Inositol phosphatase SHIP1 is a primary target of miR-155

MicroRNA-155 (miR-155) has emerged as a critical regulator of immune cell development, function, and disease. However, the mechanistic basis for its impact on the hematopoietic system remains largely unresolved. Because miRNAs function by repressing specific mRNAs through direct 3′UTR interactions, we have searched for targets of miR-155 implicated in the regulation of hematopoiesis. In the present study, we identify Src homology-2 domain-containing inositol 5-phosphatase 1 (SHIP1) as a direct target of miR-155, and, using gain and loss of function approaches, show that miR-155 represses SHIP1 through direct 3′UTR interactions that have been highly conserved throughout evolution. Repression of endogenous SHIP1 by miR-155 occurred following sustained over-expression of miR-155 in hematopoietic cells both in vitro and in vivo, and resulted in increased activation of the kinase Akt during the cellular response to LPS. Furthermore, SHIP1 was also repressed by physiologically regulated miR-155, which was observed in LPS-treated WT versus miR-155^−/− primary macrophages. In mice, specific knockdown of SHIP1 in the hematopoietic system following retroviral delivery of a miR-155-formatted siRNA against SHIP1 resulted in a myeloproliferative disorder, with striking similarities to that observed in miR-155-expressing mice. Our study unveils a molecular link between miR-155 and SHIP1 and provides evidence that repression of SHIP1 is an important component of miR-155 biology.     

Basaloid squamous carcinoma of the esophagus with analysis by in situ nick end labeling and PCNA immunostaining

BACKGROUND/AIMS: Basaloid squamous carcinoma (BSC) of the esophagus has been associated with poor outcome after surgery. In this study, apoptosis and cell proliferation of BSC were studied to evaluate the malignant potential of this cancer. METHODOLOGY: In tissue samples from 4 out of 5 BSC patients (1 patient with pre-operative radiotherapy was excluded) and from 30 patients with squamous cell carcinomas (SCCs) of the esophagus, apoptosis was studied by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL), and cell proliferation was evaluated by the immunostaining of proliferating cell nuclear antigen (PCNA). Two of the 5 patients with BSC died of recurrence and the remaining 2 patients are alive without recurrence. RESULTS: TUNEL-positive cells and PCNA-positive cells were observed to exist randomly and independently in the BSC samples. In the well- and moderately differentiated SCCs, TUNEL-positive cells and PCNA-positive cells were locally observed. The apoptotic index was significantly lower in the BSC than in the SCC tissues, and the PCNA labeling index was significantly higher in BSC than in SCC. CONCLUSIONS: These results suggest that BSC of the esophagus has great proliferative activity and weak apoptosis. These characteristics may reflect the aggressive behavior and poor prognosis of esophageal BSC. Early detection of this tumor, as in the present 2 cases, will contribute to a better prognosis.     

MiR-205 is downregulated in hereditary hemorrhagic telangiectasia and impairs TGF-beta signaling pathways in endothelial cells

Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant disorder characterized by arteriovenous malformations and hemorrhages. This vascular disease results mainly from mutations in 2 genes involved in the TGF-β pathway (ENG and ALK1) that are exclusively expressed by endothelial cells. The present study identified miR-27a and miR-205 as two circulating miRNAs differentially expressed in HHT patients. The plasma levels of miR-27a are elevated while those of miR-205 are reduced in both HHT1 and HHT2 patients compared to healthy controls. The role of miR-205 in endothelial cells was further investigated. Our data indicates that miR-205 expression displaces the TGF-β balance towards the anti-angiogenic side by targeting Smad1 and Smad4. In line, overexpression of miR-205 in endothelial cells reduces proliferation, migration and tube formation while its inhibition shows opposite effects. This study not only suggests that detection of circulating miRNA (miR-27a and miR-205) could help for the screening of HHT patients but also provides a functional link between the deregulated expression of miR-205 and the HHT phenotype.     

miR-195 regulates SIRT1-mediated changes in diabetic retinopathy

Aims/hypothesis

Endothelial cell (EC) damage is a key mechanism causing retinal microvascular injury in diabetes. Several microRNAs (miRNAs) have been found to regulate sirtuin 1 (SIRT1, which is involved in regulation of the cell cycle, survival and metabolism) in various tissues and disease states, but no studies have been conducted on the role of miRNA in regulation of SIRT1 in diabetic retinopathy. Here we investigated the effect of miRNA-195 (miR-195), a SIRT1-targeting miRNA, on the development of diabetes-induced changes in ECs and retina.

