miRNA-processing Enzyme Research Articles

Expression Profiling and Structural Characterization of MicroRNAs in Adipose Tissues of Hibernating Ground Squirrels

MicroRNAs (miRNAs) are small non-coding RNAs that are important in regulating metabolic stress. In this study, we determined the expression and structural characteristics of 20 miRNAs in brown (BAT) and white adipose tissue (WAT) during torpor in thirteen-lined ground squirrels. Using a modified stem-loop technique, we found that during torpor, expression of six miRNAs including let-7a, let-7b, miR-107, miR-150, miR-222 and miR-31 was significantly downregulated in WAT (P<0.05), which was 16%–54% of euthermic non-torpid control squirrels, whereas expression of three miRNAs including miR-143, miR-200a and miR-519d was found to be upregulated by 1.32–2.34-fold. Similarly, expression of more miRNAs was downregulated in BAT during torpor. We detected reduced expression of 6 miRNAs including miR-103a, miR-107, miR-125b, miR-21, miR-221 and miR-31 (48%–70% of control), while only expression of miR-138 was significantly upregulated (2.91±0.8-fold of the control, P<0.05). Interestingly, miRNAs found to be downregulated in WAT during torpor were similar to those dysregulated in obese humans for increased adipogenesis, whereas miRNAs with altered expression in BAT during torpor were linked to mitochondrial β-oxidation. miRPath target prediction analysis showed that miRNAs downregulated in both WAT and BAT were associated with the regulation of mitogen-activated protein kinase (MAPK) signaling, while the miRNAs upregulated in WAT were linked to transforming growth factor β (TGFβ) signaling. Compared to mouse sequences, no unique nucleotide substitutions within the stem-loop region were discovered for the associated pre-miRNAs for the miRNAs used in this study, suggesting no structure-influenced changes in pre-miRNA processing efficiency in the squirrel. As well, the expression of miRNA processing enzyme Dicer remained unchanged in both tissues during torpor. Overall, our findings suggest that changes of miRNA expression in adipose tissues may be linked to distinct biological roles in WAT and BAT during hibernation and may involve the regulation of signaling cascades.

Prognostic value of the MicroRNA regulators Dicer and Drosha in non-small-cell lung cancer: co-expression of Drosha and miR-126 predicts poor survival.

BackgroundDicer and Drosha are important enzymes for processing microRNAs. Recent studies have exhibited possible links between expression of different miRNAs, levels of miRNA processing enzymes, and cancer prognosis. We have investigated the prognostic impact of Dicer and Drosha and their correlation with miR-126 expression in a large cohort of non-small cell lung cancer (NSCLC) patients. We aimed to find patient groups within the cohort that might have an advantage of receiving adjunctive therapies.MethodsDicer expression in the cytoplasm and Drosha expression in the nucleus were evaluated by manual immunohistochemistry of tissue microarrays (TMAs), including tumor tissue samples from 335 patients with resected stages I to IIIA NSCLC. In addition, in situ hybridizations of TMAs for visualization of miR-126 were performed. Kaplan–Meier analysis was performed, and the log-rank test via SPSS v.22 was used for estimating significance levels.ResultsIn patients with normal performance status (ECOG = 0, n = 197), high Dicer expression entailed a significantly better prognosis than low Dicer expression (P = 0.024). Dicer had no significant prognostic value in patients with reduced performance status (ECOG = 1–2, n = 138). High Drosha expression was significantly correlated with high levels of the microRNA 126 (miR-126) (P = 0.004). Drosha/miR-126 co-expression had a significant negative impact on the disease-specific survival (DSS) rate (P < 0.001). Multivariate analyses revealed that the interaction Dicer*Histology (P = 0.049) and Drosha/miR-126 co-expression (P = 0.033) were independent prognostic factors.ConclusionsIn NSCLC patients with normal performance status, Dicer is a positive prognostic factor. The importance of Drosha as a prognostic factor in our material seems to be related to miR-126 and possibly other microRNAs.Electronic supplementary materialThe online version of this article (doi:10.1186/1472-6890-14-45) contains supplementary material, which is available to authorized users.

Identification and validation of a novel microRNA-like molecule derived from a cytoplasmic RNA virus antigenome by bioinformatics and experimental approaches.

