Distinct microRNA Expression Profile in Prostate Cancer Patients with Early Clinical Failure and the Impact of let-7 as Prognostic Marker in High-Risk Prostate Cancer

Background The identification of additional prognostic markers to improve risk stratification and to avoid overtreatment is one of the most urgent clinical needs in prostate cancer (PCa). MicroRNAs, being important regulators of gene expression, are promising biomarkers in various cancer entities, though the impact as prognostic predictors in PCa is poorly understood. The aim of this study was to identify specific miRNAs as potential prognostic markers in high-risk PCa and to validate their clinical impact. Methodology and Principal Findings We performed miRNA-microarray analysis in a high-risk PCa study group selected by their clinical outcome (clinical progression free survival (CPFS) vs. clinical failure (CF)). We identified seven candidate miRNAs (let-7a/b/c, miR-515-3p/5p, -181b, -146b, and -361) that showed differential expression between both groups. Further qRT-PCR analysis revealed down-regulation of members of the let-7 family in the majority of a large, well-characterized high-risk PCa cohort (n = 98). Expression of let-7a/b/and -c was correlated to clinical outcome parameters of this group. While let-7a showed no association or correlation with clinical relevant data, let-7b and let-7c were associated with CF in PCa patients and functioned partially as independent prognostic marker. Validation of the data using an independent high-risk study cohort revealed that let-7b, but not let-7c, has impact as an independent prognostic marker for BCR and CF. Furthermore, we identified HMGA1, a non-histone protein, as a new target of let-7b and found correlation of let-7b down-regulation with HMGA1 over-expression in primary PCa samples. Conclusion Our findings define a distinct miRNA expression profile in PCa cases with early CF and identified let-7b as prognostic biomarker in high-risk PCa. This study highlights the importance of let-7b as tumor suppressor miRNA in high-risk PCa and presents a basis to improve individual therapy for high-risk PCa patients.

Comprehensive MicroRNA expression profiling identifies novel markers in follicular variant of papillary thyroid carcinoma

BACKGROUND Follicular variant of papillary thyroid carcinoma (FVPTC) shares features of papillary (PTC) and follicular (FTC) thyroid carcinomas on a clinical, morphological, and genetic level. MicroRNA (miRNA) deregulation was extensively studied in PTCs and FTCs. However, very limited information is available for FVPTC. The aim of this study was to assess miRNA expression in FVPTC with the most comprehensive miRNA array panel and to correlate it with the clinicopathological data. METHODS Forty-four papillary thyroid carcinomas (17 FVPTC, 27 classic PTC) and eight normal thyroid tissue samples were analyzed for expression of 748 miRNAs using Human Microarray Assays on the ABI 7900 platform (Life Technologies, Carlsbad, CA). In addition, an independent set of 61 tumor and normal samples was studied for expression of novel miRNA markers detected in this study. RESULTS Overall, the miRNA expression profile demonstrated similar trends between FVPTC and classic PTC. Fourteen miRNAs were deregulated in FVPTC with a fold change of more than five (up/down), including miRNAs known to be upregulated in PTC (miR-146b-3p, -146-5p, -221, -222 and miR-222-5p) and novel miRNAs (miR-375, -551b, 181-2-3p, 99b-3p). However, the levels of miRNA expression were different between these tumor types and some miRNAs were uniquely dysregulated in FVPTC allowing separation of these tumors on the unsupervised hierarchical clustering analysis. Upregulation of novel miR-375 was confirmed in a large independent set of follicular cell derived neoplasms and benign nodules and demonstrated specific upregulation for PTC. Two miRNAs (miR-181a-2-3p, miR-99b-3p) were associated with an adverse outcome in FVPTC patients by a Kaplan-Meier (p < 0.05) and multivariate Cox regression analysis (p < 0.05). CONCLUSIONS Despite high similarity in miRNA expression between FVPTC and classic PTC, several miRNAs were uniquely expressed in each tumor type, supporting their histopathologic differences. Highly upregulated miRNA identified in this study (miR-375) can serve as a novel marker of papillary thyroid carcinoma, and miR-181a-2-3p and miR-99b-3p can predict relapse-free survival in patients with FVPTC thus potentially providing important diagnostic and predictive value.

