127,037 research outputs found
MicroRNAs in cardiac arrhythmia: DNA sequence variation of MiR-1 and MiR-133A in long QT syndrome.
No full textLong QT syndrome (LQTS) is a genetic cardiac condition associated with prolonged ventricular repolarization, primarily a result of perturbations in cardiac ion channels, which predisposes individuals to life-threatening arrhythmias. Using DNA screening and sequencing methods, over 700 different LQTS-causing mutations have been identified in 13 genes worldwide. Despite this, the genetic cause of 30-50% of LQTS is presently unknown. MicroRNAs (miRNAs) are small (∼ 22 nucleotides) noncoding RNAs which post-transcriptionally regulate gene expression by binding complementary sequences within messenger RNAs (mRNAs). The human genome encodes over 1800 miRNAs, which target about 60% of human genes. Consequently, miRNAs are likely to regulate many complex processes in the body, indeed aberrant expression of various miRNA species has been implicated in numerous disease states, including cardiovascular diseases. MiR-1 and MiR-133A are the most abundant miRNAs in the heart and have both been reported to regulate cardiac ion channels. We hypothesized that, as a consequence of their role in regulating cardiac ion channels, genetic variation in the genes which encode MiR-1 and MiR-133A might explain some cases of LQTS. Four miRNA genes (miR-1-1, miR-1-2, miR-133a-1 and miR-133a-2), which encode MiR-1 and MiR-133A, were sequenced in 125 LQTS probands. No genetic variants were identified in miR-1-1 or miR-133a-1; but in miR-1-2 we identified a single substitution (n.100A> G) and in miR-133a-2 we identified two substitutions (n.-19G> A and n.98C> T). None of the variants affect the mature miRNA products. Our findings indicate that sequence variants of miR-1-1, miR-1-2, miR-133a-1 and miR-133a-2 are not a cause of LQTS in this cohort
miR-132/212 knockout mice reveal roles for these miRNAs in regulating cortical synaptic transmission and plasticity
No full textmiR-132 and miR-212 are two closely related miRNAs encoded in the same intron of a small non-coding gene, which have been suggested to play roles in both immune and neuronal function. We describe here the generation and initial characterisation of a miR-132/212 double knockout mouse. These mice were viable and fertile with no overt adverse phenotype. Analysis of innate immune responses, including TLR-induced cytokine production and IFNβ induction in response to viral infection of primary fibroblasts did not reveal any phenotype in the knockouts. In contrast, the loss of miR-132 and miR-212, while not overtly affecting neuronal morphology, did affect synaptic function. In both hippocampal and neocortical slices miR-132/212 knockout reduced basal synaptic transmission, without affecting paired-pulse facilitation. Hippocampal long-term potentiation (LTP) induced by tetanic stimulation was not affected by miR-132/212 deletion, whilst theta burst LTP was enhanced. In contrast, neocortical theta burst-induced LTP was inhibited by loss of miR-132/212. Together these results indicate that miR-132 and/or miR-212 play a significant role in synaptic function, possibly by regulating the number of postsynaptic AMPA receptors under basal conditions and during activity-dependent synaptic plasticity
NEW INSIGHTS OF MIR-145 FUNCTION AND REGULATION IN HUMAN BREAST CANCER.
No full textmiR-145 is down-regulated in the majority of human cancers, including breast cancer (BC).
However, its role remains largely unknown. Here, I provide evidence for miR-145 induced
anti-proliferative and pro-apoptotic effect in several BC cell lines, which was not detected in
BC cells lacking a functional TP53 gene and exhibiting an estrogen receptor alfa (ESR1)
negative status. I found that miR-145 anti-proliferative effects were dependent upon TP53
activation and that activation of TP53 could in turn stimulates miR-145 expression. I also
found that miR-145 could repress the expression of ESR1 protein by direct interaction with
two sites within its gene coding sequence. My findings support the existence of a positive
regulatory loop where miR-145 directly targets ESR1 and indirectly activates TP53, which in
turn sustains miR-145 expression and reinforces miR-145 overall effects on proliferation and
apoptosis
MiR-30b and miR-30c are downregulated after <i>B</i>. <i>pseudomallei</i> infection.