BackgroundIt is generally believed that RNA virus replicating in the cell cytoplasm would not encode microRNAs (miRNAs) due to nucleus inaccessibility. Recent studies have described cytoplasmic RNA virus genome-derived miRNAs in West Nile virus (WNV) and Dengue virus (DENV). However, naturally occurring miRNAs derived from the antigenome of a cytoplasmic RNA virus have not been described.MethodsHepatitis A virus (HAV) was served as a model virus to investigate whether the antigenome of a cytoplasmic RNA virus would be processed into miRNAs or miRNA-like small RNAs upon infection. HAV antigenome was queried for putative miRNA precursors (pre-miRNA) with the VMir analyzer program. Mature miRNA prediction was performed using MatureBayes and Bayes-SVM-MiRNA web server v1.0. Finally, multiple experimental approaches, including cloning and sequencing-, RNAi-, plasmid-based miRNA expression- and luciferase reporter assays, were performed to identify and validate naturally occurring viral antigenome-derived miRNAs.ResultsUsing human HAV genotype IA (isolate H2) (HAVH2), a virally encoded miRNA-like small RNA was detected on the antigenome and named hav-miR-N1-3p. Transcription of viral pre-miRNA in KMB17 and HEK293T cells led to mature hav-miR-N1-3p production. In addition, silencing of the miRNA-processing enzyme Dicer or Drosha caused a dramatic reduction in miRNA levels. Furthermore, artificial target of hav-miR-N1-3p was silenced by synthesized viral miRNA mimics and the HAVH2 naturally-derived hav-miR-N1-3p.ConclusionThese results suggested that the antigenome of a cytoplasmic RNA virus could be processed into functional miRNAs. Our findings provide new evidence supporting the hypothesis that cytoplasmic RNA viruses naturally encode miRNAs through cellular miRNA processing machinery.

Deletion of Dicer in late erythroid cells results in impaired stress erythropoiesis in mice

MicroRNAs (miRNAs) have been shown to influence erythroid lineage commitment and differentiation; however, our knowledge of miRNA function in terminal erythropoiesis remains limited. To address this issue, we generated a novel animal model, where the miRNA-processing enzyme, Dicer, is selectively inactivated in erythropoietin receptor positive erythroid cells beginning with CFU-e/proerythroblast cells. This results in significant depletion of all miRNAs from the proerythroblast stage onwards, with one exception, miR-451, which is processed by Ago2 in a Dicer-independent manner. We observed that mature Dicer-dependent miRNAs, like miR-451, are dispensable under steady-state conditions, but these mutants have an impaired response to stress erythropoiesis, as demonstrated by a delay in recovery from anemia. This defect was specific to later maturing erythroid cells, as progenitor numbers were unaffected. In addition to generating a novel mouse model to study miRNA function in late erythroid cells, we conclude that miRNAs (both Dicer-dependent and independent) act primarily to regulate the optimal response to stress among late erythroid cells.

Silencing of Dicer1 temporally separates pro- and anti-apoptotic signaling and confers susceptibility to chemotherapy in p53 mutated cells

miRNAs are critically implicated in the initiation process of and progression through cancerogenesis. The mechanisms, however, by which miRNAs interfere with the signalosomes of human cancer cells, are still obscure. We utilized the p53-mutated human keratinocyte cell line HACAT to investigate the biological significance and extent to which miRNAs regulate proliferation, cell growth, and apoptosis in transformed phenotypes. Silencing of the miRNA-processing enzyme Dicer1 resulted in cell cycle arrest at the G1/S border, along with restoration of CDK inhibitor p21CIPexpression. Employing a cell cycle-wide phospho-proteomic approach, we detected neglectable changes in abundance and schedule of overall and cell cycle periodic protein expression despite cell cycle arrest of Dicer1-depleted cells. Instead, we found substantially delayed post-translational modifications of some, but not all, signaling nodes. Phospho-site-specific analyses revealed that pro-apoptotic information elicited by Myc, β-catenin, and other mitotic pathways early in G1 are absorbed and balanced by anti-apoptotic signaling from AKT and NFκB in Dicer1-competent cells. The absence of regulatory miRNAs, however, led to a substantial delay of anti-apoptotic signaling, leaving pro-apoptotic stress unbalanced in Dicer1-deprived cells. We here show that this temporal separation of pro- and anti-apoptotic signaling induced by inhibition of Dicer1 is synergistic and synthetic lethal to low-dose 5-FU chemotherapy in p53-mutated HACAT cells. The findings reported here contribute to the understanding of the complex interactions of miRNAs with the signalosom of transformed phenotypes and may help to design novel strategies to fight cancer.

