Up-regulation Of miR-145 Research Articles

AMP-Activated Protein Kinase Regulates Endothelial Cell Angiotensin-Converting Enzyme Expression via p53 and the Post-Transcriptional Regulation of microRNA-143/145

High-angiotensin-converting enzyme (ACE)-levels are associated with cardiovascular disease, but little is known about the regulation of its expression. To assess the molecular mechanisms regulating endothelial ACE expression focusing on the role of the AMP-activated protein kinase (AMPK) and miR-143/145. Shear stress decreased ACE expression in cultured endothelial cells, an effect prevented by downregulating AMPKα2 but not AMPKα1. AMPKα2(-/-) mice expressed higher ACE levels than wild-type littermates resulting in impaired hindlimb vasodilatation to the ACE substrate, bradykinin. The latter response was also evident in animals lacking the AMPKα2 subunit only in endothelial cells. In cultured endothelial cells, miR-143/145 levels were increased by shear stress in an AMPKα2-dependent manner, and miR-143/145 overexpression decreased ACE expression. The effect of shear stress was unrelated to an increase in miR-143/145 promoter activity and transcription but could be attributed to post-transcriptional regulation of precursor-miR-143/145 by AMPKα2. The AMPK substrate, p53, can enhance the post-transcriptional processing of several microRNAs, including miR-143/145. We found that shear stress elicited the AMPKα2-dependent phosphorylation of p53 (on Ser15), and that p53 downregulation prevented the shear stress-induced decrease in ACE expression. Streptozotocin-induced diabetes mellitus in mice was studied as a pathophysiological model of altered AMPK activity. Diabetes mellitus increased tissue phosphorylation of the AMPK substrates, p53 and acetyl-coenzyme A carboxylase, changes that correlated with increased miR-143/145 levels and decreased ACE expression. AMPKα2 suppresses endothelial ACE expression via the phosphorylation of p53 and upregulation of miR-143/145. Post-transcriptional regulation of miR-143/145 may contribute to the vascular complications associated with diabetes mellitus.

Tumor suppressive microRNA-424 inhibits osteosarcoma cell migration and invasion via targeting fatty acid synthase

Numerous studies have recently suggested that miRNAs contribute to the development of various types of human cancer as well as to their invasive and metastatic capacities. The aim of this study was to investigate the functional significance of miR-424 and to identify its possible target genes in osteosarcoma (OS) cells. Previously, inhibition of fatty acid synthase (FASN) has been shown to suppress OS cell proliferation, invasion and migration. The prediction was made using the microRNA.org and TargetScan.human6.0.database. The results showed that FASN is a promising target gene of miR-424. FASN may be a direct target of miR-424 as shown by the luciferase reporter assays. Furthermore, miR-424 expression was increased in osteosarcoma cells by transfection with has-miR-424. FASN mRNA and protein expression levels were measured by RT-PCR and western blot analysis. Cell migration and invasion was measured using Transwell migration and Transwell invasion assays. Expression levels of FASN mRNA and protein were greatly decreased in U2OS cells transfected with has-miR-424. The migration and invasion of cells was significantly decreased by the upregulation of miR-424. These findings suggested that miR-424 plays a key role in inhibiting OS cell migration and invasion through targeting FASN.

Overexpression of microRNA-145 in atherosclerotic plaques from hypertensive patients

Background: MicroRNAs (miRNAs) are endogenous, non-coding, short, single-stranded RNAs and represent a new class of gene regulators. Recent evidence supports a role for miRNAs in cardiovascular pathophysiology and atherosclerosis development. We have previously demonstrated that miR-145 is widely expressed in human atherosclerotic lesions and its downregulation has been correlated with vascular smooth muscle cell dedifferentiation, a cardinal step in the development of atherosclerosis. However, no evidences are available at this time about modulation of miR-145 in the setting of hypertension. Thus, the aim of this study was to investigate the expression of miR-145 in complicated hypertension.Materials and methods: Atherosclerotic plaques were obtained from 22 patients undergoing carotid endarterectomy for high-grade internal carotid artery stenosis. Plaques were subdivided into hypertension (n = 15) and control (n = 7) groups according to the presence or absence of hypertension (as defined by blood pressure > 140/90 mmHg or current antihypertensive treatment). In study plaques, miR-145 values were evaluated using real-time PCR. The level of induction has been tested by using ΔΔ cycle threshold method.Results: We found that miR-145 was significantly more expressed in atherosclerotic plaques of hypertensive patients than in control plaques (1.201 ± 0.260 vs 0.483 ± 0.148 fold induction ± SE; p = 0.026). Moreover, a post-hoc analysis showed that treatment with angiotensin receptor blockers may be associated with the maximum increase in miR-145 levels, although these data did not show any statistical significance probably due to the limited sample size.Conclusions: To the best of our knowledge, this study is the first demonstration that hypertension may upregulate miR-145 expression in human atherosclerotic plaques. Future investigations will be necessary to establish the molecular readout of miR-145 upregulation in atherosclerotic lesions in hypertension.