Methods

The level of miR-195 was measured in human retinal and dermal microvascular ECs (HRECs, HMECs) following exposure to 25 mmol/l glucose (high glucose, HG) and 5 mmol/l glucose (normal glucose, NG). SIRT1 and fibronectin levels were examined following transfection with miR-195 mimic or antagomir or forced expression of SIRT1. Retinal tissues from diabetic rats were similarly studied following intravitreal injection of an miR-195 antagomir or mimic. In situ hybridisation was used to localise retinal miR-195.

Results

HG caused increased miR-195 levels and decreased SIRT1 expression (compared with NG) in both HRECs and HMECs. Transfection with miR-195 antagomir and forced expression of SIRT1 prevented such changes, whereas transfection with miR-195 mimic produced HG-like effects. A luciferase assay confirmed the binding of miR-195 to the 3′ untranslated region of SIRT1. miR-195 expression was upregulated in retinas of diabetic rats and intravitreal injection of miR-195 antagomir ameliorated levels of SIRT1.

Conclusions/interpretation

These studies identified a novel mechanism whereby miR-195 regulates SIRT1-mediated tissue damage in diabetic retinopathy.     

MYC stimulates EZH2 expression by repression of its negative regulator miR-26a

The MYC oncogene, which is commonly mutated/amplified in tumors, represents an important regulator of cell growth because of its ability to induce both proliferation and apoptosis. Recent evidence links MYC to altered miRNA expression, thereby suggesting that MYC-regulated miRNAs might contribute to tumorigenesis. To further analyze the impact of MYC-regulated miRNAs, we investigated a murine lymphoma model harboring the MYC transgene in a Tet-off system to control its expression. Microarray-based miRNA expression profiling revealed both known and novel MYC targets. Among the miRNAs repressed by MYC, we identified the potential tumor suppressor miR-26a, which possessed the ability to attenuate proliferation in MYC-dependent cells. Interestingly, miR-26a was also found to be deregulated in primary human Burkitt lymphoma samples, thereby probably being of clinical relevance. Although today only few miRNA targets have been identified in human disease, we could show that ectopic expression of miR-26a influenced cell cycle progression by targeting the bona fide oncogene EZH2, a Polycomb protein and global regulator of gene expression yet unknown to be regulated by miRNAs. Thus, in addition to directly targeting protein-coding genes, MYC modulates genes important to oncogenesis via deregulation of miRNAs, thereby vitally contributing to MYC-induced lymphomagenesis.     

Expression of cyclooxygenase-2 (COX-2) in human esophageal cancer and in vitro inhibition by a specific COX-2 inhibitor, NS-398

BACKGROUND: The purpose of the present paper was to study the expression of cyclooxygenase-2 (COX-2) in normal squamous epithelium, squamous dysplasia and squamous cell carcinoma (SCC) of the esophagus, to elucidate the role of COX-2 in esophageal carcinogenesis, and to evaluate the in vitro effect and mechanism of a COX-2 inhibitor, NS-398, in inducing growth inhibition and apoptosis of human esophageal cancer cells. METHODS: Biopsy specimens of esophageal dysplasia (n = 21), and surgical resections of SCC (n = 37) were compared with normal esophagus (n = 37) and analyzed by RT-PCR. Human esophageal cells were used for the study. Anti-proliferative effect was measured by MTT, apoptosis was determined by DNA fragmentation assay. RESULTS: Marked COX-2 expression was shown in SCC and esophageal squamous dysplasia, and no marked COX-2 expression was observed in the normal squamous epithelium, respectively. NS-398 could inhibit esophageal cells growth in a dose-dependent manner, induce apoptosis, and elevate caspase-3 activity in vitro. CONCLUSIONS: This study provides evidence that COX-2 is upregulated in the majority of cases of squamous dysplasia and SCC of esophagus, and that NS-398 can inhibit growth and induce apoptosis via activating caspase-3 activity in vitro. These results suggest that selective inhibitors of COX-2 may be an effective preventive and therapeutic option for esophageal carcinoma.     

