MicroRNAs Regulate Pituitary Development,and MicroRNA 26b Specifically Targets Lymphoid Enhancer Factor 1 (Lef-1), Which Modulates Pituitary Transcription Factor 1 (Pit-1) Expression

To understand the role of microRNAs (miRNAs) in pituitary development, a group of pituitary-specific miRNAs were identified, and Dicer1 was then conditionally knocked out using the Pitx2-Cre mouse, resulting in the loss of mature miRNAs in the anterior pituitary. The Pitx2-Cre/Dicer1 mutant mice demonstrate growth retardation, and the pituitaries are hypoplastic with an abnormal branching of the anterior lobe, revealing a role for microRNAs in pituitary development. Growth hormone, prolactin, and thyroid-stimulating hormone β-subunit expression were decreased in the Dicer1 mutant mouse, whereas proopiomelanocortin and luteinizing hormone β-subunit expression were normal in the mutant pituitary. Further analyses revealed decreased Pit-1 and increased Lef-1 expression in the mutant mouse pituitary, consistent with the repression of the Pit-1 promoter by Lef-1. Lef-1 directly targets and represses the Pit-1 promoter. miRNA-26b (miR-26b) was identified as targeting Lef-1 expression, and miR-26b represses Lef-1 in pituitary and non-pituitary cell lines. Furthermore, miR-26b up-regulates Pit-1 and growth hormone expression by attenuating Lef-1 expression in GH3 cells. This study demonstrates that microRNAs are critical for anterior pituitary development and that miR-26b regulates Pit-1 expression by inhibiting Lef-1 expression and may promote Pit-1 lineage differentiation during pituitary development.     

MicroRNAs as a therapeutic target for cardiovascular diseases

MicroRNAs (miRNAs) are tiny, endogenous, conserved, non-coding RNAs that negatively modulate gene expression by either promoting the degradation of mRNA or down-regulating the protein production by translational repression. They maintain optimal dose of cellular proteins and thus play a crucial role in the regulation of biological functions. Recent discovery of miRNAs in the heart and their differential expressions in pathological conditions provide glimpses of undiscovered regulatory mechanisms underlying cardiovascular diseases. Nearly 50 miRNAs are overexpressed in mouse heart. The implication of several miRNAs in cardiovascular diseases has been well documented such as miRNA-1 in arrhythmia, miRNA-29 in cardiac fibrosis, miRNA-126 in angiogenesis and miRNA-133 in cardiac hypertrophy. Aberrant expression of Dicer (an enzyme required for maturation of all miRNAs) during heart failure indicates its direct involvement in the regulation of cardiac diseases. MiRNAs and Dicer provide a particular layer of network of precise gene regulation in heart and vascular tissues in a spatiotemporal manner suggesting their implications as a powerful intervention tool for therapy. The combined strategy of manipulating miRNAs in stem cells for their target directed differentiation and optimizing the mode of delivery of miRNAs to the desired cells would determine the future potential of miRNAs to treat a disease. This review embodies the recent progress made in microRNomics of cardiovascular diseases and the future of miRNAs as a potential therapeutic target - the putative challenges and the approaches to deal with it.     

Common micro RNAs (miRNAs) target complement factor H (CFH) regulation in Alzheimer’s disease (AD) and in age-related macular degeneration (AMD)