No full text(A) Total RNA from RAW264.7 cells were infected with B. pseudomallei for 4 h to perform microarray assay. Hierarchical clustering analysis was performed to show downregulated miRNAs by B. pseudomallei infection. (B) The region of the mouse Rab32 mRNA 3′UTR predicted to be targeted by miR-30b, miR-30c, miR-30d, and miR-30e, respectively (TargetScan 6.2). (C and D) Confirmation of microarray results by qRT-PCR. qRT-PCR analysis of the expression levels of miR-30b, miR-30c, miR-30d, and miR-30e in RAW264.7 cells infected with B. pseudomallei (MOI = 10) for 0, 1, 2, 4, 6, and 8 h, or at MOI = 0, 1, 10, 20, 50, and 100 for 4 h. (E) Expression of miR-30b and miR-30c in RAW264.7 cells infected with B. thailandensis, S. typhimurium and E. coli (MOI = 10). *PP<0.01. Experiments performed in triplicates showed consistent results.</p
The miR-590 C57T SNP reduces levels of miR-590-5p and miR-590-3p, without affecting the levels of pri-miR-590 and pre-miR-590.
No full text(A) Quantification of pri-miR-590 by qRT-PCR normalized by GAPDH. Mean ± SD (n = 3). HEK293T cells were transfected with the pri-miR-590-WT or pri-miR-590-SNP plasmids. The empty plasmid was used as negative control. (B-F) Northern blot images for pre-miRNA, miRNA, and U6 RNA using total RNA prepared from HEK293T cells transfected with the pri-miR-590-WT or pri-miR-590-SNP plasmids. The empty plasmid was used as negative control. Four biological replicates were analyzed for each transfection plasmid. Northern probes used are perfectly complementary to miR-590-5p (A), miR-590-3p-WT (B), miR-590-3p-SNP (C), miR-16-5p (D), and U6 RNA (E). The miR-590-3p-WT probe weakly cross-hybridized to miR-590-3p-SNP, and vice versa. (G) The abundance of pre-miR-590, miR-590-5p and miR-590-3p-(WT/SNP) relative to the mean value of miR-590-5p in the WT miR-590 gene plasmid transfection conditions. (H) The abundance of miR-16-5p normalized to the mean value of the pri-miR-590-WT plasmid transfection conditions. Mean ± SD (n = 4).</p
Role and regulation of miR-483 in cancer
No full textThe hsa-mir-483 locus is located at chromosome 11p15.5 within intron 2 of the IGF2 locus. Because of its location, de-regulated in Wilms’ tumor and other neoplasia, I hypothesized that this microRNA had a potential role in tumors. By analyzing 19 Wilms’ tumors, I proved that miR-483-3p is indeed over-expressed in 100% of the cases and a co-regulation with the over-expression of IGF2 was found.
However, several other types of common adult cancers exhibit high or even extremely high levels of miR-483-3p expression without IGF2 over-expression. Indeed, independently from IGF2, the expression of the miR-483-3p could also be induced by the oncoprotein β-catenin through a novel interaction with the basic Helix-Loop-Helix protein upstream stimulatory transcription factor 1 (USF1).
I also show that β-catenin itself is a target of miR-483-3p, triggering a negative regulative loop that becomes ineffective in cells harbouring activating mutations of β-catenin pathway.