MicroRNA miR-145 inhibits proliferation, invasiveness, and stem cell phenotype of an in vitro endometriosis model by targeting multiple cytoskeletal elements and pluripotency factors

To study the function of miR-145, known to be dysregulated in endometriosis, and to identify its target genes in an invitro endometriosis model. Experimental laboratory study. University medical centers. Primary endometrial stroma cells were derived from eutopic endometrium of three American Society for Reproductive Medicine stage III endometriosis patients and from ectopic lesions of four patients with deep infiltrating endometriosis. The human endometriotic cell line 12Z and primary eutopic and ectopic endometrial stroma cells were transiently transfected with miR-145 precursors or anti-miR-145 inhibitors and investigated for posttranscriptional regulation of predicted target genes and changes in cell behavior. Predicted target expression was measured by quantitative reverse transcription-polymerase chain reaction and Western blotting, and altered cell behavior was monitored by cell proliferation assays. The 12Z cells were additionally investigated by Matrigel invasion assays, cell cycle analysis, side population analysis, and aldehyde dehydrogenase activity assays. In all cells investigated, miR-145 overexpression inhibited cell proliferation and induced down-regulation of FASCIN-1, SOX2, and MSI2. In 12Z cells miR-145 upregulation increased Matrigel invasiveness and reduced side population and aldehyde dehydrogenase-1 activity. Additional down-regulated targets in 12Z cells included OCT4, KLF4, PODXL, JAM-A, and SERPINE1/PAI-1. ACTG2 and TAGLN were up-regulated upon pre-miR-145 transfection. JAM-A, FASCIN-1, and PAI-I down-regulation in 12Z cells were confirmed by Western blotting. miR-145 inhibits endometriotic cell proliferation, invasiveness, and stemness by targeting multiple pluripotency factors, cytoskeletal elements, and protease inhibitors.

Inducible miR-145 expression by HIF-1a protects cardiomyocytes against apoptosis via regulating SGK1 in simulated myocardial infarction hypoxic microenvironment.

Myocardial infarction (MI) is partly due to myocardial cell damage caused by hypoxia. MicroRNAs (miRNAs) have been proved to be closely related to the development and progression of many cardiovascular diseases. This study investigated the role of miR-145 in cardiomyocytes under hypoxic condition. The quantitative real-time polymerase chain reaction (qRT-PCR) was performed to test miR-145 expression in H9c2 cells with hypoxia-inducible factor (HIF)-a abnormal expression under hypoxic condition. The 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyltetrazolium bromide (MTT), Tran-swell assay and flow cytometry were used to investigate the effects of miR-145 on cell viability, migration and apoptosis under normoxic or hypoxic condition, respectively. Meanwhile, reactive oxygen species (ROS) content in hypoxic H9c2 cells was analyzed. Western blotting was used to explore the potential mechanism of miR-145 protective effects on cardiomyocytes. Expression levels of miR-145 and SGK1 in rat MI model were also assessed. Results showed that miR-145 was upregulated in H9c2 and HL-1 cells under hypoxic condi-tion, which was promoted by HIF-1a. MiR-145 overexpression enhanced cell viability and migration under normoxic condition. Under hypoxic condition, miR-145 overexpression promoted cell viability, inhibited apoptosis and ROS activity. Western blotting results proved that miR-145 overexpression inhibited the activation of apoptotic related factors, and promoted activation of PI3K/AKT signaling pathway via SGK1 upregulation. Expression levels of miR-145 and SGK1 were both upregulated in rat MI models. HIF-1a could induce miR-145 upregulation in hypoxic H9c2 and HL-1 cells. MiR-145 protected H9c2 cells against hypoxic damage. SGK1 upregulation and activated PI3K/AKT may have participated in the protective effects of miR-145 on cardiomyocytes.

