Number Of microRNAs Research Articles

TGFβ1 Suppresses Neuronal IGF1 via a Let-7F-Dependent Mechanism During Azoxymethane-Induced Hepatic Encephalopathy in Mice

Background and Aim: Hepatic encephalopathy (HE) is a neurological complication that arises after a loss of liver function. HE is associated with increased cerebral edema, neuroinflammation and the onset of cognitive decline. We have previously shown that there is increased expression and secretion of transforming growth factor β1 (TGFβ1) from hepatocytes during the azoxymethane (AOM) model of acute liver failure and HE. Increased circulating TGFβ1 was shown to promote blood-brain barrier permeability and also led to progressive neurological complications via increased activation of its receptor TGFβR2. Insulin-like growth factor 1 (IGF1) is a neuroprotective peptide that can be suppressed by TGFβ1 signaling in other organs. IGF1 expression is regulated by a number of microRNAs, including Let-7f, which is positively regulated by TGFβ1. Therefore, we hypothesize that circulating hepatic-derived TGFβ1 suppresses neural IGF1 via a mechanism involving Let-7f during HE, which subsequently exacerbates neurological decline. Methods: In order to determine the exact contribution of TGFβ1 liver-brain axis signaling in this model, male C57Bl/6 (WT) mice, hepatocyte-specific TGFβ1 knockout mice (floxed TGFβ1 × albumin-Cre) or neuron-specific TGFβR2 knockout mice (floxed TGFβR2 × Thy1-Cre) were injected with the hepatotoxin azoxymethane (AOM). In parallel, WT mice were treated with an anti-TGFβ neutralizing antibody 1 h prior to AOM, or were infused with rIGF1 into the lateral ventricle for 3 days prior to AOM injection. Cognitive impairment was monitored and livers, serum and whole brains were collected at coma. IGF1, TGFβ1, Let-7f and proinflammatory cytokine expression were assessed by immunoblotting, immunohistochemistry and/or qPCR. Microglia were stained by IBA1 and cortex field staining and cell morphology were assessed. In vitro, mouse neurons were transfected with a specific Let-7f inhibitor prior to treatment with rTGFβ1 and the expression of Let-7f, IGF1 and CCL2 were measured. Results: Mice injected with AOM had increased Let-7f and suppressed cortical IGF1 expression. Strategies to inhibit TGFβ1 signaling in the brain (ie hepatocyte-specific TGFβ1 knockout, neuron-specific TGFβR2 knockout or pretreatment with TGFβ neutralizing antibody) attenuated the (i) induction of Let-7f expression, (ii) suppression of cortical IGF1, (iii) neuroinflammation and (iv) neurological decline when compared to WT mice treated with AOM alone. In vitro, treatment of neurons with rTGFβ1 increased the expression of Let-7f and reduced the expression of IGF1 in a Let-7f-dependent manner, while also inducing CCL2 expression. Lastly, rIGF1 infusion in the brain delayed AOM-induced neurological decline and reduced neuroinflammation. Conclusion: Elevated TGFβ1 signaling during HE leads to increased cortical Let-7f expression, decreased IGF1 expression and worse outcomes in AOM-treated mice. These deleterious effects can be reversed by inhibiting neuronal TGFβ1 signaling or by increasing IGF1 concentrations in the brain, indicating that elevated TGFβ1 and suppressed IGF1 contribute to the progression of HE. The authors have none to declare.

MiR-600 inhibits cell proliferation, migration and invasion by targeting p53 in mutant p53-expressing human colorectal cancer cell lines.

Mutations of the tumor protein p53 gene, a tumor suppressor, are one of the most frequent genetic alterations observed in cancer. It has been reported that mutations in p53 result in the loss of wild-type p53 activity, and the gain of novel oncogenic properties that promote tumor growth and progression. Recent studies have demonstrated that a number of microRNAs (miRs) are involved in the post-transcriptional regulation of p53. The present study demonstrates that miR-600 is a direct negative regulator of p53 through binding a site in the 3′ untranslated region of p53 mRNA in human colorectal cancer (CRC) cells. Overexpression of miR-600 by lentiviral-mediated transduction decreased endogenous levels of p53 protein and inhibited cell proliferation, migration and invasion in mutant p53-expressing human CRC cell lines (SW480, SW620 and DLD-1) in vitro. In addition, silencing of p53 with small interfering RNA led to a similar phenotype. Furthermore, overexpression of miR-600 or p53 knockdown suppressed the expression of matrix metalloproteinase 9, and promoted the expression of E-cadherin and β-catenin. The results of the current study demonstrate that miR-600 is an important negative regulator of p53, and suggest that targeting mutant p53 using lentiviral-mediated miR-600 overexpression is a promising therapeutic strategy for the treatment of CRCs with p53 mutations.