The potential oncogenic role of miR-483-3p was supported by the findings that its ectopic expression protects cells from apoptosis and, conversely, its inhibition increase the level of apoptosis. To understand the mechanisms of its action, I investigated potential gene targets. Among these, an important pro-apoptotic protein, Puma, were inhibited by miR-483-3p. My results indicate that miR-483-3p functions as an anti-apoptotic oncogene, coordinately over-expressed with IGF2 in Wilms’ tumors or induced by β-catenin activation in other tumor types
Functional microRNA high throughput screening reveals miR-9 as a central regulator of liver oncogenesis by affecting the PPARA-CDH1 pathway
No full textBackground: Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related deaths, reflecting the aggressiveness of this type of cancer and the absence of effective therapeutic regimens. MicroRNAs have been involved in the pathogenesis of different types of cancers, including liver cancer. Our aim was to identify microRNAs that have both functional and clinical relevance in HCC and examine their downstream signaling effectors. Methods: MicroRNA and gene expression levels were measured by quantitative real-time PCR in HCC tumors and controls. A TargetScan algorithm was used to identify miR-9 downstream direct targets. Results: A high-throughput screen of the human microRNAome revealed 28 microRNAs as regulators of liver cancer cell invasiveness. MiR-9, miR-21 and miR-224 were the top inducers of HCC invasiveness and also their expression was increased in HCC relative to control liver tissues. Integration of the microRNA screen and expression data revealed miR-9 as the top microRNA, having both functional and clinical significance. MiR-9 levels correlated with HCC tumor stage and miR-9 overexpression induced SNU-449 and HepG2 cell growth, invasiveness and their ability to form colonies in soft agar. Bioinformatics and 3’UTR luciferase analyses identified E-cadherin (CDH1) and peroxisome proliferator-activated receptor alpha (PPARA) as direct downstream effectors of miR-9 activity. Inhibition of PPARA suppressed CDH1 mRNA levels, suggesting that miR-9 regulates CDH1 expression directly through binding in its 3’UTR and indirectly through PPARA. On the other hand, miR-9 inhibition of overexpression suppressed HCC tumorigenicity and invasiveness. PPARA and CDH1 mRNA levels were decreased in HCC relative to controls and were inversely correlated with miR-9 levels. Conclusions: Taken together, this study revealed the involvement of the miR-9/PPARA/CDH1 signaling pathway in HCC oncogenesis
MiR-30b and miR-30c affect phagosome maturation and modulate <i>B</i>. <i>pseudomallei</i> intracellular survival in macrophages.
No full text(A) BMDMs transfected with control, miR-30b mimic, and miR-30c mimic were incubated with 50 nM Lysotracker (red) for 1 h before infection with B. pseudomallei for 2 h and fixed, stained with an anti-CTSD (red) or anti-B. pseudomallei (green). Scale bar is 5 μm. (B and C) Percent of B. pseudomallei phagosomes that colocalized with Lysotracker and CTSD at 2 h post-infection as in A. Results represented are of three independent experiments. (D) BMDMs were transfected by control, miR-30b inhibitor or miR-30c inhibitor for 24 h, followed by B. pseudomallei infection for 2 h, and were labeled with Lysotracker (red) or stained with an anti-CTSD (red). Scale bar is 5 μm. (E and F) Quantification showing the percentage of association of the bacteria phagosomes with Lysotracker and CTSD as in D. (G) BMDMs were transfected with control, miR-30b mimic, miR-30c mimic, miR-30b inhibitor, and miR-30c inhibitor for 24 h and then infected with B. pseudomallei. The expression of CTSD was determined by Western blot analysis. (H) BMDMs were transfected with control, miR-30b mimic, and miR-30c mimic at 100 nM for 24 h, and were then infected with B. pseudomallei for different periods of time (0, 1, 2, 4, and 6 h). Intracellular bacterial counts were determined. (I) After BMDMs were transfected with control, miR-30b inhibitor or miR-30c inhibitor for 24 h, intracellular survival of B. pseudomallei was detected as in H. Data are representative of at least three independent experiments (*PP<0.01).</p