Comprehensive microRNA Profiling of Prostate Cancer

MicroRNAs are small non-coding RNA molecules that have been shown to regulate the expression of genes linked to cancer. The relevance of microRNAs in the development, progression and prognosis of prostate cancer is not fully understood. It is also possible that these specific molecules may assist in the recognition of aggressive tumors and the development of new molecular targets. Our study investigated the importance of several microRNAs in cases of prostate cancer from 37 patients that were manually microdissected to obtain pure populations of tumor cells, normal epithelium and adjacent stroma. MicroRNA was extracted for PCR array profiling. Differentially expressed miRNAs for each case were used to compare tumor vs. normal epithelium and tumor-adjacent stroma samples.Loss of 18 miRNAs (e.g.miR-34c, miR-29b, miR-212 and miR-10b) and upregulation of miR-143 and miR-146b were significantly found in all the tumors in comparison with normal epithelium and/or stroma (p≤ 0.001). A different signature was found in the high grade tumors (Gleason score ≥ 8) when compared with tumors Gleason score 6. Upregulation of miR-122, miR-335, miR-184, miR-193, miR-34, miR-138, miR-373, miR-9, miR-198, miR-144 and miR-215 and downregulation of miR-96, miR-222, miR-148, miR-92, miR-27, miR-125, miR-126, miR-27 were found in the high grade tumors.MicroRNA profiling in prostate cancer appears to have unique expression patterns in comparison with normal tissue. These differential expressed miRNAs may provide novel diagnostic and prognostic tools that will assist in the recognition of prostate cancers with aggressive behavior.

Microrna-143 Blocks ERK5 Signaling During Granulocytic Differentiation of Hematopoietic Stem Cells and Is Downregulated in AML

AbstractAbstract 3516Mitogen-activated protein kinase (MAPK) pathways are a family of related and sometimes interconnected pathways and one of the most studied. Over the last years, extensive work has established that these proteins play a critical role in G-CSF mediated maturation of neutrophil granulocytes. Understanding the mechanisms by which the MAPK pathways are regulated represents an important area of investigation. MicroRNAs, a class of small non-coding RNAs, have been found to play an important role in the regulation of diverse cellular processes by binding to target mRNAs leading to their translational repression. Deregulation of certain microRNAs, thereby, may lead to disrupted signal pathways, such as MAPK-signaling, and to tumorigenesis. However, the role of microRNAs in hematopoietic differentiation and development of leukemia remains largely unknown. In this study we performed a global screen to identify microRNAs involved in G-CSF-regulated MAPK-pathways in primary human CD34+ hematopoietic progenitor cells. Here we found microRNA-143 (miR-143) to be frequently upregulated in G-CSF stimulated CD34+ cells with a strong correlation to CD15 expression. We could also show the granulopoietic association of miR-143 in several hematopoietic cell line models and acute myeloid leukemia (AML) patient samples. Especially, AML patient samples FAB M4 and M5, which show monocytic phenotypes, had a significant lower expression level of miR-143 compared to the AML FAB types M0, M1, M2, and M3. In general, miR-143 expression was shown to be downregulated in AML patient samples in comparison to normal CD34+ hematopoietic progenitor cells. Most interestingly, we show that miR-143 expression is upregulated in acute promyelocytic leukemia (APL) patients after ATRA treatment. By in silico prediction we found MAPK protein family members (eg. MAPK1, MAPK3 and MAPK7) as predicted targets of miR-143. Western blot analysis of AML patient samples and G-CSF stimulated CD34+ cells clearly show an inverse correlation of miR-143 and MAPK7 (ERK5) protein expression. Finally, by transient overexpression of miR-143 we could show a strong downregulation of ERK protein expression in NB4 cells. Our study suggest that miR-143 upregulation by G-CSF may be an important regulatory step for permitting neutrophil differentiation. MicroRNA-143 blocks ERK5 signaling in G-CSF-induced granulopoiesis of CD34+ hematopoietic stem cells, is downregulated in myelo-monocytic acute myeloid leukemia subtypes, and upregulated after ATRA treatment in APL patients. This information may prove useful for the understanding of conditions in which neutrophil proliferation/differentiation balancing is dysregulated, such as in myeloid leukemia and myelodysplastic disorders. Disclosures:No relevant conflicts of interest to declare.