MicroRNA-302d downregulates TGFBR2 expression and promotes hepatocellular carcinoma growth and invasion.

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-associated mortality in China and the third leading cause worldwide. A number of microRNAs (miRNAs) have been implicated in cell cycle progression, growth, apoptosis, angiogenesis and metastasis in HCC. In the present study, reverse transcription-quantitative polymerase chain reaction analysis was used to detect the levels of miR-302d expression in the tissues of 30 patients with HCC. Cell cycle, growth, apoptosis and migration were analyzed using a cell counting kit, flow cytometry and a Transwell migration assay. Dual-luciferase reporter assays and western blotting were also used to analyze the expression levels of transforming growth factor beta type II receptor (TGFBR2) in HCC cells. The present study evaluated the role of miR-302d in the development and progression of HCC. Abnormally high expression of miR-302d was observed in 80% of HCC specimens. Moreover, patients with lower levels of miR-302d expression experienced a longer survival time than those with higher levels of miR-302d expression. It was demonstrated that miR-302d promoted HCC cell growth and migration, suppressed cell apoptosis and affected cell cycle distribution in vitro, and augmented tumorigenicity in vivo. Furthermore, TGFBR2, which is a tumor suppressor, was confirmed as a target of miR-302d in HCC cells. Dual-luciferase reporter assays indicated that TGFBR2 expression was negatively regulated by miR-302d. Taken together, the results of the present study suggest that miR-302d may serve as a valuable tool for predicting the prognosis of patients with HCC.

Comparison of microRNA expression profiles in K562-cells-derived microvesicles and parental cells, and analysis of their roles in leukemia.

Microvesicles (MVs) are 30-1,000-nm extracellular vesicles that are released from a multitude of cell types and perform diverse cellular functions, including intercellular communication, antigen presentation, and transfer of proteins, messenger RNA and microRNA (also known as miR). MicroRNAs have been demonstrated to be aberrantly expressed in leukemia, and the overall microRNA expression profile may differentiate normal blood cells vs. leukemia cells. MVs containing microRNAs may enable intercellular cross-talk in vivo. This prompted us to investigate specific variations of microRNA expression patterns in MVs derived from leukemia cells. The present study examined the microRNA expression profile of MVs from chronic myeloid leukemia K562 cells and that of MVs from normal human volunteers' peripheral blood cells. The potential targets of the differentially expressed microRNAs were predicted using computational searches. Bioinformatic analyses of the predicted target genes were performed for further evaluation. The present study analyzed microRNAs of MVs derived from leukemia and normal cells, and characterized specific microRNAs expression. The results revealed that MVs derived from K562 cells expressed 181 microRNAs of the 888 microRNAs assessed. Further analysis revealed that 16 microRNAs were downregulated, while 7 were upregulated in these MVs. In addition, significant differences in microRNA expression profiles between MVs derived from K562 cells and K562 cells were identified. The present results revealed that 77 and 122 microRNAs were only expressed in MVs derived from K562 cells and in K562 cells, respectively. There were 104 microRNAs co-expressed in MVs derived from K562 cells and in K562 cells. Target gene-related pathway analyses demonstrated that the majority of the dysregulated microRNAs were involved in pathways associated with leukemia, particularly the mitogen-activated protein kinase (MAPK) and the p53 signaling pathways. By further conducting microRNA gene network analysis, the present study revealed that the miR-15a/b, miR-16, miR-17 and miR-30 families were likely to play a role in the regulation of the MAPK signaling pathway. Since K562 cells presented the t(9;22) translocation, the current study further examined the predicted function of 12 microRNAs located in chromosomes 9 [Homo sapiens (hsa)-let-7a, hsa-let-7f, miR-126, miR-126*, miR-23b, miR-24, miR-27b and miR-7] and 22 (hsa-let-7b, miR-1249, miR-130b and miR-185), which were expressed both in MVs derived from K562 cells and in K562 cells. The present study identified microRNAs of MVs from leukemia and normal cells, and characterized the expression of specific microRNAs. The current study is also the first to identify and characterize distinct microRNA expression between MVs derived from K562 cells and K562 cells. These findings highlight that a number of microRNAs from leukemia-derived MVs may contribute to the development of hematopoietic malignancies. Further investigation may reveal the function of these differentially expressed microRNAs and may provide potential targets for novel therapeutic strategies.