Dicer Regulates Differentiation and Viability during Mouse Pancreatic Cancer Initiation

miRNA levels are altered in pancreatic ductal adenocarcinoma (PDA), the most common and lethal pancreatic malignancy, and intact miRNA processing is essential for lineage specification during pancreatic development. However, the role of miRNA processing in PDA has not been explored. Here we study the role of miRNA biogenesis in PDA development by deleting the miRNA processing enzyme Dicer in a PDA mouse model driven by oncogenic Kras. We find that loss of Dicer accelerates Kras driven acinar dedifferentiation and acinar to ductal metaplasia (ADM), a process that has been shown to precede and promote the specification of PDA precursors. However, unconstrained ADM also displays high levels of apoptosis. Dicer loss does not accelerate development of Kras driven PDA precursors or PDA, but surprisingly, we observe that mouse PDA can develop without Dicer, although at the expense of proliferative capacity. Our data suggest that intact miRNA processing is involved in both constraining pro-tumorigenic changes in pancreatic differentiation as well as maintaining viability during PDA initiation.

Comment on ‘Strong reduction of AGO2 expression in melanoma and cellular consequences’

Sir, We would like to congratulate Voller et al (2013) on their recent study showing an unchanged mRNA AGO2 expression in melanoma and a strong reduction of AGO2 expression on protein level. This goes along with the findings in our study on the miRNA processing machinery performed in melanoma, which likewise has previously shown an unchanged mRNA AGO2 expression level in melanoma (Sand et al, 2012c). Interestingly, in contrast to melanoma skin cancer, mRNA AGO2 expression levels in epithelial skin cancer (both cutaneous squamous cell and basal cell carcinoma) were significantly higher (P<0.05) compared with healthy controls (Sand et al, 2010, 2012a, 2012b). Although we do agree with the authors that a deregulation of microRNA (miRNA) was observed in several types of cancer and particularly in skin cancer, with reference to our previous studies we do not agree that changes in the miRNA processing enzymes have not been analysed until today as stated in their abstract (Sand et al, 2009, 2011, 2012d, 2012e, 2013; 2014; Sand and Skrygan, 2014).

MicroRNA Regulation in Systemic Lupus Erythematosus Pathogenesis.

MicroRNAs (miRNAs) are endogenous small RNA molecules best known for their function in post-transcriptional gene regulation. Immunologically, miRNA regulates the differentiation and function of immune cells and its malfunction contributes to the development of various autoimmune diseases including systemic lupus erythematosus (SLE). Over the last decade, accumulating researches provide evidence for the connection between dysregulated miRNA network and autoimmunity. Interruption of miRNA biogenesis machinery contributes to the abnormal T and B cell development and particularly a reduced suppressive function of regulatory T cells, leading to systemic autoimmune diseases. Additionally, multiple factors under autoimmune conditions interfere with miRNA generation via key miRNA processing enzymes, thus further skewing the miRNA expression profile. Indeed, several independent miRNA profiling studies reported significant differences between SLE patients and healthy controls. Despite the lack of a consistent expression pattern on individual dysregulated miRNAs in SLE among these studies, the aberrant expression of distinct groups of miRNAs causes overlapping functional outcomes including perturbed type I interferon signalling cascade, DNA hypomethylation and hyperactivation of T and B cells. The impact of specific miRNA-mediated regulation on function of major immune cells in lupus is also discussed. Although research on the clinical application of miRNAs is still immature, through an integrated approach with advances in next generation sequencing, novel tools in bioinformatics database analysis and new in vitro and in vivo models for functional evaluation, the diagnostic and therapeutic potentials of miRNAs may bring to fruition in the future.

MicroRNA-132 is enriched in developing axons, locally regulates Rasa1 mRNA, and promotes axon extension.