Characterization of the potential role for RNA-binding protein FUS/TLS in DNA damage response: A quantitative proteomic approach

FUS/TLS (fused in sarcoma/translocated in liposarcoma) is a ubiquitously expressed RNA-binding protein of the hnRNP family, that has been discovered as fused to transcription factors, through chromosomal translocations, in several human sarcomas and found in protein aggregates in neurons of patients with an inherited form of Amyotrophic Lateral Sclerosis (ALS) [1]. To date, FUS/TLS has been implicated in a variety of cellular processes such as gene expression control, transcriptional regulation, pre-mRNA splicing and miRNA processing [2]. In addition, some evidences link FUS/TLS to genome stability control and DNA damage response. In fact, mice lacking FUS/TLS are hypersensitive to ionizing radiation (IR) and show high levels of chromosome instability and in response to double-strand breaks, FUS/TLS gets phosphorylated by the protein kinase ATM [3,4,5]. Furthermore, the inducible depletion of FUS/TLS in a neuroblastoma cell line (SH-SY5Y FUS/TLS TET-off iKD) subjected to genotoxic stress (IR) resulted in an increased phosphorylation of γH2AX respect to control cells, suggesting an higher activation of the DNA damage response. The study aims to investigate the specific role of FUS/TLS in DNA damage response through the characterization of the proteomic profile of SH-SY5Y FUS/TLS iKD cells subjected to DNA damage stress, by mass spectrometry-based quantitative proteomics (e.g. SILAC). Preliminary results of mass spectrometric identification of FUS/TLS interacting proteins in HEK293 cells, expressing a recombinant flag-tagged FUS/TLS protein, highlighted the interactions with several proteins involved in DNA damage response, such as DNA-PK, XRCC-5/-6, and ERCC-6, raising the possibilities that FUS/TLS is involved in this pathway, even thou its exact role still need to be addressed.

Unravelling the multiple roles of FUS in RNA processing and on ALS pathogenesis

Two genes with related functions in RNA biogenesis were recently reported in patients with familial ALS: the FUS/TLS gene at the ALS6 locus and the TARDBP/TDP-43 gene at the ALS10 locus [1, 2]. FUS has been implicated to function in several steps of gene expression, including transcription regulation [3], RNA splicing [4, 5], mRNA transport in neurons [6] and, interestingly, in microRNA (miRNA) processing [7]. The goal of this project is to identify the molecular mechanisms leading to the development of FUS mutations-associated ALS. Specifically, we want to test the hypothesis that these FUS mutations misregulate miRNA levels that in turn affect the expression of genes critical for motor neuron survival. In addition we want to test whether misregulation of the miRNA profile is a common feature in ALS. We have performed immunoprecipitations from total extracts of 293T cells expressing FLAG-tagged FUS to characterize its interactome by mass spectrometry. This proteomic study not only revealed a strong interaction of FUS with splicing factors, but shows that FUS might be involved in many, quite different pathways. To map which parts of the FUS protein contribute to the interaction with splicing factors, we have performed a set of experiments with a series of missense and deletion mutants. With this approach, we will not only gain information on the binding partners of FUS along with a map of the required domains for the interactions, but it will also help to unravel whether certain ALS-associated FUS mutations lead to a loss or gain of function due to gain or loss of interactors. Additionally, we have performed quantitative interactomics using SILAC to identify interactome differences of ALS-associated FUS mutants. To this end we have performed immunoprecipitations of total extract from 293T cells, stably transduced with constructs expressing wild-type FUS-FLAG as well as three different ALS-associated mutants (G156E, R244C, P525L). First results indicate striking differences in the interactome with certain RNA binding proteins. We are now validating these candidates in order to reveal the importance of these differential interactions in the context of ALS.

Unravelling the impact of microRNA on Amyotrophic Lateral Sclerosis (ALS) pathogenesis