MiR-24 regulates the proliferation and invasion of glioma by ST7L via β-catenin/Tcf-4 signaling

MicroRNAs are strongly implicated as affecting glioma, but their specific roles and functions have yet to be fully elucidated. In this study, we defined the expression and function of miR-24, which we found to be upregulated in glioma samples and glioma cells by qRT-PCR. Downregulation of miR-24 in glioma cell lines inhibited proliferation and invasion and induced apoptosis. Using computational and expression analysis, ST7L was identified as a candidate target of miR-24. A reporter assay with the 3′UTR of ST7L cloned downstream of a luciferase gene showed increased luciferase activity in the absence of miR-24, providing strong evidence that miR-24 is a direct regulator of ST7L. Furthermore, we observed that restoration of ST7L activity resulted in effects that were similar to those from transfecting a miR-24 inhibitor into glioma cells. Mechanistic investigation revealed that the deletion of miR-24 suppressed β-catenin/Tcf-4 transcription activity by targeting ST7L. In conclusion, our study demonstrates that miR-24 upregulation is common in glioma and that suppression of miR-24 expression inhibits cell proliferation and invasion, suggesting that miR-24 may act as an oncogene in glioma.

MicroRNA‐24 regulates cardiac fibrosis after myocardial infarction

Cardiac fibrosis after myocardial infarction (MI) has been identified as a key factor in the development of heart failure. Although dysregulation of microRNA (miRNA) is involved in various pathophysiological processes in the heart, the role of miRNA in fibrosis regulation after MI is not clear. Previously we observed the correlation between fibrosis and the miR-24 expression in hypertrophic hearts, herein we assessed how miR-24 regulates fibrosis after MI. Using qRT-PCR, we showed that miR-24 was down-regulated in the MI heart; the change in miR-24 expression was closely related to extracellular matrix (ECM) remodelling. In vivo, miR-24 could improve heart function and attenuate fibrosis in the infarct border zone of the heart two weeks after MI through intramyocardial injection of Lentiviruses. Moreover, in vitro experiments suggested that up-regulation of miR-24 by synthetic miR-24 precursors could reduce fibrosis and also decrease the differentiation and migration of cardiac fibroblasts (CFs). TGF-β (a pathological mediator of fibrotic disease) increased miR-24 expression, overexpression of miR-24 reduced TGF-β secretion and Smad2/3 phosphorylation in CFs. By performing microarray analyses and bioinformatics analyses, we found furin to be a potential target for miR-24 in fibrosis (furin is a protease which controls latent TGF-β activation processing). Finally, we demonstrated that protein and mRNA levels of furin were regulated by miR-24 in CFs. These findings suggest that miR-24 has a critical role in CF function and cardiac fibrosis after MI through a furin–TGF-β pathway. Thus, miR-24 may be used as a target for treatment of MI and other fibrotic heart diseases.

NEDD9, a novel target of miR-145, increases the invasiveness of glioblastoma.

miR-145 is an important repressor of pluripotency in embryonic stem cells and a tumor suppressor in different cancers. Here, we found that miR-145 is strongly down-regulated in glioblastoma (GB) specimens and corresponding glioblastomaneurospheres (GB-NS, containing GB stem-like cells) compared to normal brain (NB) and to low-grade gliomas (LGG). We observed a direct correlation between miR-145 expression and the progression-free survival (PFS) in LGG patients and overall survival (OS) in GB patients. Using microarray analysis, we identified relevant differences in gene expression profiles between GB-NS over-expressing miR-145 (miRover-NS) and GB-NS Empty (Empty-NS). We focused our attention on HEF1/Cas-L/NEDD9, a scaffold protein involved in invasion in several types of cancer. We confirmed a significant down-regulation of NEDD9 in miRover-NS and we found a higher expression in GB and GB-NS compared to NB. Approximately 50% of LGG patients expressed higher levels of NEDD9 than NB, and the PFS of such patients was shorter than in patients expressing lower levels of NEDD9. We observed that intracranial injection of GB-NS over-expressing miR-145 delays significantly tumor development :deriving tumors showed a significant down-regulation of NEDD9. In addition, we demonstrated a significant inhibition of invasion in silencing experiments with GB-NS shNEDD9 (shNEDD9), and an up-regulation of miR-145 in shNEDD9, suggesting a doublenegative feedback loop between miR-145 and NEDD9. Our results demonstrate the critical role of miR-145 and NEDD9 in regulating glioblastoma invasion and suggest a potential role of NEDD9 as a biomarker for glioma progression.