Serum and urinary free microRNA level in patients with systemic lupus erythematosus

MicroRNAs circulating in body fluid have been suggested as biomarkers of various diseases. We studied the serum and urinary level of several miRNA species (miR-200 family, miR-205 and miR-192) in patients with systemic lupus erythematosus (SLE). We studied 40 SLE patients. Serum and urinary miRNA levels were determined and compared with that of healthy controls. The serum levels of miR-200a, miR-200b, miR-200c, miR-429, miR-205 and miR-192, and urinary miR-200a, miR-200c, miR-141, miR-429 and miR-192 of SLE patients were lower than those of controls. Glomerular filtration rate (GFR) correlated with serum miR-200b (r = 0.411, p = 0.008), miR-200c (r = 0.343, p = 0.030), miR-429 (r = 0.347, p = 0.028), miR-205 (r?=?0.429, p?=?0.006) and miR-192 (r = 0.479, p = 0.002); proteinuria inversely correlated with serum miR-200a (r?=?-0.375, p?=?0.017) and miR-200c (r =?-0.347, p =?0.029). SLE disease activity index (SLEDAI) inversely correlated with serum miR-200a (r?=?-0.376, p?=?0.017). Serum miR-200b (r = 0.455, p = 0.003) and miR-192 (r?=?0.589, p?相似文献   

Reciprocal effects of micro-RNA-122 on expression of heme oxygenase-1 and hepatitis C virus genes in human hepatocytes

BACKGROUND & AIMS: Heme oxygenase-1 (HO-1) is an antioxidant defense and key cytoprotective enzyme, which is repressed by Bach1. Micro-RNA-122 (miR-122) is specifically expressed and highly abundant in human liver and required for replication of hepatitis C virus (HCV) RNA. This study was to assess whether a specific miR-122 antagomir down-regulates HCV protein replication and up-regulates HO-1. METHODS: We transfected antagomir of miR-122, 2'-O-methyl-mimic miR-122, or nonspecific control antagomir, into wild-type (WT) Huh-7 cells or Huh-7 stably replicating HCV subgenomic protein core through nonstructural protein 3 of HCV (NS3) (CNS3 replicon cells) or NS3-5B (9-13 replicon cells). RESULTS: Antagomir of miR-122 reduced the abundance of HCV RNA by 64% in CNS3 and by 84% in 9-13 cells. Transfection with 2'-O-methlyl-mimic miR-122 increased HCV levels up to 2.5-fold. Antagomir of miR-122 also decreased Bach1 and increased HO-1 mRNA levels in CNS3, 9-13, and WT Huh-7 cells. Increasing HO-1 by silencing Bach1 with 50 nmol/L Bach1-short interfering RNA or by treatment with 5 mumol/L cobalt protoporphyrin or heme (known inducers of HO-1) decreased HCV RNA and protein by 50% in HCV replicon cells. CONCLUSIONS: Down-regulation of HCV replication using an antagomir targeted to miR-122 is effective, specific, and selective. Increasing HO-1, by silencing the Bach1 gene or by treatment with cobalt protoporphyrin or heme, decreases HCV replication. Thus, miR-122 plays an important role in the regulation of HCV replication and HO-1/Bach1 expression in hepatocytes. Down-regulation of miR-122 and up-regulation of HO-1 may be new strategies for anti-HCV intervention and cytoprotection.     