Developing axons can locally synthesize proteins, with roles in axon growth, guidance, and regeneration, but the mechanisms that regulate axonal mRNA translation are not well understood. MicroRNAs (miRNAs) are important regulators of translation but have still been little characterized in developing axons. Here we study mouse dorsal root ganglion (DRG) axons and show that their extension is impaired by conditional deficiency of the miRNA-processing enzyme Dicer in vitro and in vivo. A screen for axonal localization identifies a specific set of miRNAs preferentially enriched within the developing axon. High axonal expression and preferential localization were observed for miR-132, a miRNA previously known for roles in dendrites and dysregulation in major neurologic diseases. miR-132 knockdown reduced extension of cultured DRG axons, whereas overexpression increased extension. Mechanistically, miR-132 regulated the mRNA for the Ras GTPase activator Rasa1, a novel target in neuronal function. Moreover, miR-132 regulation of Rasa1 translation was seen in severed axons, demonstrating miRNA function locally within the axon. miR-132 expression in DRGs peaked in the period of maximum axon growth in vivo, consistent with its effect on axon growth, and suggesting a role as a developmental timer. Together, these findings identify miR-132 as a positive regulator of developing axon extension, acting through repression of Rasa1 mRNA, in a mechanism that operates locally within the axon.

Strong reduction of AGO2 expression in melanoma and cellular consequences

Background:Processing of microRNAs (miRNAs) is a highly controlled process. Deregulation of miRNA expression was observed in several types of cancer but changes in the miRNA-processing enzymes have not been analysed until today. In this study, we analysed Argonaute2 (AGO2, EIF2C2), as one main factor of the miRNA processing ensemble, in the context of cancer development, especially in melanoma.Methods:We determined the AGO2 expression level in melanoma, as well as in other cancers, with biochemical approaches (qRT–PCR, western blot and immunofluorescence studies) and analysed the cell behaviour in migration assays.Results:Specifically in melanoma, we revealed a strong reduction of AGO2 expression compared with primary melanocytes. The reduction of AGO2 expression was only found on protein level, whereas the mRNA level stayed unchanged hinting to post-transcriptional regulation. We could show that re-expression of AGO2 in melanoma leads to a strong improvement of regulatory effects due to increased functionality of small-interfering RNAs and short hairpin RNAs.Conclusion:We identified melanoma-specific downregulation of AGO2 and corresponding reduced RNAi efficiency. These findings will help to understand the molecular basis of malignant melanoma and can potentially lead to an improvement of therapeutic strategies.

Cell Survival Following Radiation Exposure Requires miR-525-3p Mediated Suppression of ARRB1 and TXN1

BackgroundmicroRNAs (miRNAs) are non-coding RNAs that alter the stability and translation efficiency of messenger RNAs. Ionizing radiation (IR) induces rapid and selective changes in miRNA expression. Depletion of the miRNA processing enzymes Dicer or Ago2 reduces the capacity of cells to survive radiation exposure. Elucidation of critical radiation-regulated miRNAs and their target proteins offers a promising approach to identify new targets to increase the therapeutic effectiveness of the radiation treatment of cancer.Principal FindingsExpression of miR-525-3p is rapidly up-regulated in response to radiation. Manipulation of miR-525-3p expression in irradiated cells confirmed that this miRNA mediates the radiosensitivity of a variety of non-transformed (RPE, HUVEC) and tumor-derived cell lines (HeLa, U2-Os, EA.hy926) cell lines. Thus, anti-miR-525-3p mediated inhibition of the increase in miR-525-3p elevated radiosensitivity, while overexpression of precursor miR-525-3p conferred radioresistance. Using a proteomic approach we identified 21 radiation-regulated proteins, of which 14 were found to be candidate targets for miR-525-3p-mediated repression. Luciferase reporter assays confirmed that nine of these were indeed direct targets of miR-525-3p repression. Individual analysis of these direct targets by RNAi-mediated knockdown established that ARRB1, TXN1 and HSPA9 are essential miR-525-3p-dependent regulators of radiation sensitivity.ConclusionThe transient up-regulation of miR-525-3p, and the resultant repression of its direct targets ARRB1, TXN1 and HSPA9, is required for cell survival following irradiation. The conserved function of miR-525-3p across several cell types makes this microRNA pathway a promising target for modifying the efficacy of radiotherapy.