Alzheimer’s disease (AD) and age-related macular degeneration (AMD) are complex and progressive inflammatory degenerations of the human neocortex and retina. Recent molecular, genetic and epigenetic evidence indicate that at least 4 micro RNAs (miRNAs) - including the NF-кB-regulated miRNA-9, miRNA-125b, miRNA-146a and miRNA-155 - are progressively up-regulated in both AD and AMD. This quartet of up-regulated miRNAs in turn down-regulate a small brain- and retinal-cell-relevant family of target mRNAs, including that encoding complement factor H (CFH), a major negative regulator of the innate immune and inflammatory response. Together miRNA-146a and miRNA-155 recognize an overlapping miRNA regulatory control (MiRC) region in the CFH 3’-untranslated region (3’- UTR; 5’-TTTAGTATTAA-3’) to which either of these miRNAs may interact. Progressive, pathogenic increases in specific miRNA binding to the entire 232 nucleotide CFH 3’-UTR appears to be a major regulator of CFH expression down-regulation, and the inflammatory pathology that characterizes both AMD and AD. The data presented in this report provides evidence that up-regulation of brain- and retinal- abundant miRNAs, including miRNA-9, miRNA-125b, miRNA-146a and miRNA-155, are common to the pathogenetic mechanism of CFH deficiency that drives inflammatory neurodegeneration, and for the first time indicates multiple, independent miRNA-mediated regulation of the CFH mRNA 3’-UTR.     

let-7 microRNAs induce tamoxifen sensitivity by downregulation of estrogen receptor α signaling in breast cancer

MicroRNAs (miRNAs) play an important regulatory role in breast tumorigenesis. Previously, we found that let-7 miRNAs were downregulated significantly in formalin-fixed paraffin-embedded (FFPE) breast cancer tissues. In this study, we further found that endogenous levels of let-7b and let-7i miRNAs are inversely correlated with levels of estrogen receptor (ER)-a36, a new variant of ER-α66, in the FFPE tissue set. Bioinformatic analysis suggested that ER-α36 may be another target of let-7 miRNAs. To test this hypothesis, cotransfection of let-7 mimics or inhibitors together with full-length or a fragment of ER-α36 3'UTR luciferase construct was performed, and we found that let-7b and let-7i mimics suppressed the activity of reporter gene significantly, which was enhanced remarkably by let-7b and let-7i inhibitors. Both mRNA and protein expression of ER-α36 were inhibited by let-7 mimics and enhanced by let-7 inhibitors. Furthermore, ER-α36 mediated nongenomic MAPK and Akt pathways were weakened by let-7b and let-7i mimics in triple negative breast cancer cell line MDA-MB-231. The reverse correlation between let-7 miRNAs and ER-α36 also exists in Tamoxifen (Tam)-resistant MCF7 cell line. Transfection of let-7 mimics to Tam-resistant MCF7 cells downregulated ER-α36 expression and enhanced the sensitivity of MCF7 cells to Tam in estrogen-free medium, which could be restored by overexpression of ER-α36 constructs without 3'UTR. Our results suggested a novel regulatory mechanism of let-7 miRNAs on ER-α36 mediated nongenomic estrogen signal pathways and Tam resistance.     

In vivo structure-function analysis of human Dicer reveals directional processing of precursor miRNAs

Dicer is an RNase III family endoribonuclease and haploinsufficient tumor suppressor that processes mature miRNAs from the 5' (5p) or 3' (3p) arm of hairpin precursors. In murine Dicer knockout fibroblasts, we expressed human Dicer with point mutations in the RNase III, helicase, and PAZ domains and characterized miRNA expression by Northern blot and massively parallel sequencing of small RNAs. We report that inactivation of the RNase IIIA domain results in complete loss of 3p-derived mature miRNAs, but only partial reduction in 5p-derived mature miRNAs. Conversely, inactivation of the RNase IIIB domain by mutation of D1709, a residue mutated in a subset of nonepithelial ovarian cancers, results in complete loss of 5p-derived mature miRNAs, including the tumor-suppressive let-7 family, but only partial reduction in 3p-derived mature miRNAs. Mutation of the PAZ domain results in global reduction of miRNA processing, while mutation of the Walker A motif in the helicase domain of Dicer does not alter miRNA processing. These results provide insight into the biochemical activity of human Dicer in vivo and, furthermore, suggest that mutation of the clinically relevant residue D1709 within the RNase IIIB results in a uniquely miRNA-haploinsufficient state in which the let-7 family of tumor suppressor miRNAs is lost while a complement of 3p-derived miRNAs remains expressed.     