ALS is the most common adult neurodegenerative disease that specifically affects upper and lower neurons leading to progressive paralysis and death. There is currently no effective treatment. Thus, identification of the signaling pathways and cellular mediators of ALS remains a major challenge in the search for novel therapeutics. Recent studies have shown that noncoding RNA molecules have a significant impact on normal CNS development and on causes and progression of human neurological disorders. To investigate the hypothesis that expression of the mutant SOD1 protein, which is one of the genetic causes of ALS, may alter expression of miRNAs thereby contributing to the pathogenesis of familial ALS, we compared miRNA expression in SH-SY5Y expressing either the wild type or the SOD1 protein using small RNA deep-sequencing followed by RT-PCR validation. This strategy allowed us to find a group of up and down regulated miRNAs, which are predicted to play a role in the motorneurons physiology and pathology. The aim of my work is to understand if these modulators of gene expression may play a causative role in disease onset or progression. To this end I have checked the expression level of these misregulated miRNAs derived from RNA-deep sequencing by qPCR on cDNA derived from ALS mice models at early onset of the disease. Thus, I’m looking for the most up-regulated one even in Periferal Blood Mononuclear Cell (PBMC) of sporadic ALS patients. Furthermore I’m functionally characterizing the most up-regulated miRNAs through the validation of bioinformatic-predicted targets by analyzing endogenous targets levels after microRNA transfection and by UTR-report luciferase assays. Thereafter I’ll analyze the effect of misregulated targets on pathogenesis or progression of ALS by loss of functions or gain of functions experiments, based on the identified up/down-regulation of the specific target by miRNAs. In the end I would define the mechanisms responsible for the miRNAs level misregulation, by silencing or stimulating the signal transduction pathways putatively involved in miRNA regulation.

Characterization of the potential role for RNA-binding protein FUS in DNA damage response: A quantitative proteomic approach

FUS/TLS (fused in sarcoma/translocated in liposarcoma) is a ubiquitously expressed protein of the hnRNP family, that has been discovered as fused to transcription factors in several human sarcomas and found in protein aggregates in neurons of patients with an inherited form of Amyotrophic Lateral Sclerosis [Vance C. et al., 2009]. FUS is a 53 kDa nuclear protein that contains structural domains, such as a RNA Recognition Motif (RRM) and a zinc finger motif, that give to FUS the ability to bind to both RNA and DNA sequences. It has been implicated in a variety of cellular processes, such as pre-mRNA splicing, miRNA processing, gene expression control and transcriptional regulation [Fiesel FC. and Kahle PJ., 2011]. Moreover, some evidences link FUS to genome stability control and DNA damage response: mice lacking FUS are hypersensitive to ionizing radiation (IR) and show high levels of chromosome instability and, in response to double-strand breaks, FUS is phosphorylated by the protein kinase ATM [Kuroda M. et al., 2000; Hicks GG. et al., 2000; Gardiner M. et al., 2008]. Furthermore, preliminary results of mass spectrometric identification of FUS interacting proteins in HEK293 cells, expressing a recombinant flag-tagged FUS protein, highlighted the interactions with proteins involved in DNA damage response, such as DNA-PK, XRCC-5/-6, and ERCC-6, raising the possibilities that FUS is involved in this pathway, even though its role still needs to be clarified. This study aims to investigate the biological roles of FUS in human cells and in particular the putative role in DNA damage response through the characterization of the proteomic profile of the neuroblastoma cell line SH-SY5Y upon FUS inducible depletion, by a quantitative proteomic approach. The SH-SY5Y cell line that will be used in this study expresses, in presence of tetracycline, a shRNA that targets FUS mRNA, leading to FUS protein depletion (SH-SY5Y FUS iKD cells). To quantify changes in proteins expression levels a SILAC strategy (Stable Isotope Labeling by Amino acids in Cell culture) will be conducted on SH-SY5Y FUS iKD cells and a control SH-SY5Y cell line (that expresses a mock shRNA) and the relative changes in proteins levels will be evaluated after five and seven days upon FUS depletion, by nanoliquid chromatography coupled to tandem mass spectrometry (nLC-MS/MS) and bioinformatics analysis. Preliminary experiments demonstrated that the SH-SY5Y FUS iKD cells, when subjected to genotoxic stress (high dose of IR), upon inducible depletion of FUS, showed a increased phosphorylation of gH2AX with respect to control cells, suggesting an higher activation of the DNA damage response.

Development and Validation of a Symptom-Based Activity Index for Adults with Eosinophilic Esophagitis