Steatotic hepatocyte-derived extracellular vesicles promote β-cell apoptosis and diabetes via microRNA-126a-3p

Extracellular vesicles (EVs) have emerged as a unique mediator of interorgan communications, playing important roles in the pathophysiologic process of various diseases, including diabetes and other metabolic diseases. Here, we reported that the EVs released by steatotic hepatocytes exerted a detrimental effect on pancreatic β cells, leading to β-cell apoptosis and dysfunction. The effect was profoundly attributable to an up-regulation of miR-126a-3p in the steatotic hepatocyte-derived EVs. Accordingly, overexpression of miR-126a-3p promoted, whereas inhibition of miR-126a-3p prevented β-cell apoptosis, through a mechanism related to its target gene, insulin receptor substrate-2. Moreover, inhibition of miR-126a-3p by its specific antagomir was able to partially reverse the loss of β-cell mass and ameliorate hyperglycaemia in diabetic mice. Thus, the findings reveal a novel pathogenic role of steatotic hepatocyte-derived EVs, which mechanistically links nonalcoholic fatty liver disease to the development of diabetes.     

SHIP inhibits Akt activation in B cells through regulation of Akt membrane localization

Activation of the serine/threonine kinase Akt and the regulation of its activation are recognized as critical in controlling proliferative/survival signals via many hematopoietic receptors. In B lymphocytes, the B-cell receptor (BCR)-mediated activation of Akt is attenuated by co-cross-linking of BCR with the inhibitory receptor Fc gamma RIIB1, and the binding of the SH2 domain-containing inositol phosphatase, SHIP, to Fc gamma RIIB1. Because SHIP dephosphorylates phosphatidylinositol 3,4,5-trisphosphate (PIP3) and activation of Akt requires PIP3, the destruction of this phospholipid has been proposed as the mechanism for Akt inhibition. However, upstream kinases that activate Akt, such as PDK1, also require PIP3 for activation. In this report, we addressed whether SHIP inhibits Akt directly at the level of Akt recruitment to the membrane, indirectly through PDK recruitment/phosphorylation of Akt, or both. We generated stable B-cell lines expressing a regulatable, but constitutively membrane-bound Akt that still required PDK-dependent phosphorylation for activation. Several lines of evidence suggested that activation of this membrane-targeted Akt is not inhibited by Fc gamma RIIB1/SHIP and that PDK is not a target for SHIP-mediated inhibition. These data demonstrate that SHIP inhibits Akt primarily through regulation of Akt membrane localization. We also observed during these studies that Fc gamma RIIB1/SHIP does not inhibit p70(S6k) activation, even though several other PIP3-dependent events were down-regulated. Because the enhanced activation of Akt in the absence of SHIP correlates with hyperproliferation in the myeloid lineage, our data have implications for SHIP and Akt-dependent regulation of proliferation in the hematopoietic lineage. (Blood. 2000;96:1449-1456)     

MiRNA profile in esophageal squamous cell carcinoma: downregulation of miR-143 and miR-145

AIM:To investigate the expression profile of miRNA in esophageal squamous cell carcinoma(ESCC).METHODS:The expression profile of miRNA in ESCC tissues was analyzed by miRNA microarray.The expression levels of miR-143 and miR-145 in 86 ESCC patients were determined by real-time polymerase chain reaction(PCR) using TaqMan assay.The mobility effect was estimated by wound-healing using esophageal carcinoma cells transfected with miRNA expression plasmids.RESULTS:A set of miRNAs was found to be deregulated in th...     

miR-29a up-regulation in AR42J cells contributes to apoptosis via targeting TNFRSF1A gene

