different human Argonaute proteins

MicroRNAs (miRNAs) are small noncoding RNAs that function in literally all cellular processes. miRNAs interact with Argonaute (Ago) proteins and guide them to specific target sites located in the 3′-untranslated region (3′-UTR) of target mRNAs leading to translational repression and deadenylation-induced mRNA degradation. Most miRNAs are processed from hairpin-structured precursors by the consecutive action of the RNase III enzymes Drosha and Dicer. However, processing of miR-451 is Dicer independent and cleavage is mediated by the endonuclease Ago2. Here we have characterized miR-451 sequence and structure requirements for processing as well as sorting of miRNAs into different Ago proteins. Pre-miR-451 appears to be optimized for Ago2 cleavage and changes result in reduced processing. In addition, we show that the mature miR-451 only associates with Ago2 suggesting that mature miRNAs are not exchanged between different members of the Ago protein family. Based on cloning and deep sequencing of endogenous miRNAs associated with Ago1–3, we do not find evidence for miRNA sorting in human cells. However, Ago identity appears to influence the length of some miRNAs, while others remain unaffected.     

IL-4 Up-Regulates MiR-21 and the MiRNAs Hosted in the CLCN5 Gene in Chronic Lymphocytic Leukemia

Interleukin 4 (IL-4) induces B-cell differentiation and survival of chronic lymphocytic leukemia (CLL) cells. MicroRNAs (miRNAs) regulate mRNA and protein expression, and several miRNAs, deregulated in CLL, might play roles as oncogenes or tumor suppressors. We have studied the miRNA profile of CLL, and its response to IL-4, by oligonucleotide microarrays, resulting in the detection of a set of 129 mature miRNAs consistently expressed in CLL, which included 41 differentially expressed compared to normal B cells (NBC), and 6 significantly underexpressed in ZAP-70 positive patients. IL-4 stimulation brought about up-regulation of the 5p and 3p mature variants of the miR-21 gene, which maps immediately downstream to the VMP1 gene, and of the mature forms generated from the miR-362 (3p and 5p), miR-500a (3p), miR-502 (3p), and miR-532 (3p and 5p) genes, which map within the third intron of the CLCN5 gene. Both genes are in turn regulated by IL-4, suggesting that these miRNAs were regulated by IL-4 as passengers from their carrier genes. Their levels of up-regulation by IL-4 significantly correlated with cytoprotection. MiR-21 has been reported to be leukemogenic, associated to bad prognosis in CLL, and the miRNA more frequently overexpressed in human cancer. Up-regulation by IL-4 of miR-21 and the miRNAs hosted in the CLCN5 locus may contribute to evasion of apoptosis of CLL cells. These findings indicate that the IL-4 pathway and the miRNAs induced by IL-4 are promising targets for the development of novel therapies in CLL.     

Biaryl modification of the 5'-terminus of one strand of a microRNA duplex induces strand specificity

MicroRNAs (miRNAs) are single-stranded non-coding RNAs composed of 20-23 nucleotides. They are initially transcribed in the nucleus as pri-miRNAs. After processing, one strand from the miRNA duplex (miR-5p/miR-3p duplex) is loaded onto the RNA-induced silencing complex (RISC) to produce a functional, mature miRNA that inhibits the expression of multiple target genes. In the case of some miRNAs, both strands can be equally incorporated into the RISC as single strands, and both strands can function as mature miRNAs. Thus, a technique for selective expression of miR-5p and miR-3p strands is required to identify distinct targets of miRNAs. In this Letter, we report the synthesis and properties of miRNA duplexes carrying biaryl units at the 5'-terminus of one strand. We found that incorporation of biaryl units at the 5'-terminus of one strand of miRNA duplexes induced strand specificity in these duplexes. Further, we succeeded in identifying endogenous mRNA targets for each strand of the duplex by using the biaryl-modified miRNA duplexes.     