Circulation Research “In This Issue” Anthology

Circulation Research “In This Issue” Anthology

Role of c-MYC/miRNA-548/HDAC6 axis in drug resistance and lymphoma survival

The B cell lymphoproliferative malignancies (1) arise from B cells that require interaction with non-malignant cells and stromal cells during lymphomagenesis (2,3). There has been improvement in overall survival in these lymphomas (4), however, a significant proportion (30–40%) of cases relapse even with the introduction of rituximab in therapeutic regimens (3). Gene expression profiling studies reveal signatures originating from the microenvironment at diagnosis are significantly associated with response to therapy in follicular lymphoma (FL) and diffuse large B cell lymphomas (DLBCL) (5). Moreover, the diffuse distribution of follicular dendritic cells (FDCs) in mantle cell lymphoma (MCL) is associated with worse clinical outcome (5,6). These observations indicate that interaction between stroma and B cell lymphoma cells contributes to drug resistance and support neoplastic cell survival. A number of microRNAs (miRNAs) are dysregulated when lymphoma cells adhere to lymph node stromal cells in MCL and other B cell lymphomas (7). The dysregulated miRNAs critically influence the tumor development, progression and have been reported to be the important prognosticators in human cancers including lymphomas (8). Recently, prognostic miRNAs have identified in several NHL including DLBCL, MCL, FL and PTCL (8,9).

Effect of miRNA-205 on VEGFA and FGF2 and on resistance to chemotherapeutics in breast cancer.

1028Background: Multidrug resistance (MDR) remains one of the most significant obstacles in breast cancer treatment, and this process often involves dysregulation of a great number of microRNAs (miRNAs). miR-205 has been reported to be negatively correlated with proliferation and metastasis of many types of cancer, while mechanism of chemoresistance mediated by miR-205 in breast cancer have not been fully elucidated. Methods: Taqman RT-PCR analysis was utilized to examine miR-205 expression in tumor tissues from the breast cancer patients before neoadjuvant chemotherapy treatment, as well as in two drug-resistant derivatives of MCF-7 and Cal51 cells. Experimental modulation of miR expression has been obtained by lentiviral transfection. Taxol and doxorubicin IC50 values were obtained by short-term drug sensitivity assays. Apoptosis was determined by flow cytometry after annexin V staining, by caspase 3/7 and caspase 9 activity assays and the levels of apoptosis-related protein cleaved PARP and cleaved cas...

MicroRNA-222 regulates muscle alternative splicing through Rbm24 during differentiation of skeletal muscle cells.

A number of microRNAs have been shown to regulate skeletal muscle development and differentiation. MicroRNA-222 is downregulated during myogenic differentiation and its overexpression leads to alteration of muscle differentiation process and specialized structures. By using RNA-induced silencing complex (RISC) pulldown followed by RNA sequencing, combined with in silico microRNA target prediction, we have identified two new targets of microRNA-222 involved in the regulation of myogenic differentiation, Ahnak and Rbm24. Specifically, the RNA-binding protein Rbm24 is a major regulator of muscle-specific alternative splicing and its downregulation by microRNA-222 results in defective exon inclusion impairing the production of muscle-specific isoforms of Coro6, Fxr1 and NACA transcripts. Reconstitution of normal levels of Rbm24 in cells overexpressing microRNA-222 rescues muscle-specific splicing. In conclusion, we have identified a new function of microRNA-222 leading to alteration of myogenic differentiation at the level of alternative splicing, and we provide evidence that this effect is mediated by Rbm24 protein.