Proteomic identification of target proteins following Drosha knockdown in cervical cancer

The nuclear microRNA (miRNA) processing enzyme Drosha is upregulated in cervical cancer, and its overexpression is related to an invasive tumour phenotype. However, the mechanisms that underlie this effect remain poorly understood. The aim of this study was to identify the potential targets of Drosha in cervical cancer. Here, we demonstrated that Drosha knockdown (Drosha-KD) inhibited proliferation, colony formation and the migration of cervical cancer cells invitro. A global upregulation of proteins in Drosha-KD cells was revealed by two-dimensional gel electrophoresis (2-DE). Eighteen proteins were identified by liquid chromatography and tandem mass spectrometry technology (LC-MS/MS) from 21 selected protein spots that exhibited significant alterations in Drosha-KD cells. The majority of the identified proteins have been previously associated with tumour formation. The downregulation of tubulin5β in Drosha-KD cervical cancer cells was further confirmed by western blotting. Our results suggest that Drosha affects the biological activity of cervical cancer cells by regulating the expression of numerous tumour-associated proteins.

Expression of genes for microRNA‐processing enzymes is altered in advanced non‐alcoholic fatty liver disease

Recently, microRNAs (miRNA) have been linked to the pathogenesis of non-alcoholic fatty liver disease (NAFLD) and its progression to non-alcoholic steatohepatitis (NASH). First transcribed as pri-miRNA, these molecules are further processed by a complex of endonuclear and cytosolic RNA binding molecules to form mature miRNAs. The aim of this study is to investigate mechanisms of miRNA regulation in the visceral adipose of obese NAFLD patients via measuring expression of miRNA processing enzymes and pri-miRNA. Total RNAs were extracted from visceral adipose tissue (VAT) samples collected from patients undergoing bariatric surgery. All patients had biopsy-proven NAFLD (NASH patients [n = 12] and non-NASH NAFLD [n = 12]). For each patient, we profiled mRNA levels for three miRNA processing elements (Drosha, DGCR8, and Dicer1) and seven pri-miRNAs (pri-miR-125b-2, pri-miR-16-2, pri-miR-26a-1, pri-miR-26a-2, pri-miR-7-1, pri-miR-7-2, and pri-miR-7-3). Expression of Dicer1, Drosha and DGCR8 was significantly increased within the NASH cohort along with expression of pri-miR-7-1. The presence of focal necrosis on the liver biopsy correlated significantly with levels of Dicer1 and DGRC8. Both NASH and ballooning degeneration of hepatocytes correlated negatively with the expression levels of hsa-miR-125b. Histologic NASH correlated positively with the expression levels of pri-miR-16-2 and pri-miR-7-1. The presence of the hepatocyte's ballooning degeneration in the liver biopsy correlated positively with pri-miR-26a-1 and pri-miR-7-1. The expression profile of pri-miR-125b-2 also correlated positively with body mass index. Our findings support the hypothesis that VAT-derived miRNA may contribute to the pathogenesis of NASH in obese patients.

The use of miRNA microarrays for the analysis of cancer samples with global miRNA decrease.

Recent studies have established that mutations or deletions in microRNA (miRNA) processing enzymes resulting in a global decrease of miRNA expression are frequent across cancers and can be associated with a poorer prognosis. While very popular in miRNA profiling studies, it remains unclear whether miRNA microarrays are suited or not to accurately detecting global miRNA decreases seen in cancers. In this work, we analyzed the miRNA profiles of samples with global miRNA decreases using Affymetrix miRNA microarrays following the inducible genetic deletion of Dicer1. Surprisingly, up to a third of deregulated miRNAs identified upon Dicer1 depletion were found to be up-regulated following standard robust multichip average (RMA) background correction and quantile normalization, indicative of normalization bias. Our comparisons of five preprocess steps performed at the probe level demonstrated that the use of cyclic loess relying on non-miRNA small RNAs present on the Affymetrix platform significantly improved specificity and sensitivity of detection of decreased miRNAs. These findings were validated in samples from patients with prostate cancer, where conjugation of robust normal-exponential background correction with cyclic loess normalization and array weights correctly identified the greatest number of decreased miRNAs, and the lowest amount of false-positive up-regulated miRNAs. These findings highlight the importance of miRNA microarray normalization for the detection of miRNAs that are truly differentially expressed and suggest that the use of cyclic loess based on non-miRNA small RNAs can help to improve the sensitivity and specificity of miRNA profiling in cancer samples with global miRNA decrease.