MicroRNA-21 Regulates PI3K/Akt/mTOR Signaling by Targeting TGFβI during Skeletal Muscle Development in Pigs

MicroRNAs (miRNAs), which are short (22–24 base pairs), non-coding RNAs, play critical roles in myogenesis. Using Solexa deep sequencing, we detected the expression levels of 229 and 209 miRNAs in swine skeletal muscle at 90 days post-coitus (E90) and 100 days postnatal (D100), respectively. A total of 138 miRNAs were up-regulated on E90, and 31 were up-regulated on D100. Of these, 9 miRNAs were selected for the validation of the small RNA libraries by quantitative RT-PCR (RT-qPCR). We found that miRNA-21 was down-regulated by 17-fold on D100 (P<0.001). Bioinformatics analysis suggested that the transforming growth factor beta-induced (TGFβI) gene was a potential target of miRNA-21. Both dual luciferase reporter assays and western blotting demonstrated that the TGFβI gene was regulated by miRNA-21. Co-expression analysis revealed that the mRNA expression levels of miRNA-21 and TGFβI were negatively correlated (r = -0.421, P = 0.026) in skeletal muscle during the 28 developmental stages. Our results revealed that more miRNAs are expressed in prenatal than in postnatal skeletal muscle. The miRNA-21 is a novel myogenic miRNA that is involved in skeletal muscle development and regulates PI3K/Akt/mTOR signaling by targeting the TGFβI gene.     

Short loop-targeting oligoribonucleotides antagonize Lin28 and enable pre-let-7 processing and suppression of cell growth in let-7-deficient cancer cells

MicroRNAs (miRNAs) originate from stem-loop-containing precursors (pre-miRNAs, pri-miRNAs) and mature by means of the Drosha and Dicer endonucleases and their associated factors. The let-7 miRNAs have prominent roles in developmental differentiation and in regulating cell proliferation. In cancer, the tumor suppressor function of let-7 is abrogated by overexpression of Lin28, one of several RNA-binding proteins that regulate let-7 biogenesis by interacting with conserved motifs in let-7 precursors close to the Dicer cleavage site. Using in vitro assays, we have identified a binding site for short modified oligoribonucleotides (‘looptomirs’) overlapping that of Lin28 in pre-let-7a-2. These looptomirs selectively antagonize the docking of Lin28, but still permit processing of pre-let-7a-2 by Dicer. Looptomirs restored synthesis of mature let-7 and inhibited growth and clonogenic potential in Lin28 overexpressing hepatocarcinoma cells, thereby demonstrating a promising new means to rescue defective miRNA biogenesis in Lin28-dependent cancers.     

Atherosclerosis-associated endothelial cell apoptosis by miRNA let7-b-mediated downregulation of HAS-2

MicroRNAs (miRNAs) play essential roles in the regulation and pathophysiology of various types of human diseases including atherosclerosis. Increasing numbers of miRNAs have been identified to be important regulators in the progression of atherosclerosis by regulating gene expression. However, functional miRNAs and the underlying mechanisms involved in atherosclerosis need fully elucidation. In the present study, the function of miRNA let-7b was investigated in human aortic endothelial cells (HAECs). The results showed that downregulation of let-7b in the high-fat diet mice and HAECs was inversely correlated with the expression level of HAS-2. upregulation of let-7b significantly reduced apoptosis of HAECs. The results also revealed that HAS-2 was a target gene of let-7b and HAS-2 reduction reversed the antiapoptotic effect of let-7b through regulation of the P13K/Akt pathway. These results together suggest the potential of regulating the let-7b expression and endothelial apoptosis against development and progression of atherosclerosis.     

Suppression of Induced microRNA-15b Prevents Rapid Loss of Cardiac Function in a Dicer Depleted Model of Cardiac Dysfunction

Background

Dicer endonuclease, critical for maturation of miRNAs, is depleted in certain forms of cardiomyopathy which results in differential expression of certain microRNAs. We sought to elucidate the mechanisms underlying the rapid loss of cardiac function following cardiac-specific Dicer depletion in adult mice.