A Role for miR-145 in Pulmonary Arterial Hypertension

Rationale: Despite improved understanding of the underlying genetics, pulmonary arterial hypertension (PAH) remains a severe disease. Extensive remodeling of small pulmonary arteries, including proliferation of pulmonary artery smooth muscle cells (PASMCs), characterizes PAH. MicroRNAs (miRNAs) are noncoding RNAs that have been shown to play a role in vascular remodeling. Objective: We assessed the role of miR-145 in PAH. Methods and Results: We localized miR-145 in mouse lung to smooth muscle. Using quantitative PCR, we demonstrated increased expression of miR-145 in wild-type mice exposed to hypoxia. PAH was evaluated in miR-145 knockout and mice treated with anti-miRs via measurement of systolic right ventricular pressure, right ventricular hypertrophy, and percentage of remodeled pulmonary arteries. miR-145 deficiency and anti-miR–mediated reduction resulted in significant protection from the development of PAH. In contrast, miR-143 anti-miR had no effect. Furthermore, we observed upregulation of miR-145 in lung tissue of patients with idiopathic and heritable PAH compared with unaffected control subjects and demonstrated expression of miR-145 in SMC of remodeled vessels from such patients. Finally, we show elevated levels of miR-145 expression in primary PASMCs cultured from patients with BMPR2 mutations and also in the lungs of BMPR2 -deficient mice. Conclusions: miR-145 is dysregulated in mouse models of PAH. Downregulation of miR-145 protects against the development of PAH. In patient samples of heritable PAH and idiopathic PAH, miR-145 is expressed in remodeled vessels and mutations in BMPR2 lead to upregulation of miR-145 in mice and PAH patients. Manipulation of miR-145 may represent a novel strategy in PAH treatment.

Role of microRNA in 3,3-diindolylmethane (DIM)-mediated amelioration of SEB-induced acute lung injury (173.6)

Abstract Staphylococcal enterotoxin B (SEB) is a CDC select agent of bioterrorism and a public health threat during nosocomial infections. A deleterious effect of SEB exposure is acute lung injury (ALI) and acute respiratory distress syndrome characterized by pulmonary infiltration. 3,3-diindolylmethane (DIM) found in cruciferous vegetables, has been shown to have anti-inflammatory properties. We studied the mechanism underlying the ameliorative effects of DIM on SEB-induced ALI. Mice were exposed to SEB [50 mg; intranasal] and administered DIM [100 mg/kg] via oral gavage. Forty-eight hours after SEB exposure, serum, lung tissue and lung infiltrating cells were collected. DIM treatment led to a decrease in lymphocytic infiltration, specifically CD4+, CD8+ and Vβ8+ T cells as well as NKT cells. DIM also led to a reduction of fluid leak in lungs. DIM [10, 25, 50 µM] induced apoptosis in splenocytes and CD3+ T cells treated in vitro with SEB in a dose-dependent manner. DIM treatment led to downregulation in microRNA, miR-706 and miR-494 that target pro-apoptotic genes, and upregulation of miR-22 and miR-34a targeting anti-apoptotic genes in lung-infiltrating cells. Thus, studies are underway to examine whether in T cells, DIM treatment leads to modulation in the expression of genes involved in apoptosis. Together, these studies demonstrate that DIM treatment may suppress SEB-mediated ALI by induction of apoptosis in T cells via alteration in expression of miRNA.

MiR‐145 is differentially regulated by TGF‐β1 and ischaemia and targets Disabled‐2 expression and wnt/β‐catenin activity

The effect of wnt/β-catenin signalling in the response to acute myocardial infarction (AMI) remains controversial. The membrane receptor adaptor protein Disabled-2 (Dab2) is a tumour suppressor protein and has a critical role in stem cell specification. We recently demonstrated that down-regulation of Dab2 regulates cardiac protein expression and wnt/β-catenin activity in mesenchymal stem cells (MSC) in response to transforming growth factor-β1 (TGF-β1). Although Dab2 expression has been shown to have effects in stem cells and tumour suppression, the molecular mechanisms regulating this expression are still undefined. We identified putative binding sites for miR-145 in the 3′-UTR of Dab2. In MSC in culture, we observed that TGF-β1 treatment led to rapid and sustained up-regulation of pri–miR-145. Through gain and loss of function studies we demonstrate that miR-145 up-regulation was required for the down-regulation of Dab2 and increased β-catenin activity in response to TGF-β1. To begin to define how Dab2 might regulate wnt/β-catenin in the heart following AMI, we quantified myocardial Dab2 as a function of time after left anterior descending ligation. There was no significant Dab2 expression in sham-operated myocardium. Following AMI, Dab2 levels were rapidly up-regulated in cardiac myocytes in the infarct border zone. The increase in cardiac myocyte Dab2 expression correlated with the rapid and sustained down-regulation of myocardial pri–miR-145 expression following AMI. Our data demonstrate a novel and critical role for miR-145 expression as a regulator of Dab2 expression and β-catenin activity in response to TGF-β1 and hypoxia.