BACKGROUND & Aims: Standardized instruments are needed to assess the activity of eosinophilic esophagitis (EoE), to provide endpoints for clinical trials and observational studies. We aimed to develop and validate a patient-reported outcome (PRO) instrument and score, based on items that could account for variations in patients' assessments of disease severity. We also evaluated relationships between patients' assessment of disease severity and EoE-associated endoscopic, histologic, and laboratory findings. METHODS We collected information from 186 patients with EoE in Switzerland and the US (69.4% male; median age, 43 years) via surveys (n = 135), focus groups (n = 27), and semi-structured interviews (n = 24). Items were generated for the instruments to assess biologic activity based on physician input. Linear regression was used to quantify the extent to which variations in patient-reported disease characteristics could account for variations in patients' assessment of EoE severity. The PRO instrument was prospectively used in 153 adult patients with EoE (72.5% male; median age, 38 years), and validated in an independent group of 120 patients with EoE (60.8% male; median age, 40.5 years). RESULTS Seven PRO factors that are used to assess characteristics of dysphagia, behavioral adaptations to living with dysphagia, and pain while swallowing accounted for 67% of the variation in patients' assessment of disease severity. Based on statistical consideration and patient input, a 7-day recall period was selected. Highly active EoE, based on endoscopic and histologic findings, was associated with an increase in patient-assessed disease severity. In the validation study, the mean difference between patient assessment of EoE severity and PRO score was 0.13 (on a scale from 0 to 10). CONCLUSIONS We developed and validated an EoE scoring system based on 7 PRO items that assesses symptoms over a 7-day recall period. Clinicaltrials.gov number: NCT00939263.

Role of MicroRNA Modulation in the Interferon-α/Ribavirin Suppression of HIV-1 In Vivo.

BACKGROUND Interferon-α (IFN-α) treatment suppresses HIV-1 viremia and reduces the size of the HIV-1 latent reservoir. Therefore, investigation of the molecular and immunologic effects of IFN-α may provide insights that contribute to the development of novel prophylactic, therapeutic and curative strategies for HIV-1 infection. In this study, we hypothesized that microRNAs (miRNAs) contribute to the IFN-α-mediated suppression of HIV-1. To inform the development of novel miRNA-based antiretroviral strategies, we investigated the effects of exogenous IFN-α treatment on global miRNA expression profile, HIV-1 viremia, and potential regulatory networks between miRNAs and cell-intrinsic anti-HIV-1 host factors in vivo. METHODS Global miRNA expression was examined in longitudinal PBMC samples obtained from seven HIV/HCV-coinfected, antiretroviral therapy-naïve individuals before, during, and after pegylated interferon-α/ribavirin therapy (IFN-α/RBV). We implemented novel hybrid computational-empirical approaches to characterize regulatory networks between miRNAs and anti-HIV-1 host restriction factors. RESULTS miR-422a was the only miRNA significantly modulated by IFN-α/RBV in vivo (p<0.0001, paired t test; FDR<0.037). Our interactome mapping revealed extensive regulatory involvement of miR-422a in p53-dependent apoptotic and pyroptotic pathways. Based on sequence homology and inverse expression relationships, 29 unique miRNAs may regulate anti-HIV-1 restriction factor expression in vivo. CONCLUSIONS The specific reduction of miR-422a is associated with exogenous IFN-α treatment, and likely contributes to the IFN-α suppression of HIV-1 through the enhancement of anti-HIV-1 restriction factor expression and regulation of genes involved in programmed cell death. Moreover, our regulatory network analysis presents additional candidate miRNAs that may be targeted to enhance anti-HIV-1 restriction factor expression in vivo.

Identification of gunshots to the head by detection of RNA in backspatter primarily expressed in brain tissue

Traces of backspatter recovered from the inside of the barrel of a gun that was used to deliver suicidal or homicidal contact shots may be a source of valuable forensic evidence and first systematic investigations of the persistence of victim DNA from inside firearms have been presented. The aim of the present study was to include victim RNA in such analyses to determine the origin of tissues in addition and parallel to standard DNA profiling for forensic identification purposes. In a first step, suitable mRNA (C1orf61) and micro-RNAs (miR-124a and miR-124*) that are primarily expressed in brain tissue were selected from potential candidates and confirmed using quantitative PCR (qPCR). Secondly, a co-extraction procedure for RNA and DNA was established and brain differentiability of the selected RNAs was demonstrated via qPCR using samples from experimental shots at ballistic models. In a third step, this procedure was successfully applied to analyse samples from real casework comprising eight cases of suicidal contact shots. In this pilot study, we are first to report the possibility of co-extracting mRNA, miRNA and DNA from ballistic trace samples collected from the inside of firearms and we demonstrate that RNA and DNA based analyses can be performed in parallel to produce informative and highly complementary evidence.

MicroRNA profile of poorly differentiated thyroid carcinomas: new diagnostic and prognostic insights.