Expression and localization of microRNAs in perinatal rat pancreas: role of miR-21 in regulation of cholesterol metabolism

Objective

To investigate the expression of pancreatic microRNAs (miRNAs) during the period of perinatal beta-cell expansion and maturation in rats, determine the localization of these miRNAs and perform a pathway analysis with predicted target mRNAs expressed in perinatal pancreas.

Research Design and Methods

RNA was extracted from whole pancreas at embryonic day 20 (E20), on the day of birth (P0) and two days after birth (P2) and hybridized to miRNA microarrays. Differentially expressed miRNAs were verified by northern blotting and their pancreatic localization determined by in situ hybridization. Pathway analysis was done using regulated sets of mRNAs predicted as targets of the miRNAs. Possible target genes were tested using reporter-gene analysis in INS-1E cells.

Results

Nine miRNAs were differentially expressed perinatally, seven were confirmed to be regulated at the level of the mature miRNA. The localization studies showed endocrine localization of six of these miRNAs (miR-21, -23a, -29a, -125b-5p, -376b-3p and -451), and all were expressed in exocrine cells at one time point at least. Pathways involving metabolic processes, terpenoid and sterol metabolism were selectively affected by concomitant regulation by miRNAs and mRNAs, and Srebf1 was validated as a target of miR-21.

Conclusions

The findings suggest that miRNAs are involved in the functional maturation of pancreatic exocrine and endocrine tissue following birth. Pathway analysis of target genes identify changes in sterol metabolism around birth as being selectively affected by differential miRNA expression during this period.     

The miR-15/107 Group of MicroRNA Genes: Evolutionary Biology, Cellular Functions, and Roles in Human Diseases

The miR-15/107 group of microRNA (miRNA) gene is increasingly appreciated to serve key functions in humans. These miRNAs regulate gene expression involved in cell division, metabolism, stress response, and angiogenesis in vertebrate species. The miR-15/107 group has also been implicated in human cancers, cardiovascular disease and neurodegenerative disease, including Alzheimer's disease. Here we provide an overview of the following: (1) the evolution of miR-15/107 group member genes; (2) the expression levels of miRNAs in mammalian tissues; (3) evidence for overlapping gene-regulatory functions by different miRNAs; (4) the normal biochemical pathways regulated by miR-15/107 group miRNAs; and (5) the roles played by these miRNAs in human diseases. Membership in this group is defined based on sequence similarity near the mature miRNAs' 5′ end: all include the sequence AGCAGC. Phylogeny of this group of miRNAs is incomplete; thus, a definitive taxonomic classification (e.g., designation as a “superfamily”) is currently not possible. While all vertebrates studied to date express miR-15a, miR-15b, miR-16, miR-103, and miR-107, mammals alone are known to express miR-195, miR-424, miR-497, miR-503, and miR-646. Multiple different miRNAs in the miR-15/107 group are expressed at moderate to high levels in human tissues. We present data on the expression of all known miR-15/107 group members in human cerebral cortical gray matter and white matter using new miRNA profiling microarrays. There is extensive overlap in the mRNAs targeted by miR-15/107 group members. We show new data from cultured H4 cancer cells that demonstrate similarities in mRNAs targeted by miR-16 and miR-103 and also support the importance of the mature miRNAs' 5′ seed region in mRNA target recognition. In conclusion, the miR-15/107 group of miRNA genes is a fascinating topic of study for evolutionary biologists, miRNA biochemists, and clinically oriented translational researchers alike.     

Genome-Wide Analyses of Radioresistance-Associated miRNA Expression Profile in Nasopharyngeal Carcinoma Using Next Generation Deep Sequencing

Background

Rapidly growing evidence suggests that microRNAs (miRNAs) are involved in a wide range of cancer malignant behaviours including radioresistance. Therefore, the present study was designed to investigate miRNA expression patterns associated with radioresistance in NPC.

Methods

The differential expression profiles of miRNAs and mRNAs associated with NPC radioresistance were constructed. The predicted target mRNAs of miRNAs and their enriched signaling pathways were analyzed via biological informatical algorithms. Finally, partial miRNAs and pathways-correlated target mRNAs were validated in two NPC radioreisitant cell models.