MyoD is regulated by the miR-29a-Tet1 pathway in C2C12 myoblast cells.

Skeletal myogenesis is regulated by a considerable number of microRNAs (miRNAs). miRNA regulatory networks are complicated, and details of how they operate remain unclear. In this study, MTT assays confirmed that miR-29a is the most effective miR-29 paralog. Microarray analysis demonstrated upregulation of ten-eleven translocation enzyme-1 (Tet1) mRNA in response to miR-29a inhibition in C2C12 murine myoblast cells. We investigated the factors acting downstream in the miR-29a-Tet1 signal pathway using real-time RT-PCR. MyoD expression was upregulated by Tet1 inhibition and downregulated by miR-29a inhibition, whereas expression of cyclin-dependent kinase 6 (Cdk6) was regulated in an opposite manner. These results suggest that the miR-29a-Tet1 pathway upregulates MyoD expression and conversely downregulates Cdk6 expression. However, changes in the expression of other myogenic factors such as serum response factor (Srf), the myocyte enhancer factor 2 family (Mef2a, b and c), myogenin, myogenic regulatory factor 4 (Mrf4), muscle creatine kinase (Mck), and other cell cycle regulators such as Cdk4 and thymine DNA glycosylase (Tdg) cannot be explained in terms of the miR-29a-Tet1 pathway alone. The miR29a-Tet1 pathway may be part of a complex myogenic regulatory network in C2C12 cells. (J Oral Sci 58, 219-229, 2016).

Role of miR-146a in Enforcing Effect of Specific Immunotherapy on Allergic Rhinitis

ABSTRACTAllergic rhinitis (AR) is one of the common disorders in airway allergic inflammation. The pathogenesis of AR is unclear. It is accepted that immune deregulation is associated with the pathogenesis of AR. Recent reports suggest that a large number of micro RNAs (miR) can regulate immune functions. This study aims to investigate the role of miR-146a in an enforcing immunotherapy of AR. In this study, a mouse AR model was created. The levels of miR-146a in the mouse nasal mucosa were assessed by real time RT-PCR. A specific immunotherapy was performed in AR mice. The results showed that the AR mice had an AR-like inflammation in the nasal mucosa. Compared with naïve mice, markedly lower levels of miR-146a were detected in AR mice. The co-administration with miR-146a significantly enforced the effect of ovalbumin (OVA)-specific immunotherapy on inhibition of AR inflammation in the nasal mucosa. Further analysis showed that miR-146a induced transforming growth factor-β in dendritic cells; the latter induced naïve CD4+ T cells to differentiate into regulatory T cells. In conclusion, miR-146a can enforce OVA-specific immunotherapy via inducing antigen-specific regulatory T cells. miR-146a may have therapeutic potential to be used in the immunotherapy of allergic diseases.

MicroRNA-147 suppresses proliferation, invasion and migration through the AKT/mTOR signaling pathway in breast cancer.

The Akt/mTOR pathway is considered to be the most frequently aberrantly activated pathway in human tumors. It is activated in a variety of types of tumor, and is therefore an attractive target for study, including it's potential regulation by microRNAs. A number of microRNAs (miRs) have been demonstrated to target the Akt/mTOR pathway. A previous study reported that miR-147 targets the EGFR-driven cell-cycle protein network in breast cancer. EGFR serves a crucial role upstream to Akt/mTOR. To define the function and mechanism of miR-147 in breast cancer, the present study assessed miR-147 expression in a normal mammary epithelial cell line and three breast cancer cell lines, and observed that miR-147 expression was markedly low in the highly invasive cell line, MDA-MB-231. Ectopic expression of miR-147 in MDA-MB-231 resulted in a reduction of the phosphorylation of crucial molecules in the Akt/mTOR pathway and the proliferation, invasion and migration of the cell line was also reduced. The effects of miR-147 expression are similar to that of shRNA which is specifically designed to silence the expression of Akt. The findings of the present study indicate that miR-147 suppressed the proliferation, invasion and migration of breast cancer cells through targeting the Akt/mTOR signaling pathway. As a new microRNA targeting Akt/mTOR pathway. Using miR-147 may therefore provide an effective therapeutic approach to suppress tumorigenicity in breast cancer.

