Wen-Jun Yuan

Endothelial-specific intron-derived miR-126 is down-regulated in human breast cancer and targets both VEGFA and PIK3R2

Endothelial cells are the key components of vascular intima and play pivotal roles in vasculogenesis, angiogenesis, and tumor growth. Using Northern blot and real-time PCR, we confirmed that miR-126 and its host gene EGF-like domain 7 (EGFL7) were widely expressed in rat tissues but strictly expressed in endothelial cells. In mammals, miR-126 gene is embedded in intron7 of EGFL7. To explore the biogenesis of miR-126, plasmid EGFL7(126)-pEGFPc1 containing segment of exon7-intron7-exon8 of EGFL7 was constructed and expressed in 293T. Expression of spliced exon7–8 and excised mature miR-126 was detected by PCR and Northern blot. Knocking-down of endothelial endogenous miR-126 did not affect EGFL7 expression at mRNA or protein level. To investigate the possible roles of miR-126, PicTar, miRBase, miRanda, Bibiserv, and Targetscan were used to screen the targets. VEGFA and PIK3R2 were confirmed as the targets of miR-126 by luciferase reporter assay and Western blot. Interestingly, Northern blot and western blot showed that miR-126 was down-regulated in breast tumors where the VEGF/PI3K/AKT signaling pathway was activated. Introduction of miR-126 mimics into MCF-7 could effectively decrease VEGF/PI3K/AKT signaling activity. In summary, miR-126 was strictly expressed in endothelial cells and excised from EGFL7 pre-mRNA without affecting splicing and expression of its host gene. In addition, miR-126 could target both VEGFA and PIK3R2, and its expression was decreased in human breast cancer, implying that miR-126 may play a role in tumor genesis and growth by regulating the VEGF/PI3K/AKT signaling pathway.

Attenuation of microRNA-1 derepresses the cytoskeleton regulatory protein twinfilin-1 to provoke cardiac hypertrophy

MicroRNAs are involved in several aspects of cardiac hypertrophy, including cardiac growth, conduction, and fibrosis. However, their effects on the regulation of the cardiomyocyte cytoskeleton in this pathological process are not known. Here, with microRNA microarray and small RNA library sequencing, we show that microRNA-1 (miR-1) is the most abundant microRNA in the human heart. By applying bioinformatic target prediction, a cytoskeleton regulatory protein twinfilin-1 was identified as a potential target of miR-1. Overexpression of miR-1 not only reduced the luciferase activity of the reporter containing the 3′ untranslated region of twinfilin-1 mRNA, but also suppressed the endogenous protein expression of twinfilin-1, indicating that twinfilin-1 is a direct target of miR-1. miR-1 was substantially downregulated in the rat hypertrophic left ventricle and phenylephrine-induced hypertrophic cardiomyocytes, and accordingly, the protein level of twinfilin-1 was increased. Furthermore, overexpression of miR-1 in hypertrophic cardiomyocytes reduced the cell size and attenuated the expression of hypertrophic markers, whereas silencing of miR-1 in cardiomyocytes resulted in the hypertrophic phenotype. In accordance, twinfilin-1 overexpression promoted cardiomyocyte hypertrophy. Taken together, our results demonstrate that the cytoskeleton regulatory protein twinfilin-1 is a novel target of miR-1, and that reduction of miR-1 by hypertrophic stimuli induces the upregulation of twinfilin-1, which in turn evokes hypertrophy through the regulation of cardiac cytoskeleton.

Overexpression of microRNA-378 attenuates ischemia-induced apoptosis by inhibiting caspase-3 expression in cardiac myocytes

MicroRNAs (miRNAs) are a novel class of powerful, endogenous regulators of gene expression. In an intact rat model of myocardial ischemia caused by coronary artery ligation, this study identified 17 miRNAs that changed more than 1.5-fold in the myocardium subjected to 4-h ischemia. Using miRNA microarray analysis, most of these aberrantly expressed miRNAs were confirmed by quantitative RT-PCR. MiR-378, a significantly down-regulated miRNA, was selected for further function study. In serum deprived rat H9c2 cardiomyocytes exposed to hypoxia (1% O2), miR-378 expression was down-regulated as well. The overexpression of miR-378 resulting from miR-378 mimic transfection significantly enhanced cell viability, reduced lactate dehydrogenase release, and inhibited apoptosis and necrosis. By contrast, miR-378 deficiency resulting from miR-378 inhibitor transfection aggravated the hypoxia-induced apoptosis and cell injury. In accordance, miR-378 inhibitor caused significant apoptosis and cell injury to cardiomyocytes cultured under normoxia. Using bioinformatic algorithms, caspase-3, a key apoptosis executioner, was predicted as a putative target of miR-378. The quantitative RT-PCR showed no effects of miR-378 mimic or inhibitor on caspase-3 mRNA level. However, the amount of caspase-3 proteins was reduced by miR-378 mimic, whereas increased by miR-378 inhibitor. Furthermore, the luciferase reporter assay confirmed caspase-3 to be a target of miR-378, and the apoptosis and cell injury caused by miR-378 inhibitor in both normoxic and hypoxic cells were abolished by a caspase-3 inhibitor. This study first showed that miR-378 inhibited caspase-3 expression and attenuated ischemic injury in cardiomyocytes. It may represent a potential novel treatment for apoptosis and ischemic heart disease.