Abstract LB-465: The miRNA cluster 424/503 is an integral part of the TGFb-mediated cytostatic response in human mammary epithelial cells

Abstract TGF-beta plays a major role during development and normal tissue homeostasis. Indeed and focusing in mammary tissue biology TGF-beta has been unmasked as a key component during epithelium cell proliferation, lactancy and involution. However, and despite most of the TGF-beta actions are well known for decades, its dichotomic effects depending on cell type, context and disease states and the mechanisms that lie underneath are still not fully understood. In this study we uncover a novel player in the mechanism through which TGF-beta induces cell cycle arrest in mammary epithelial cells. Using microRNA array expression profile we found a strong correlation between the expression of miRs 424 and 503 and TGF-beta treatment in MCF10A cells. Interestingly, this microRNAs are located clustered in the long arm of the X chromosome, regulated by a region located upstream containing a canonical SMAD3/4 binding site and expressed as a policistronic pri-miRNA. Ectopic upregulation of miR-424/503 induces cell cycle arrest at G1 phase mimicking TGF-beta actions. Our mechanistic studies have unveiled that this miRNA cluster exerts its function by modulating the expression of CDC25A through binding to its 3′UTR. Altogether our results point to miRs 424/503 as a new player in the cytostatic program mediated by TGF-beta in mammary epithelial cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-465. doi:1538-7445.AM2012-LB-465

Upregulation of miR-22 Promotes Osteogenic Differentiation and Inhibits Adipogenic Differentiation of Human Adipose Tissue-Derived Mesenchymal Stem Cells by RepressingHDAC6Protein Expression

Mesenchmal stem cells (MSCs) can be differentiated into either adipocytes or osteoblasts, and a reciprocal relationship exists between adipogenesis and osteogenesis. Multiple transcription factors and signaling pathways have been reported to regulate adipogenic or osteogenic differentiation, respectively, yet the molecular mechanism underlying the cell fate alteration between adipogenesis and osteogenesis still remains to be illustrated. MicroRNAs are important regulators in diverse biological processes by repressing protein expression of their targets. Here, miR-22 was found to regulate adipogenic and osteogenic differentiation of human adipose tissue-derived mesenchymal stem cells (hADMSCs) in opposite directions. Our data showed that miR-22 decreased during the process of adipogenic differentiation but increased during osteogenic differentiation. On one hand, overexpression of miR-22 in hADMSCs could inhibit lipid droplets accumulation and repress the expression of adipogenic transcription factors and adipogenic-specific genes. On the other hand, enhanced alkaline phosphatase activity and matrix mineralization, as well as increased expression of osteo-specific genes, indicated a positive role of miR-22 in regulating osteogenic differentiation. Target databases prediction and validation by Dual Luciferase Reporter Assay, western blot, and real-time polymerase chain reaction identified histone deacetylase 6 (HDAC6) as a direct downstream target of miR-22 in hADMSCs. Inhibition of endogenous HDAC6 by small-interfering RNAs suppressed adipogenesis and stimulated osteogenesis, consistent with the effect of miR-22 overexpression in hADMSCs. Together, our results suggested that miR-22 acted as a critical regulator of balance between adipogenic and osteogenic differentiation of hADMSCs by repressing its target HDAC6.