Abstract IA21: The MYC-microRNA pathway in B-cell neoplasms

Abstract MYC is a noncanonical transcription factor that binds to thousands of genomic loci and affects over 15% of the human transcriptome, with surprisingly little overlap between MYC-bound and MYC-regulated genes. This discordance raises the question whether MYC chooses its targets based on their individual biological effects (“a la carte”) or by virtue of belonging to a certain group of genes (on a “prix fixe” basis). Over the last few years, we have developed a model wherein MYC initially deregulates a select number of microRNAs. These microRNAs then target a broad spectrum of genes based solely on the presence in their 3' UTRs (untranslated regions) of distinct “seed” sequences. Existing evidence suggests that there are significant microRNA components to all key MYC-driven phenotypes, including cell-cycle progression, apoptosis, metabolism, angiogenesis, metastasis, stemness, and last but not least – hematopoiesis (1). For example in a human B-lymphoid cell line, Myc-repressed genes are significantly enriched for predicted binding sites of Myc-regulated miRNAs, primarily those comprising the Myc-activated miR-17-92 cluster. Notably, gene set enrichment analysis demonstrates that miR-17-92 is a major regulator of B-cell receptor (BCR) pathway components. Many of them are immunoreceptor tyrosine inhibitory motif (ITIM)-containing proteins, and ITIM proteins CD22 and FCGR2B were found to be direct targets of miR-17-92. Consistent with the propensity of ITIM proteins to recruit phosphatases, either MYC or miR-17-92 expression is necessary to sustain phosphorylation of spleen tyrosine kinase (SYK) and the B-cell linker protein (BLNK) upon ligation of the BCR. Further downstream, stimulation of the BCR response by miR-17-92 resulted in the enhanced calcium flux, activation of the CD19-PI3K axis, and elevated levels of Myc itself (2). Notably, inhibition of the miR-17-92 cluster in diffuse large B-cell lymphoma (DLBCL) cell lines diminished the BCR response as measured by SYK and BLNK phosphorylation. Conversely, human DLBCLs of the BCR subtype express higher Myc and mir17hg transcript levels than other subtypes. Hence, the Myc-miR-17-92-BCR axis, frequently affected by genomic rearrangements, constitutes a novel lymphomagenic feed-forward loop (3).

Regulation of gene expression in rats with spinal cord injury based on microarray data.

The present study aimed to investigate the molecular mechanisms of spinal cord injury (SCI) in rats. First, the differentially expressed genes (DGEs) were screened based on GSE45006 microarray data downloaded from Gene Expression Omnibus using the significant analysis of microarray (SAM) method. Screening was performed for DEGs which were negatively or possibly correlated with time and subsequently subjected to gene ontology (GO) functional annotation. Furthermore, pathway enrichment analysis using the Kyoto Encyclopedia of Genes and Genomes was also performed. In addition, a protein-protein interaction (PPI) network was constructed using the Search Tool for the Retrieval of Interacting Genes/Proteins database. Finally, GeneCodis was used to seek transcription factors and microRNAs that are involved in the regulation of DEGs. A total of 806 DEGs were upregulated and 549 DEGs were downregulated in the rats with SCI. Cholesterol metabolism-associated genes (e.g. HMGCS1, FDFT1 and IDI1) were negatively correlated with time, while injury genes (e.g. SERPING1, C1S and RAB27A) were positively correlated with time after SCI. PCNA, MCM2, JUN and SNAP25 were the hub proteins of the PPI network. The transcription factors LEF1 and SP1 were observed to be associated with the regulation of two DEGs that were involved in the cholesterol-associated metabolism as well as injury responses. A number of microRNAs (e.g. miR210, miR-487b and miR-16) were observed to target cholesterol metabolism-associated DGEs. The hub genes PCNA, MCM2, JUN and SNAP25 presumably have critical roles in rats with SCI, and the transcription factors LEF1 and SP1 may be important for the regulation of cholesterol metabolism and injury responses following SCI.

Moving from discovery to validation in circulating microRNA research.