MiRNA-205 modulates cellular invasion and migration via regulating zinc finger E-box binding homeobox 2 expression in esophageal squamous cell carcinoma cells
No full textBackground: Esophageal squamous cell carcinoma (ESCC) is often diagnosed at later stages until they are incurable. MicroRNA (miR) is a small, non-coding RNA that negatively regulates gene expression mainly via translational repression. Accumulating evidence indicates that deregulation of miR is associated with human malignancies including ESCC. The aim of this study was to identify miR that could be specifically expressed and exert distinct biological actions in ESCC. Methods: Total RNA was extracted from ESCC cell lines, OE21 and TE10, and a non-malignant human esophageal squamous cell line, Het-1A, and subjected to microarray analysis. Expression levels of miR that showed significant differences between the 2 ESCC and Het-1A cells based on the comprehensive analysis were analyzed by the quantitative reverse transcriptase (RT)-PCR method. Then, functional analyses, including cellular proliferation, apoptosis and Matrigel invasion and the wound healing assay, for the specific miR were conducted. Using ESCC tumor samples and paired surrounding non-cancerous tissue obtained endoscopically, the association with histopathological differentiation was examined with quantitative RT-PCR. Results: Based on the miR microarray analysis, there were 14 miRs that showed significant differences (more than 2-fold) in expression between the 2 ESCC cells and non-malignant Het-1A. Among the significantly altered miRs, miR-205 expression levels were exclusively higher in 5 ESCC cell lines examined than any other types of malignant cell lines and Het-1A. Thus, miR-205 could be a specific miR in ESCC. Modulation of miR-205 expression by transfection with its precursor or anti-miR-205 inhibitor did not affect ESCC cell proliferation and apoptosis, but miR-205 was found to be involved in cell invasion and migration. Western blot revealed that knockdown of miR-205 expression in ESCC cells substantially enhanced expression of zinc finger E-box binding homeobox 2, accompanied by reduction of E-cadherin, a regulator of epithelial mesenchymal transition. The miR-205 expression levels were not associated with histological differentiation of human ESCC. Conclusions: These results imply that miR-205 is an ESCC-specific miR that exerts tumor-suppressive activities with EMT inhibition by targeting ZEB2.Kayoko Matsushima... Gregory J Goodall... et al
miR-218-5p directly targets HMGB2 and CMPK1 3'UTR.
No full text(A). Luciferase activity in HEK293T cells cotransfected with miR-218-5p mimic (miR-218-5p; 10 nM) or its control (NC of mimic; 10 nM) and the reporter constructs for 48 h. (B). Luciferase activity in HEK293T cells cotransfected with increasing amounts of miR-218-5p mimic (miR-218-5p; 5, 10, and 15 nM) or its control (NC of mimic), and the pGL-3-HMGB2 3'UTR reporter or pGL-3-CMPK1 3'UTR reporter for 48 h. (C). Western blotting of HMGB2 and CMPK1 expression in HUVECs transfected with increasing amounts of miR-218-5p mimic (10, 20 and 40 nM) or its control for 48 h. (D). Western blotting of HMGB2 and CMPK1 expression in HUVECs transfected with a miR-218-5p inhibitor for 48 h. (E). Putative binding site of miR-218-5p in the 3'UTR region of HMGB2 and mutagenesis of target site in miR-218-5p. (F). Putative binding site of miR-218-5p in the 3'UTR region of CMPK1 and mutagenesis of target site in miR-218-5p. (G). Luciferase activity in HEK293T cells cotransfected with miR-218-5p mimic (miR-218-5p; 10 nM), miR-218-5p mutant mimic (miR-218-5p mut 1; 10 nM) or a negative control (NC of mimic; 10 nM), and the HMGB2 3'UTR or CMPK1 3'UTR reporter construct for 48 h. (H). Western-blotting of HMGB2 and CMPK1 expression in HUVECs transfected with a negative control mimic (NC of mimic; 20 nM), miR-218-5p mimic (miR-218-5p mimic; 20 nM) or miR-218-5p mutant mimic (miR-218-5p mut 1; 20 nM) for 48 h, respectively. The quantified results represent mean ± SD. Results are from three independent experiments, each with quadruple technical replicates, were performed. * P P P ### P n.s, not significant.</p
- …