MicroRNA-143 is a critical regulator of cell cycle activity in stem cells with co-overexpression of Akt and angiopoietin-1 via transcriptional regulation of Erk5/cyclin D1 signaling

We report that simultaneous expression of Akt and angiopoietin-1 (Ang-1) transgenes supported mitogenesis in stem cells with a critical role for microRNA-143 (miR-143) downstream of FoxO1 transcription factor. Mesenchymal stem cells (MSC) from young male rats were transduced with Ad-vectors encoding for Akt (AktMSC) and Ang-1 (Ang-1MSC) transgenes for their individual or simultaneous overexpression (AAMSC; > 5-fold gene level and > 4-fold Akt and Ang-1 protein expression in AAMSC vs. Ad-Empty transduced MSC; EmpMSC). AAMSC had higher phosphorylation of FoxO1, which activated Erk5, a distinct mitogen-induced MAPK that drove transcriptional activation of cyclin D1 and Cdk4. Flow cytometry showed > 10% higher S-phase cell population that was confirmed by BrdU assay (15%) and immunohistology for Ki67 (11%) in AAMSC using EmpMSC as controls. miR array supported by real-time PCR showed induction of miR-143 in AAMSC (4.73-fold vs.. EmpMSC). Luciferase assay indicated a dependent relationship between miR-143 and Erk5 in AAMSC. FoxO1-specific siRNA upregulated miR-143, whereas inhibition of miR-143 did not change FoxO1 activation. However, miR-143 inhibition repressed phosphorylation of Erk5 and abrogated cyclin D1 with concomitant reduction in cells entering cell cycle. During in vivo studies, male GFP+ AAMSC transplanted into wild-type female infarcted rat hearts showed significantly higher number of Ki67 expressing cells (p < 0.05 vs. EmpMSC) 7 days after engraftment (n = 4 animals/group). In conclusion, co-overexpression of Akt and Ang-1 in MSC activated cell cycle progression by upregulation of miR-143 and stimulation of FoxO1 and Erk5 signaling.

Attenuation of MicroRNA‐22 derepressed PTEN to effectively protect rat cardiomyocytes from hypertrophy

Cardiac hypertrophy, which is characterized by the enlargement of cell size, reactivation of fetal genes, remains one of the most important triggers to heart failure. Increasing evidence shows that microRNA (miRNA) is extensively involved in the pathogenesis of cardiac hypertrophy. But the effects of miRNAs on cardiomyocyte hypertrophy have not been completely solved yet. Here, we showed that a collection of miRNAs was aberrantly expressed in hypertrophic cardiomyocytes induced by phenylephrine (PE) or angiotensin II (Ang II). Among them, miR-22 was the most strikingly up-regulated miRNA. To investigate the role of miR-22 in hypertrophy, both over-expression and knock-down assays were performed on cardiomyocytes. The results showed that up-regulation of miR-22 significantly increased the cell size and markedly influenced the expression of hypertrophic markers, including induction of nppa and reduction of myh6. In contrast, reduction of miR-22 level attenuated either PE- or Ang II-induced hypertrophic reaction. Furthermore, several genes, including PTEN, were identified as potential targets of miR-22 by bioinformatic algorithms. Using luciferase analysis, miR-22 could significantly suppress the luciferase activity of reporter fused with 3' untranslated region of PTEN mRNA. Furthermore, up-regulation of miR-22 could suppress the protein level of PTEN and reduction of miR-22 level markedly increased the protein level of PTEN in cardiomyocytes by Western blot analysis, suggesting that the contribution of miR-22 to cardiomyocyte hypertrophy may be partially through targeting PTEN. Taken together, miRNAs were dynamically regulated in cardiomyocyte hypertrophy and attenuation of miR-22 in rat cardiomyocytes efficiently protected from hypertrophic effects through derepressing PTEN.

Up-regulated miR-145 Expression Inhibits Porcine Preadipocytes Differentiation by Targeting IRS1

Generally, most miRNAs that were up-regulated during differentiation promoted adipogenesis, but our research indicated that up-regulation of miR-145 in porcine preadipocytes did not promote but inhibit adipogenesis. In this study, miR-145 was significantly up-regulated during porcine dedifferentiated fat (DFAT) cells differentiation. In miR-145 overexpressed DFAT cells, adipogenesis was inhibited and triglycerides accumulation was decreased after hormone stimulation (P<0.05). Furthermore, up-regulation of miR-145 expression repressed induction of mRNA levels of adipogenic markers, such as CCAAT/enhancer-binding protein α (C/EBPα), and peroxisome proliferator-activated receptor γ2 (PPARγ2). These effects caused by miR-145 overexpression were mediated by Insulin receptor substrate 1 (IRS1) as a mechanism. These data suggested that induced miR-145 expression during differentiation could inhibit adipogenesis by targeting IRS1, and miR-145 may be novel agent for adipose tissue engineering.