Results

Conditional Dicer deletion in the adult murine myocardium demonstrated compromised heart function, mitochondrial dysfunction and oxidant stress. Elevated miR-15b was observed as an early response to Dicer depletion and was found to silence Pim-1 kinase, a protein responsible for maintaining mitochondrial integrity and function. Anti-miRNA based suppression of induced miRNA-15b rescued the function of Dicer-depleted adult heart and attenuated hypertrophy.

Conclusions

Anti-miRNA based suppression of inducible miRNA-15b can prevent rapid loss of cardiac function in a Dicer-depleted adult heart and can be a key approach worthy of therapeutic consideration.     

MiRNA在特发性肺纤维化中的作用

近年来研究发现微RNA（microRNA,miRNA）与机体人部分生理、病理过程均有密切关系,如：组织的发育和分化、组织再生、病毒防御以及细胞增殖与凋亡等。miRNA在特发性肺纤维化（IPF）中的作用也日渐为研究者所重视,在IPF中有些miRNA上调（如miR-155、miR-21）,有些下调（如let-7、miR-29、miR-200）。这一发现为寻找IPF治疗方法提供了一个新的突破口。本文对近年来miRNA在IPF中作用的研究进展进行了综述,并对miRNA-21、let-7d、miRNA-155、miRNA-29以及miRNA-200在肺纤维化中的作用分别进行了阐述,为研究miRNA征IPF中的作用及机制提供一定参考。     

Expression Profiling and Structural Characterization of MicroRNAs in Adipose Tissues of Hibernating Ground Squirrels

Micro RNAs（mi RNAs） are small non-coding RNAs that are important in regulating metabolic stress. In this study, we determined the expression and structural characteristics of 20 mi RNAs in brown（BAT） and white adipose tissue（WAT） during torpor in thirteen-lined ground squirrels. Using a modified stem-loop technique, we found that during torpor, expression of six mi RNAs including let-7a, let-7b, mi R-107, mi R-150, mi R-222 and mi R-31 was significantly downregulated in WAT（P 〈 0.05）, which was 16%–54% of euthermic non-torpid control squirrels,whereas expression of three mi RNAs including mi R-143, mi R-200 a and mi R-519 d was found to be upregulated by 1.32–2.34-fold. Similarly, expression of more mi RNAs was downregulated in BAT during torpor. We detected reduced expression of 6 mi RNAs including mi R-103 a, mi R-107, mi R-125 b, mi R-21, mi R-221 and mi R-31（48%–70% of control）, while only expression of mi R-138 was significantly upregulated（2.91 ± 0.8-fold of the control, P 〈 0.05）. Interestingly,mi RNAs found to be downregulated in WAT during torpor were similar to those dysregulated in obese humans for increased adipogenesis, whereas mi RNAs with altered expression in BAT during torpor were linked to mitochondrial b-oxidation. mi RPath target prediction analysis showed that mi RNAs downregulated in both WAT and BAT were associated with the regulation of mitogen-activated protein kinase（MAPK） signaling, while the mi RNAs upregulated in WAT were linked to transforming growth factor b（TGFb） signaling. Compared to mouse sequences, no unique nucleotide substitutions within the stem-loop region were discovered for the associated pre-mi RNAs for the mi RNAs used in this study, suggesting no structure-influenced changes in pre-mi RNA processing efficiency in the squirrel. As well, the expression of mi RNA processingenzyme Dicer remained unchanged in both tissues during torpor. Overall, our findings suggest that changes of mi RNA expression in adipose tissues may be linked     

MicroRNA-5p and -3p co-expression and cross-targeting in colon cancer cells

Background

Two mature miRNA species may be generated from the 5’ and 3’ arms of a pre-miRNA precursor. In most cases, only one species remains while the complementary species is degraded. However, co-existence of miRNA-5p and -3p species is increasingly being reported. In this work, we aimed to systematically investigate co-expression of miRNA-5p/3p in colon cancer cells in a genome-wide analysis, and to examine cross-targeting of the dysregulated miRNAs and 5p/3p species.