Stage-specific regulation of natural killer cell homeostasis and response against viral infection by microRNA-155

Natural killer (NK) cells function in the recognition and destruction of host cells infected with pathogens. Many regulatory mechanisms govern the potent responses of NK cells, both at the cellular and molecular level. Ablation of microRNA (miRNA) processing enzymes demonstrated that miRNAs play critical roles in NK cell differentiation and function; however, the role of individual miRNAs requires further investigation. Using mice containing a targeted deletion of microRNA-155 (miR-155), we observed defects in NK cell maintenance and maturation at steady state, as well as in homeostatic proliferation in lymphopenic mice. In addition, we discovered that miR-155 is up-regulated in activated NK cells during mouse cytomegalovirus (MCMV) infection in response to signals from the proinflammatory cytokines IL-12 and IL-18 and through signal transducer and activator of transcription 4 (STAT4) signaling. Although miR-155 was found to be dispensable for cytotoxicity and cytokine production when triggered through activating receptors, NK cells lacking miR-155 exhibited severely impaired effector and memory cell numbers in both lymphoid and nonlymphoid tissues after MCMV infection. We demonstrate that miR-155 differentially targets Noxa and suppressor of cytokine signaling 1 (SOCS1) in NK cells at distinct stages of homeostasis and activation. NK cells constitutively expressing Noxa and SOCS1 exhibit profound defects in expansion during the response to MCMV infection, suggesting that their regulation by miR-155 promotes antiviral immunity.

Dicer Deficiency Reveals MicroRNAs Predicted to Control Gene Expression in the Developing Adrenal Cortex

MicroRNAs (miRNAs) are small, endogenous, non-protein-coding RNAs that are an important means of posttranscriptional gene regulation. Deletion of Dicer, a key miRNA processing enzyme, is embryonic lethal in mice, and tissue-specific Dicer deletion results in developmental defects. Using a conditional knockout model, we generated mice lacking Dicer in the adrenal cortex. These Dicer-knockout (KO) mice exhibited perinatal mortality and failure of the adrenal cortex during late gestation between embryonic day 16.5 (E16.5) and E18.5. Further study of Dicer-KO adrenals demonstrated a significant loss of steroidogenic factor 1-expressing cortical cells that was histologically evident as early as E16.5 coincident with an increase in p21 and cleaved-caspase 3 staining in the cortex. However, peripheral cortical proliferation persisted in KO adrenals as assessed by staining of proliferating cell nuclear antigen. To further characterize the embryonic adrenals from Dicer-KO mice, we performed microarray analyses for both gene and miRNA expression on purified RNA isolated from control and KO adrenals of E15.5 and E16.5 embryos. Consistent with the absence of Dicer and the associated loss of miRNA-mediated mRNA degradation, we observed an up-regulation of a small subset of adrenal transcripts in Dicer-KO mice, most notably the transcripts coded by the genes Nr6a1 and Acvr1c. Indeed, several miRNAs, including let-7, miR-34c, and miR-21, that are predicted to target these genes for degradation, were also markedly down-regulated in Dicer-KO adrenals. Together these data suggest a role for miRNA-mediated regulation of a subset of genes that are essential for normal adrenal growth and homeostasis.