MicroRNAs are dynamically regulated in hypertrophic hearts, and miR-199a is essential for the maintenance of cell size in cardiomyocytes†

Cardiac hypertrophy, which is characterized by an increase in cell size and reactivation of fetal genes, occurs as an adaptive response to diverse forms of stress and often results in heart failure and sudden death. Growing evidence indicates that microRNAs (miRNAs) are involved in cardiac hypertrophy, but the function of these miRNAs remains elusive. Here, using real time PCR analysis, we showed that several miRNAs were dynamically regulated in the rat hypertrophic hearts and miR‐199a was up‐regulated by 10‐fold in hypertrophic hearts after abdominal aorta constriction for 12 weeks. With tissue profiling analysis, we showed that miR‐199a was predominantly expressed in cardiomyocytes, but was also faintly detected in cardiac fibroblasts. To investigate whether miR‐199a was involved in cardiac hypertrophy, both over‐expression and knockdown of miR‐199a were performed in cultured cardiomyocytes. Over‐expression of miR‐199a in cardiomyocytes increased the cell size as measured by cell surface area, and also reduced the mRNA expression level of α‐myosin heavy chain. In accordance, knockdown of endogenous miR‐199a in cardiomyocytes reduced the cell size. Down‐regulation of miR‐199a also attenuated the phenylephrine‐induced increase of cell size. Furthermore, bioinformatic algorithms were used to predict the potential targets of miR‐199a in cardiac hypertrophy, and hypoxia‐inducible factor 1 alpha was confirmed by the luciferase reporter assay to be a potential target of miR‐199a. Taken together, our results demonstrated that miR‐199a, which was predominantly expressed in cardiomyocytes, was essential for the maintenance of cell size of cardiomyocytes and might play a role in the regulation of cardiac hypertrophy. J. Cell. Physiol. 225: 437–443, 2010.

MicroRNA-125a/b-5p inhibits endothelin-1 expression in vascular endothelial cells.

Background Endothelin-1 (ET-1) is considered to be one of the most potent and long-lasting vasoconstrictive peptides, but the mechanisms on the regulation of ET-1 expression are not fully understood. Method and results In this study, we found that microRNA (miR)-125a-5p and miR-125b-5p are highly expressed in vascular endothelial cells (VECs), which can be regulated by oxidized low-density lipoprotein (oxLDL). To explore the function of miR-125a/b-5p in VECs, we examined the roles of potential targets of miR-125a/b-5p that could influence endothelium function. We found that both miR-125a/b-5p can suppress oxLDL-induced ET-1 expression by directly targeting 3′ untranslated region of prepro-endothelin-1 (preproET-1) mRNA determined by luciferase reporter assay, western blot, and enzyme immunometric assay. Consistently, inhibitors of miR-125a/b-5p can directly enhance preproET-1 expression. The decreased expressions of miR-125a-5p and miR-125b-5p are negatively associated with upregulation of preproET-1 expression in aorta of stroke-prone spontaneously hypertensive rats (SHR-SPs). Conclusion Our finding demonstrated that endothelial miR-125a/b-5p inhibits ET-1 expression in VECs, which revealed a novel miRNA-mediated mechanism in vasomotor homeostasis.

PTEN deletion prevents ischemic brain injury by activating the mTOR signaling pathway

It is increasingly clear that the tumor suppressor PTEN (phosphatase and tensin homolog deleted on chromosome 10) is a negative regulator of neuronal cell survival. However, its molecular mechanisms remain poorly understood. Here we found that PTEN/mTOR is critical for controlling neuronal cell death after ischemic brain injury. Male rats were subjected to MCAO (middle cerebral artery occlusion) followed by pretreating with bpv (pic), a potent inhibitor for PTEN, or by intra-cerebroventricular infusion of PTEN siRNA. bpv (pic) significantly decreased infarct volume and reduced the number of TUNEL-positive cells. We further demonstrated that although bpv (pic) did not affect brain injury-induced mTOR protein expression, bpv (pic) prevented decrease in phosphorylation of mTOR, and the subsequent decrease in S6. Similarly, down-regulation of PTEN expression also reduced the number of TUNEL-positive cells, and increased phospho-mTOR. These data suggest that PTEN deletion prevents neuronal cell death resulting from ischemic brain injury and that its neuroprotective effects are mediated by increasing the injury-induced mTOR phosphorylation.

Inhibitory effect of obestatin on glucose-induced insulin secretion in rats.