MicroRNAs (miRNAs), small noncoding RNAs, are involved in tumorigenesis and in the development of various cancers. Quantitative real-time polymerase chain reaction (qPCR) is the most commonly used tool to investigate miRNA expression, and qPCR low-density arrays are increasingly being used as an experimental technique for both the identification of potentially relevant miRNAs and their subsequent validation. Due to the reduced number of microRNAs to be validated, this phase is generally performed on ad hoc customized cards for which a technical robustness is assumed similar to that of the high-throughput cards used during the identification phase. With the aim of investigating the degree of reproducibility between the 2 types of cards, we analyzed plasma-circulating miRNAs evaluated in 60 subjects enrolled in a colorectal cancer screening program. Our results showed a reproducibility between the 2 methods that was not fully satisfactory, with a concordance correlation coefficient equal to 0.69 (95% confidence interval, 0.12-0.92). This report highlights the need to add a technical validation step to the high-throughput-based miRNA identification workflow, after their discovery and before the validation step in an independent series.

Endogenous antiviral microRNAs determine permissiveness for hepatitis B virus replication in cultured human fetal and adult hepatocytes.

Superior cell culture models for hepatitis B virus (HBV) will help advance insights into host-virus interactions. To identify mechanisms regulating HBV replication, this study used cultured human HepG2 cells and adult or fetal hepatocytes transduced with adenoviral vector to express HBV upstream of green fluorescent protein. The vector efficiently transduced all cell types. In HepG2 cells, replicative viral intermediates, nucleocapsid-associated HBcAg, and HBsAg were expressed. However, in fetal or adult hepatocytes, pregenomic HBV RNA and viral RNAs were expressed, but nucleocapsid-associated HBcAg in cells or HBsAg in culture medium were absent, indicating interruptions in viral replication due to possible microRNA-related interference. MicroRNA profiling demonstrated that a large number of microRNAs with antiviral potential were differentially expressed in hepatocytes after culture. In transfection assays using HepG2 cells, candidate antiviral microRNAs, e.g., hsa-miR-24 or hsa-miR-638 decreased the levels of HBV transcripts or HBV gene products. Since candidate microRNAs could have targeted interferon response genes as an alternative explanation interferon signaling was examined. However, HBV replication in cultured hepatocytes was not restored despite successful inhibition of JAK1/2-STAT signaling by the inhibitor, ruxolitinib. Therefore, HBV was unable to complete replication in cultured hepatocytes due to expression of multiple antiviral microRNAs. This mechanism should help understand restrictions in HBV replication for developing HBV models in cultured cells while providing frameworks for pathophysiological studies of HBV replication in subsets of hepatocytes or stem/progenitor cells during hepatitis.

The microRNA feedback regulation of p63 in cancer progression.

The transcription factor p63 is a member of the p53 gene family that plays a complex role in cancer due to its involvement in epithelial differentiation, cell cycle arrest and apoptosis. MicroRNAs are a class of small, non-coding RNAs with an important regulatory role in various cellular processes, as well as in the development and progression of cancer. A number of microRNAs have been shown to function as transcriptional targets of p63. Conversely, microRNAs also can modulate the expression and activity of p63. However, the p63-microRNA regulatory circuit has not been addressed in depth so far. Here, computational genomic analysis was performed using miRtarBase, Targetscan, microRNA.ORG, DIANA-MICROT, RNA22-HSA and miRDB to analyze miRNA binding to the 3'UTR of p63. JASPAR (profile score threshold 80%) and TFSEARCH datasets were used to search transcriptional start sites for p53/p63 response elements. Remarkably, these data revealed 63 microRNAs that targeted p63. Furthermore, there were 39 microRNAs targeting p63 that were predicted to be regulated by p63. These analyses suggest a crosstalk between p63 and microRNAs. Here, we discuss the crosstalk between p63 and the microRNA network, and the role of their interactions in cancer.