AIM: To investigate the expression of mi R-29 a in rat acute pancreatitis and its functional role in AR42 J cell apoptosis.METHODS: Twelve SD rats were divided into a control group and an acute edematous pancreatitis(AEP) group randomly. AEP was induced by intraperitoneal injection of L-arginine(150 mg/kg) in the AEP group and equal volume of 0.9% Na Cl was injected in the control group. The apoptosis of acinar cells in pancreatic tissue was determined by TUNEL assay. mi RNA chip assay was performed to examine the expression of mi RNAs in two groups. Besides, to further explore the role of mi R-29 a in apoptosis in vitro, recombinant rat TNF-α(50 ng/m L) was administered to treat the rat pancreatic acinar cell line AR42 J for inducing AR42 J cell apoptosis. Quantitative real-time PCR(q RT-PCR) was adopted to measure mi R-29 a expression. Then, mi RNA mimic, mi RNA antisense oligonucleotide(AMO) and control vector were used to transfect AR42 J cells. The expression of mi R-29 a was confirmed by q RT-PCR andthe apoptosis rate of AR42 J cells was detected by flow cytometry analysis. Western blot was used to detect the expression of activated caspase3. Moreover, we used bioinformatics software and luciferase assay to test whether TNFRSF1 A was the target gene of mi R-29 a. After transfection, q RT-PCR and Western blot was used to detect the expression of TNFRSF1 A in AR42 J cells after transfection.RESULTS: The expression of mi R-29 a was much higher in the AEP group compared with the control group as displayed by the mi RNA chip assay. After inducing apoptosis of AR42 J cells in vitro, the expression of mi R-29 a was significantly increased by 1.49 ± 0.04 times in comparison with the control group. As revealed by q RT-PCR assay, the expression of mi R-29 a was 2.68 ± 0.56 times higher in the mi R-29 a mimic group relative to the control vector group, accompanied with an obviously increased acinar cell apoptosis rate(42.83 ± 1.25 vs 24.97 ± 0.15, P 0.05). Moreover, the expression of mi R-29 a in the mi RNA AMO group was 0.46 ± 0.05 times lower than the control vector group, and the cell apoptosis rate was much lower accordingly(17.27 ± 1.36 vs 24.97 ± 0.15, P 0.05). The results of bioinformatics software and luciferase assay showed that TNFRSF1 A might be a target gene of mi R-29 a. TNFRSF1 A expression was up-regulated in the mi R-29 a mimic group, while the mi R-29 a AMO group showed the reverse trend.CONCLUSION: mi R-29 a might promote the apoptosis of AR42 J cells via up-regulating the expression of its target gene TNFRSF1 A.     

miR-155 regulates IFN-γ production in natural killer cells

MicroRNAs (miRs) are small, noncoding RNA molecules with important regulatory functions whose role in regulating natural killer (NK) cell biology is not well defined. Here, we show that miR-155 is synergistically induced in primary human NK cells after costimulation with IL-12 and IL-18, or with IL-12 and CD16 clustering. Over-expression of miR-155 enhanced induction of IFN-γ by IL-12 and IL-18 or CD16 stimulation, whereas knockdown of miR-155 or its disruption suppressed IFN-γ induction in monokine and/or CD16-stimulated NK cells. These effects on the regulation of NK cell IFN-γ expression were found to be mediated at least in part via miR-155's direct effects on the inositol phosphatase SHIP1. Consistent with this, we observed that modulation of miR-155 overrides IL-12 and IL-18-mediated regulation of SHIP1 expression in NK cells. Collectively, our data indicate that miR-155 expression is regulated by stimuli that strongly induce IFN-γ in NK cells such as IL-12, IL-18, and CD16 activation, and that miR-155 functions as a positive regulator of IFN-γ production in human NK cells, at least in part via down-regulating SHIP1. These findings may have clinical relevance for targeting miR-155 in neoplastic disease.     

A rare SNP in pre-miR-34a is associated with increased levels of miR-34a in pancreatic beta cells