Results

50 known and 9 novel miRNAs with significant difference were identified, and their target mRNAs were narrowed down to 53 nasopharyngeal-/NPC-specific mRNAs. Subsequent KEGG analyses demonstrated that the 53 mRNAs were enriched in 37 signaling pathways. Further qRT-PCR assays confirmed 3 down-regulated miRNAs (miR-324-3p, miR-93-3p and miR-4501), 3 up-regulated miRNAs (miR-371a-5p, miR-34c-5p and miR-1323) and 2 novel miRNAs. Additionally, corresponding alterations of pathways-correlated target mRNAs were observed including 5 up-regulated mRNAs (ICAM1, WNT2B, MYC, HLA-F and TGF-β1) and 3 down-regulated mRNAs (CDH1, PTENP1 and HSP90AA1).

Conclusions

Our study provides an overview of miRNA expression profile and the interactions between miRNA and their target mRNAs, which will deepen our understanding of the important roles of miRNAs in NPC radioresistance.     

Aberrant expression of microRNA induced by high-fructose diet: implications in the pathogenesis of hyperlipidemia and hepatic insulin resistance

Fructose is a highly lipogenic sugar that can alter energy metabolism and trigger metabolic disorders. In the current study, microRNAs (miRNAs) altered by a high-fructose diet were comprehensively explored to elucidate their significance in the pathogenesis of chronic metabolic disorders. miRNA expression profiling using small noncoding RNA sequencing revealed that 19 miRNAs were significantly upregulated and 26 were downregulated in the livers of high-fructose-fed mice compared to chow-fed mice. Computational prediction and functional analysis identified 10 miRNAs, miR-19b-3p, miR-101a-3p, miR-30a-5p, miR-223-3p, miR-378a-3p, miR-33-5p, miR-145a-3p, miR-128-3p, miR-125b-5p and miR-582-3p, assembled as a regulatory network to potentially target key genes in lipid and lipoprotein metabolism and insulin signaling at multiple levels. qRT-PCR analysis of their potential target genes [IRS-1, FOXO1, SREBP-1c/2, ChREBP, insulin-induced gene-2 (Insig-2), microsomal triglyceride transfer protein (MTTP) and apolipoprotein B (apoB)] demonstrated that fructose-induced alterations of miRNAs were also reflected in mRNA expression profiles of their target genes. Moreover, the miRNA profile induced by high-fructose diet differed from that induced by high-fat diet, indicating that miRNAs mediate distinct pathogenic mechanisms in dietary-induced metabolic disorders. This study presents a comprehensive analysis of a new set of hepatic miRNAs, which were altered by high-fructose diet and provides novel insights into the interaction between miRNAs and their target genes in the development of metabolic syndrome.     

Deregulated microRNAs in myotonic dystrophy type 2

Myotonic Dystrophy Type-2 (DM2) is an autosomal dominant disease caused by the expansion of a CCTG tetraplet repeat. It is a multisystemic disorder, affecting skeletal muscles, the heart, the eye, the central nervous system and the endocrine system. Since microRNA (miRNA) expression is disrupted in Myotonic Dystrophy Type-1 and many other myopathies, miRNAs deregulation was studied in skeletal muscle biopsies of 13 DM2 patients and 13 controls. Eleven miRNAs were deregulated: 9 displayed higher levels compared to controls (miR-34a-5p, miR-34b-3p, miR-34c-5p, miR-146b-5p, miR-208a, miR-221-3p and miR-381), while 4 were decreased (miR-125b-5p, miR-193a-3p, miR-193b-3p and miR-378a-3p). To explore the relevance of DM2 miRNA deregulation, the predicted interactions between miRNA and mRNA were investigated. Global gene expression was analyzed in DM2 and controls and bioinformatic analysis identified more than 1,000 miRNA/mRNA interactions. Pathway and function analysis highlighted the involvement of the miRNA-deregulated mRNAs in multiple aspects of DM2 pathophysiology. In conclusion, the observed miRNA dysregulations may contribute to DM2 pathogenetic mechanisms.