Abstract 18277: Role of the miR-204/miR-145 Communication in the Etiology of Pulmonary Arterial Hypertension

Introduction: Pulmonary arterial hypertension (PAH) is characterized by distal pulmonary arteries remodeling secondary to the proliferation and resistance to apoptosis of the pulmonary artery smooth muscle (PASMC). We recently described that this phenotype is associated with 7 aberrantly expressed miRNAs in PAH-PASMC. We showed that miR-204 was the most downregulated and accounts for the activation of the neoplasic axis STAT3/Pim1/NFAT, inducing PASMC proliferation and resistance to apoptosis. Because miR-204 downregulation precedes the development of PAH in chronic hypoxic mice and monocrotaline rats, we tested whether its downregulation accounts for the deregulation of the 6 others affected miRNAs in PAH. As miR-145 is implicated in systemic vascular remodeling and is the one of the most upregulated miRNA in PAH we focused our research on the regulation of miR-145 by miR-204. Methods/Results: Using lungs biopsies from 8 PAH and 8 control patients we confirmed the downregulation of miR-204 and the upregulation of miR-145 in PAH. Moreover, both miR-204 downregulation and miR-145 upregulation correlate with PAH severity. These findings were confirmed in both lungs and distal PAs of 5-MCT and 5 Sugen/Hypoxia/Normoxia rats compare to 5 control rats in which severity was assessed by CT-scan angiogram. Time to event analysis in both monocrotaline (1; 2; 3 and 4 weeks post MCT injection) and Sugen (8 and 15 weeks post Sugen) rats showed that miR-204 downregulation precedes miR-145 upregulation. In vitro using cultured human PASMC from 5 control and 3 PAH patients we showed that miR-204 upregulation in PAH-PASMC decreases miR-145; while miR-204 downregulation in control PASMC increases miR-145. Finally, in vivo nebulized MCT rats with miR-204 mimic had significantly less miR-145 compared to rats nebulized with mimic negative control. Conclusion: We demonstrated for the first time in PAH that one miRNA (miR-204) can regulate the expression of other miRNA such as miR-145. Because miR-204 downregulation precede miR-145 upregulation and PAH development, miR-204 could be considered as an early molecular marker of PAH and might represent the molecular origin of this lethal disease.

The miR-143/145 Cluster Is a Novel Transcriptional Target of Jagged-1/Notch Signaling in Vascular Smooth Muscle Cells

Activation of Notch signaling by Jagged-1 (Jag-1) in vascular smooth muscle cells (VSMC) promotes a differentiated phenotype characterized by increased expression of contractile proteins. Recent studies show that microRNAs (miR)-143/145 regulates VSMC phenotype. The serum response factor (SRF)/myocardin complex binds to CArG sequences to activate miR-143/145 transcription, but no other regulators are known in VSMC. Using miR arrays, we found miR-143/145 induced following expression of a constitutively active Notch1 intracellular domain (N1ICD). We hypothesized that miR-143/145 is required for Jag-1/Notch-induced VSMC differentiation. Activation of Notch receptors by Jag-1 caused CBF1-dependent up-regulation of miR-143/145, increased differentiation, and decreased proliferation. Conversely, inhibiting basal Notch signaling decreased steady state levels of miR-143/145. Using SRF knockdown, we found that Jag-1/Notch induction of miR-143/145 is SRF independent, although full acquisition of contractile markers requires SRF. Using miR-143/145 promoter reporter constructs we show Jag-1/Notch increases promoter activity, and this is dependent on intact CBF1 consensus sites within the promoter. Chromatin immunoprecipitation (ChIP) assays revealed that N1ICD-containing complexes bind to CBF1 sites in the miR-143/145 promoter. We also identified N1ICD complex binding to CBF1 sites within the endogenous human miR-143/145 promoter. Using miR-143/145-interfering oligonucleotides, we demonstrate that Jag-1/Notch signaling requires induction of both miR-143 and miR-145 to promote the VSMC contractile phenotype. Thus, miR-143/145 is a novel transcriptional target of Jag-1/Notch signaling in VSMC. We propose miR-143/145 as activated independently by Jag-1/Notch and SRF in parallel pathways. Multiple pathways converging on miR-143/145 provides potential for fine-tuning or amplification of VSMC differentiation signals.