MiR-522 contributes to cell proliferation of human glioblastoma cells by suppressing PHLPP1 expression

Previous studies have shown that microRNAs play essential roles in cancer growth and progression. Although a number of microRNAs were differentially expressed in glioblastoma (GBM). In this study, we evaluated the miR-522s role in cell proliferation in GBM. Expression of miR-522 is markedly upregulated in GBM tissues and GBM cells compared with the matched non-tumor adjacent brain tissues (TAT) and normal human astrocytes (NHAs). In functional assays, miR-522 promoted GBM cell proliferation, which could be reversed by inhibitor of miR-522. We further identified PH domain leucine-rich repeats protein phosphatase-1 (PHLPP1) as a putative target of miR-522, which is likely a main contributor to the promotion of tumor cell growth observed in our assays. Our results demonstrated that miR-522 promoted tumor cell proliferation and hence may represent a novel therapeutically relevant cellular target to treatment of GBM patients.

In This Issue

HomeCirculation ResearchVol. 116, No. 1In This Issue Free AccessResearch ArticlePDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessResearch ArticlePDF/EPUBIn This Issue Ruth Williams Ruth WilliamsRuth Williams Search for more papers by this author Originally published2 Jan 2015https://doi.org/10.1161/RES.0000000000000045Circulation Research. 2015;116:2Pik3cb Links Hippo-YAP and PI3K-AKT Pathways (p 35)Download figureDownload PowerPointLin et al reveal how Yes-associated protein activates cardiomyocyte proliferation.Signaling via the Hippo kinase cascade results in the nuclear localization and activation of transcriptional co-activator YAP (Yes-associated protein). The Hippo-YAP pathway promotes cardiomyocyte proliferation in the developing fetal heart and, when over-expressed, in adult cardiomyocytes as well. The transcriptional targets of YAP that control proliferation, however, remain largely unknown. To identify the targets of this pathway, Lin and colleagues performed genome-wide chromatin immunoprecipitation experiments with YAP as well as genome-wide transcriptional profiling of cells that had a gain or loss of function of YAP. The results obtained from these experiments revealed a total of 26 genes that bound directly to YAP (together with its DNA-binding partner, TEAD), and showed increased or reduced transcriptional activity. Among the genes that were upregulated was pik3cb, a gene that encodes part of the catalytic subunit of the kinase PIK3, which has been found in other independent studies to drive cardiomyocyte proliferation. Lin and colleagues went on to show that YAP-induced proliferation required functional pik3cb, and that heart-specific depletion of YAP, which causes hypertrophy and heart failure in mice, could be rescued in part by boosting the expression of pik3cb. Taken together these results indicate that increasing YAP or pik3cb expression could be a useful strategy to stimulate cardiomyocyte proliferation and thereby promote myocardial regeneration after injury.miR-208 in Right Ventricular Failure (p 56)Download figureDownload PowerPointPaulin et al identify a right ventricle-specific mechanism in pulmonary hypertension and, with it, potential biomarkers of disease progression.Unlike systemic hypertension, which leads to left ventricular failure, pulmonary hypertension results in right ventricular failure (RVF). And even though in both conditions cardiac hypertrophy occurs as a compensatory mechanism, in RVF this brief compensation is quickly followed by a catastrophic decompensation phase and increased mortality. To investigate how RVF transitions from compensation to decompensation, Paulin and colleagues studied a rat model of pulmonary hypertension. They analyzed RVF tissue during compensation and decompensation and discovered that the latter was associated with increased inflammation and decreased expression of a right ventricle-specific transcription factor called Mef2. Upon performing an unbiased microarray screen, they found that a number of microRNAs (miRs) were misregulated during decompensation. Among the down-regulated miRs was miR208, which normally suppresses expression of a transcription factor called MED, a transcription factor that itself suppresses Mef2. Thus the downregulation of miR208 in decompensation could increase MED, which in turn could further suppress the already diminished Mef2. Exactly how these factors contribute to RVF remains to be determined, but in the meantime these changes could serve as valuable biomarkers of disease progression.α-Catenins Regulate Yap Activity in the Heart (p 70)Download figureDownload PowerPointAlpha-catenins suppress YAP and reduce proliferation in cardiomyocytes, report Li et al.Like Lin and colleagues, Li and colleagues have been studying the control of cardiomyocyte proliferation by the transcriptional co-activator YAP. But whereas Lin and colleagues searched for downstream targets of YAP, Li and colleagues have identified α-catenins as upstream regulators. Inside cells, α-catenin proteins link the junction proteins, cadherins, to the cytoskeleton, but aside from this structural role, α-catenins have been found, in some cell types, to control proliferation. It is thought that, through their interaction with the cadherin junctions, the α-catenins sense an increase in cell density and shut down cell division. Li and colleagues now show that this anti-proliferative effect also occurs in the heart. Specifically, they found that mouse hearts lacking the two types of α-catenin exhibited an approximately 23% increase in cardiomyocyte number. They also found that phosphorylation of YAP was reduced in the catenin-deficient hearts, which prevented YAP from entering the nucleus and activating proliferation genes. The team went on to show that compared with their wild-type counterparts, the mice lacking α-catenin displayed improved repair of heart tissue following myocardial infarction. The discovery lends further support to notion that the activation of YAP could be a potential regenerative therapy. Previous Back to top Next FiguresReferencesRelatedDetails January 2, 2015Vol 116, Issue 1 Advertisement Article InformationMetrics © 2015 American Heart Association, Inc.https://doi.org/10.1161/RES.0000000000000045 Originally publishedJanuary 2, 2015 PDF download Advertisement