Obestatin is a bioactive peptide encoded by the same gene that encodes ghrelin. Our aim was to investigate the effect of obestatin on insulin secretion. We evaluated the effects of obestatin on insulin secretion from rat islet cells which had been incubated overnight in the presence of 8.3, 11.1, and 22.2 mmol/l of glucose. In vivo, the serum levels of glucose and insulin were measured 0, 1, 5, 10, 20, 40, and 60 min after the intravenous administration of saline or glucose (1g/kg), with or without obestatin, and the area under the 60 min curve of insulin concentration (AUC(insulin)) was calculated. Obestatin (0.01-100 nmol/l) inhibited insulin secretion from rat islets in a dose-dependent fashion. In vivo, when administered intravenously to rats together with glucose, obestatin (10, 50, and 250 nmol/kg) inhibited both the rapid 1-min insulin response and the AUC(insulin) in a dose-dependent fashion. Our data demonstrate that under glucose-stimulated conditions, exogenous obestatin acts as a potent inhibitor of insulin secretion in anaesthetized rats in vivo as well as in cultured islets in vitro.

Different responses of circulating ghrelin, obestatin levels to fasting, re-feeding and different food compositions, and their local expressions in rats

Obestatin, a sibling of ghrelin derived from preproghrelin, opposes several physiological actions of ghrelin. Our previous study has demonstrated that both plasma ghrelin and obestatin levels were decreased significantly 2h after food intake in human. To further expand current knowledge, we investigated the temporal profiles of their levels in ad libitum fed rats, 48h fasted rats and 48h fasted rats refed 2h with a standard chow, crude fiber, 50% glucose or water, and their expressions in stomach, liver and pancreatic islets immunohistochemically. Plasma ghrelin and obestatin levels were measured by EIA. Plasma leptin, insulin and glucose levels were also evaluated. Both plasma ghrelin and obestatin levels increased significantly in fasted rats compared with ad libitum fed rats. The ingestion of standard chow produced a profound and sustained suppression of ghrelin levels, whereas plasma obestatin levels decreased significantly but recovered quickly. Intake of crude fiber or 50% glucose, however, produced a more profound and sustained suppression of obestatin levels, though they had relatively less impact on ghrelin levels. Plasma glucose was the only independent predictor of ghrelin levels, obestatin levels, and ghrelin to obestatin ratios. Obestatin immunoreactivity was detected in the fundus of stomach, liver and pancreatic islets, with roughly similar patterns of distribution to ghrelin. These data show quantitative and qualitative differences in circulating ghrelin and obestatin responses to the short-term feeding status and nutrient composition, and may support a role for obestatin in regulating metabolism and energy homeostasis.

HSP70 and GRP78 induced by endothelin-1 pretreatment enhance tolerance to hypoxia in cultured neonatal rat cardiomyocytes.

The heat shock protein 70 and glucose-regulated protein 78 have been shown to protect cells against deleterious stimuli. This study was performed to determine whether endothelin-1 pretreatment could increase cardiomyocyte tolerance to hypoxia and induce heat shock protein 70 and glucose-regulated protein 78 expression. Cultured cardiomyocytes were treated with endothelin-1 at doses of 0.01, 0.1 and 1.0 nmol/L for 10 minutes followed by 10 minutes endothelin-1-free normal medium prior to 12 hours hypoxia. Lactate dehydrogenase activity and malondialdehyde level in the medium were determined at the end of hypoxia, and myocyte heat shock protein 70 and glucose-regulated protein 78 were assayed with Western blot. Lactate dehydrogenase activity and malondialdehyde content in the medium were significantly elevated after hypoxia (P < 0.01, n = 6). Heat shock protein 70 and glucoseregulated protein 78 expression in cardiomyocytes also increased significantly after hypoxia (P < 0.01 vs control, n = 3). Endothelin- 1 pretreatment reduced lactate dehydrogenase and malondialdehyde after hypoxia, and increased heat shock protein 70 and glucoseregulated protein 78 levels during normal culture and hypoxia. Glucose-regulated protein 78 antisense oligodeoxynucleotide partially abrogated the protective effect of endothelin-1 pretreatment on hypoxic cardiomyocyte injury. This study indicated that endothelin-1 pretreatment could protect hypoxic cardiomyocytes and might exert this effect through upregulation of heat shock protein 70 and glucose-regulated protein 78.

Obestatin, obesity and diabetes

The high prevalence of obesity and diabetes will lead to higher rates of morbidity and mortality. It is well known that ghrelin plays a potential role in obesity and diabetes. Obestatin, a novel 23 amino acid amidated peptide encoded by the same gene that encodes ghrelin, was initially reported to have opposite actions to ghrelin in the regulation of food intake, emptying of the stomach and body weight. Recent work suggests that obestatin also regulate beta-cell survival and insulin secretion. The ghrelin-obestatin system is, therefore, a promising target for the developing of new drugs for the treatment of obesity and diabetes. This review summarizes the interrelationship between obestatin, obesity and diabetes.