Results

Four colon cancer cell lines were examined relative to two normal colon tissues. Of the 1,190 miRNAs analyzed, 92 and 36 were found to be up- or down-regulated, respectively, in cancer cells. Nineteen co-expressed miRNA-5p/3p pairs were further identified suggesting frequent 5p/3p co-accumulation in colon cancer cells. Of these, 14 pairs were co-up-regulated and 3 pairs were co-down-regulated indicating concerted 5p/3p dysregulation. Nine dysregulated miRNA pairs fell into three miRNA gene families, namely let-7, mir-8/200 and mir-17, which showed frequent cross-targeting in the metastasis process. Focusing on the let-7d-5p/3p pair, the respectively targeted IGF1R and KRAS were shown to be in a reverse relationship with expression of the respective miRNA, which was confirmed in transient transfection assays using let-7d mimic or inhibitor. Targeting of KRAS by let-7d was previous reported; targeting of IGF1R by let-7d-5p was confirmed in luciferase assays in this study. The findings of let-7d-5p/3p and multiple other miRNAs targeting IGF1R, KRAS and other metastasis-related factors suggest that 5p/3p miRNAs contribute to cross-targeting of multiple cancer-associated factors and processes possibly to evade functional abolishment when any one of the crucial factors are inactivated.

Conclusions

miRNA-5p/3p species are frequently co-expressed and are coordinately regulated in colon cancer cells. In cancer cells, multiple cross-targeting by the miRNAs, including the co-existing 5p/3p species, frequently occurs in an apparent safe-proof scheme of miRNA regulation of important tumorigenesis processes. Further systematic analysis of co-existing miRNA-5p/3p pairs in clinical tissues is important in elucidating 5p/3p contributions to cancer pathogenesis.

Electronic supplementary material

The online version of this article (doi:10.1186/s12929-014-0095-x) contains supplementary material, which is available to authorized users.     

Dicer insufficiency and microRNA-155 overexpression in lupus regulatory T cells: an apparent paradox in the setting of an inflammatory milieu

Systemic lupus erythematosus is a chronic autoimmune disease characterized by loss of tolerance to self-Ags and activation of autoreactive T cells. Regulatory T (Treg) cells play a critical role in controlling the activation of autoreactive T cells. In this study, we investigated mechanisms of potential Treg cell defects in systemic lupus erythematosus using MRL-Fas(lpr/lpr) (MRL/lpr) and MRL-Fas(+/+) mouse models. We found a significant increase in CD4(+)CD25(+)Foxp3(+) Treg cells, albeit with an altered phenotype (CD62L(-)CD69(+)) and with a reduced suppressive capacity, in the lymphoid organs of MRL strains compared with non-autoimmune C3H/HeOuj mice. A search for mechanisms underlying the altered Treg cell phenotype in MRL/lpr mice led us to find a profound reduction in Dicer expression and an altered microRNA (miRNA, miR) profile in MRL/lpr Treg cells. Despite having a reduced level of Dicer, MRL/lpr Treg cells exhibited a significant overexpression of several miRNAs, including let-7a, let-7f, miR-16, miR-23a, miR-23b, miR-27a, and miR-155. Using computational approaches, we identified one of the upregulated miRNAs, miR-155, that can target CD62L and may thus confer the altered Treg cell phenotype in MRL/lpr mice. In fact, the induced overexpression of miR-155 in otherwise normal (C3H/HeOuj) Treg cells reduced their CD62L expression, which mimics the altered Treg cell phenotype in MRL/lpr mice. These data suggest a role of Dicer and miR-155 in regulating Treg cell phenotype. Furthermore, simultaneous appearance of Dicer insufficiency and miR-155 overexpression in diseased mice suggests a Dicer-independent alternative mechanism of miRNA regulation under inflammatory conditions.