Acupuncture may exert its therapeutic effect through microRNA-339/Sirt2/NFκB/FOXO1 axis.

Recently, we have found that a number of microRNAs (miRNAs) and proteins are involved in the response to acupuncture therapy in hypertensive rats. Our bioinformatics study suggests an association between these miRNAs and proteins, which include miR-339 and sirtuin 2 (Sirt2). In this paper, we aimed to investigate whether Sirt2 was a direct target of miR-339 in neurons. In human SH-SY5Y cells, the luciferase assay implied that Sirt2 was likely a target of miRNA-339. Overexpression of miR-339 downregulated Sirt2 expression, while knockdown of miR-339 upregulated Sirt2 expression in human SH-SY5Y cells and rat PC12 cells. In addition, overexpression of miR-399 increased the acetylation of nuclear factor-κB (NF-κB) and forkhead box protein O1 (FOXO1) in SH-SY5Y cells, which are known targets of Sirt2. Our findings demonstrate that miR-339 regulates Sirt2 in human and rat neurons. Since Sirt2 plays a critical role in multiple important cellular functions, our data imply that acupuncture may act through epigenetic changes and subsequent action on their targets, such as miRNA-339/Sirt2/NF-κB/FOXO1 axis. Some physiological level changes of neurons after altering the miR-339 levels are needed to validate the suggested therapeutic role of miR-339/Sirt2/NF-κB/FOXO1 axis in response to acupuncture therapy in the future work.

MicroRNA-146a and -21 cooperate to regulate vascular smooth muscle cell proliferation via modulation of the Notch signaling pathway.

A number of microRNAs (miRs) have been shown to participate in the regulation of vascular smooth muscle cell (VSMC) proliferation, a key step in the formation of atherosclerotic plaque, by targeting certain genes. The aim of the present study was to investigate the roles of miR‑146a and miR‑21 in VSMC growth and to study the underlying mechanisms. The expression levels of four previously reported, differentially expressed microRNAs in atherosclerotic plaque (miR‑146a/b, miR‑21, miR‑34a and miR‑210) were measured in two groups: An atherosclerotic plaque group (n=10) and a normal control group (n=10). Polymerase chain reaction (PCR) analysis revealed that the relative expression levels of miR‑146a and miR‑21 in atherosclerotic plaque samples were significantly upregulated to ~260 and 250%, respectively, compared with those in normal controls. Notch2 and Jag1 were confirmed to be target genes of miR‑146a and miR‑21 through the use of a luciferase assay, PCR and western blot analysis. Additionally, VSMCs transfected with miR‑146a expressed significantly lower levels of Notch2 protein and presented an accelerated cell proliferation, which could be attributed to a reduction in the levels of cell cycle arrest. Cotransfection of miR‑146a and miR‑21 further promoted cell cycle progression in addition to VSMC proliferation. In conclusion, the present study revealed that miR‑146a and miR‑21 were significantly upregulated in atherosclerotic plaque, and cooperated to accelerate VSMC growth and cell cycle progression by targeting Notch2 and Jag1.