Changes in the levels of specific microRNAs (miRNAs) can reduce glucose-stimulated insulin secretion and increase beta-cell apoptosis, two causes of islet dysfunction and progression to type 2 diabetes. Studies have shown that single nucleotide polymorphisms (SNPs) within miRNA genes can affect their expression. We sought to determine whether miRNAs, with a known role in beta-cell function, possess SNPs within the pre-miRNA structure which can affect their expression. Using published literature and dbSNP, we aimed to identify miRNAs with a role in beta-cell function that also possess SNPs within the region encoding its pre-miRNA. Following transfection of plasmids, encoding the pre-miRNA and each allele of the SNP, miRNA expression was measured. Two rare SNPs located within the pre-miRNA structure of two miRNA genes important to beta-cell function (miR-34a and miR-96) were identified. Transfection of INS-1 and MIN6 cells with plasmids encoding pre-miR-34a and the minor allele of rs72631823 resulted in significantly (p < 0.05) higher miR-34a expression, compared to cells transfected with plasmids encoding the corresponding major allele. Similarly, higher levels were also observed upon transfection of HeLa cells. Transfection of MIN6 cells with plasmids encoding pre-miR-96 and each allele of rs41274239 resulted in no significant differences in miR-96 expression. A rare SNP in pre-miR-34a is associated with increased levels of mature miR-34a. Given that small changes in miR-34a levels have been shown to cause increased levels of beta-cell apoptosis this finding may be of interest to studies looking at determining the effect of rare variants on type 2 diabetes susceptibility.     

微RNA 18与B细胞易位基因2在肝癌细胞中差异表达相关性的研究

目的 研究微RNA 18(miR-18)对肝癌细胞HepG2基因表达谱的影响,预测其靶基因,初步探讨miR-18与抗增殖基因B细胞易位基因2(BTG2)两者在肝癌发生过程中差异表达的相关性.方法 利用基因表达谱芯片技术筛选HepG2的微RNA差异表达谱,应用生物信息学方法预测表达明显上调的miR-18的靶基因,初步筛选出直接调控的靶基因为抗增殖基因BTG2;应用RT-PCR和Northern blot法分析BTG2正常与肝癌组织中的表达情况.结果 生物信息学分析结果显示miR-18分子调控的下游靶基因有609个,涉及细胞增殖、分化和凋亡,转录调节等众多生理和病理过程;抗增殖基因BTG2在肝癌组织和细胞中呈明显低表达.结论 miR-18在肝癌细胞中表达明显上调,并可能负性调控抗增殖基因BTG2在肝癌细胞中的表达,两者共同在肝癌细胞增殖方面发挥着重要作用.     

Involvement of human micro-RNA in growth and response to chemotherapy in human cholangiocarcinoma cell lines

BACKGROUND & AIMS: Micro-RNA (miRNA) are endogenous regulatory RNA molecules that modulate gene expression. Alterations in miRNA expression can contribute to tumor growth by modulating the functional expression of critical genes involved in tumor cell proliferation or survival. Our aims were to identify specific miRNA involved in the regulation of cholangiocarcinoma growth and response to chemotherapy. METHODS: miRNA expression in malignant and nonmalignant human cholangiocytes was assessed using a microarray. Expression of selected miRNA and their precursors was evaluated by Northern blots and real-time polymerase chain reaction, respectively. The effect of selected miRNA on cell growth and response to chemotherapy was assessed using miRNA-specific antisense oligonucleotides to decrease miRNA expression or with precursor miRNA to increase cellular expression. RESULTS: miRNA expression was markedly different in malignant cholangiocytes, with decreased expression of many miRNA compared with nonmalignant cells. A cluster of miRNA, including miR-320, miR-200b, miR-21, miR-23a, miR-141, miR-27a, and miR-34a, were expressed in all cell lines. MiR-21, miR-141, and miR-200b were highly over-expressed in malignant cholangiocytes. Inhibition of miR-21 and miR-200b increased sensitivity to gemcitabine, whereas inhibition of miR-141 decreased cell growth. Treatment of tumor cell xenografts with systemic gemcitabine altered the expression of a significant number of miRNA. miR-21 modulates gemcitabine-induced apoptosis by phosphatase and tensin homolog deleted on chromosome 10 (PTEN)-dependent activation of PI 3-kinase signaling. Potential target genes that were modulated by selected miRNA were identified. CONCLUSIONS: Alterations in miRNA expression contribute to tumor growth and response to chemotherapy. Aberrantly expressed miRNA or their targets will provide mechanistic insight and therapeutic targets for cholangiocarcinoma.