Downregulation of the microRNA biogenesis components and its association with poor prognosis in hepatocellular carcinoma

Genetic alterations and deregulation of the miRNA biogenesis pathway components have been reported in human tumors. Tissue-specific deletion of the Dicer gene, which encodes an essential miRNA processing enzyme, promotes carcinogenesis in animal models. These features indicate that aberrant miRNA biogenesis components are directly associated with cancer. For the present study, we conducted quantitative RT-PCR of 14 genes that are related to the miRNA biogenesis pathway in 47 paired samples of primary hepatocellular carcinoma (HCC) and matched non-cancerous liver. Expression of seven genes (Dgcr8, p68, p72, Dicer, Ago3, Ago4 and Piwil4) was significantly decreased in primary HCC, especially in non-viral HCC subtypes, compared to the non-cancerous liver. Combinations of decreased expression of the miRNA biogenesis components in non-cancerous liver were related to cigarette smoking, alcohol intake and diabetes, which are known to be risk factors for HCC, and were also associated with the occurrence of multicentric tumors. Reduction of two of these genes (Dicer and p68) in HCC was associated with poor prognosis. Trimethylation of histone H3 lysine 27 in the promoters is implicated in the deregulation of these miRNA-biogenesis-related genes in non-HBV genome integrated HCC cell lines. In conclusion, deregulation of the miRNA biogenesis pathway components is frequently observed in non-viral-associated HCC and is linked to etiological risk factors and poor prognosis. Our study further showed that epigenetic regulation could be implicated in the deregulation of these genes during hepatocarcinogenesis.

MiR-145 and miR-143 Regulate Odontoblast Differentiation through Targeting Klf4 and Osx Genes in a Feedback Loop

Dentin tissue is derived from mesenchymal cells induced into the odontoblast lineage. The differentiation of odontoblasts is a complex process regulated by several transcriptional factor signaling transduction pathways. However, post-translational regulation of these factors during dentinogenesis remains unclear. To further explore the mechanisms, we investigated the role of microRNA (miRNA) during odontoblast differentiation. We profiled the miRNA expression pattern during mouse odontoblast differentiation using a microarray assay and identified that miR-145 and miR-143 were down-regulated during this process. In situ hybridization verified that the two miRNAs were gradually decreased during mouse odontoblast differentiation. Loss-of-function and gain-of-function experiments revealed that down-regulation of miR-145 and miR-143 could promote odontoblast differentiation and increased Dspp and Dmp1 expression in mouse primary dental pulp cells and vice versa. We found that miR-145 and miR-143 controlled odontoblast differentiation through several mechanisms. First, KLF4 and OSX bind to their motifs in Dspp and Dmp1 gene promoters and up-regulate their transcription thereby inducing odontoblast differentiation. The miR-145 binds to the 3'-UTRs of Klf4 and Osx genes, inhibiting their expression. Second, KLF4 repressed miR-143 transcription by binding to its motifs in miR-143 regulatory regions as detected by ChIP assay and dual luciferase reporter assay. Third, miR-143 regulates odontoblast differentiation in part through miR-145 pathway. Taken together, we for the first time showed that the miR-143 and miR-145 controlled odontoblast differentiation and dentin formation through KLF4 and OSX transcriptional factor signaling pathways.

MicroRNAs Regulate Tumor Angiogenesis Modulated by Endothelial Progenitor Cells

Bone marrow-derived endothelial progenitor cells (EPC) contribute to the angiogenesis-dependent growth of tumors in mice and humans. EPCs regulate the angiogenic switch via paracrine secretion of proangiogenic growth factors and by direct luminal incorporation into sprouting nascent vessels. miRNAs have emerged as key regulators of several cellular processes including angiogenesis; however, whether miRNAs contribute to bone marrow-mediated angiogenesis has remained unknown. Here, we show that genetic ablation of miRNA-processing enzyme Dicer, specifically in the bone marrow, decreased the number of circulating EPCs, resulting in angiogenesis suppression and impaired tumor growth. Furthermore, genome-wide deep sequencing of small RNAs revealed tumor EPC-intrinsic miRNAs including miR-10b and miR-196b, which have been previously identified as key regulators of HOX signaling and adult stem cell differentiation. Notably, we found that both miR-10b and miR-196b are responsive to vascular endothelial growth factor stimulation and show elevated expression in human high-grade breast tumor vasculature. Strikingly, targeting miR-10b and miR-196b led to significant defects in angiogenesis-mediated tumor growth in mice. Targeting these miRNAs may constitute a novel strategy for inhibiting tumor angiogenesis.

Hypothalamic regulation of energy balance: a key role for DICER miRNA processing in arcuate POMC neurons

Hypothalamic regulation of energy balance: a key role for DICER miRNA processing in arcuate POMC neurons