The diagnosis of conventional and oncocytic poorly differentiated (oPD) thyroid carcinomas is difficult. The aim of this study is to characterise their largely unknown miRNA expression profile and to compare it with well-differentiated thyroid tumours, as well as to identify miRNAs which could potentially serve as diagnostic and prognostic markers. A total of 14 poorly differentiated (PD), 13 oPD, 72 well-differentiated thyroid carcinomas and eight normal thyroid specimens were studied for the expression of 768 miRNAs using PCR-Microarrays. MiRNA expression was different between PD and oPD thyroid carcinomas, demonstrating individual clusters on the clustering analysis. Both tumour types showed upregulation of miR-125a-5p, -15a-3p, -182, -183-3p, -222, -222-5p, and downregulation of miR-130b, -139-5p, -150, -193a-5p, -219-5p, -23b, -451, -455-3p and of miR-886-3p as compared with normal thyroid tissue. In addition, the oPD thyroid carcinomas demonstrated upregulation of miR-221 and miR-885-5p. The difference in expression was also observed between miRNA expression in PD and well-differentiated tumours. The CHAID algorithm allowed the separation of PD from well-differentiated thyroid carcinomas with 73-79% accuracy using miR-23b and miR-150 as a separator. Kaplan-Meier and multivariate analysis showed a significant association with tumour relapses (for miR-23b) and with tumour-specific death (for miR-150) in PD and oPD thyroid carcinomas. MiRNA expression is different in conventional and oPD thyroid carcinomas in comparison with well-differentiated thyroid cancers and can be used for discrimination between these tumour types. The newly identified deregulated miRNAs (miR-150, miR-23b) bear the potential to be used in a clinical setting, delivering prognostic and diagnostic informations.

Proteomic characterization of the FUS/TLS interactome

FUS/TLS (fused in sarcoma/translocated in liposarcoma) is a ubiquitously expressed RNA-binding protein, that has been discovered as fused to transcription factors in several human sarcomas and found in protein aggregates in neurons of patients with an inherited form of Amyotrophic Lateral Sclerosis [1]. To date, FUS has been implicated in a variety of cellular processes such as gene expression control, transcriptional regulation, pre-mRNA splicing and miRNA processing [2]. In addition, some evidences link FUS to genome stability control and DNA damage response. In fact, mice lacking FUS are hypersensitive to ionizing radiation and show high levels of chromosome instability and in response to double-strand breaks, FUS gets phosphorylated by the protein kinase ATM [3, 4, 5]. Moreover, upon DNA damage stress, FUS mediates Ebp1 (ErbB3 receptor-binding protein) SUMOylation, a post-translational modification that is required for its onco-suppressive activity, by acting as SUMO E3 ligase [6]. The study aims to investigate the role of FUS in DNA damage response and SUMOylation, two cellular pathways tightly interconnected to each other. Moreover, we will exploit biochemical and mass spectrometry-based approaches in order to identify other potential substrates of the E3 SUMO ligase activity of FUS. Preliminary results of mass spectrometric identification of FUS interacting proteins, in HEK293 and SHSY5Y cells, highlighted the interaction of FUS with several proteins involved in DNA damage response and many of those have been described already as target of SUMOylation, such as XRCC5, DDX5, PARP1, Nucleophosmin, and others. These evidences strengthen the hypothesis that FUS might represent a link between these pathways, even thou its exact role still needs to be clearly addressed. [1] Vance C. et al. (2009) Science 323(5918): p. 1208-11 [2] Fiesel FC., Kahle PJ. (2011) FEBS J. 278(19): p. 3550-68 [3] Kuroda M. et al. (2000) Embo J. 19(3): p. 453-62 [4] Hicks GG. et al. (2000) Nat Genet. 24(2):p. 175-9 [5] Gardiner M. et al. (2008) Biochem J. 415(2): p. 297-307 [6] Oh SM. et al. (2010) Oncogene 29(7): p. 1017-30

Circulating microRNA profiling in patients with advanced non-squamous NSCLC receiving bevacizumab/erlotinib followed by platinum-based chemotherapy at progression (SAKK 19/05).

OBJECTIVES Molecular subclassification of non small-cell lung cancer (NSCLC) is essential to improve clinical outcome. This study assessed the prognostic and predictive value of circulating micro-RNA (miRNA) in patients with non-squamous NSCLC enrolled in the phase II SAKK (Swiss Group for Clinical Cancer Research) trial 19/05, receiving uniform treatment with first-line bevacizumab and erlotinib followed by platinum-based chemotherapy at progression. MATERIALS AND METHODS Fifty patients with baseline and 24 h blood samples were included from SAKK 19/05. The primary study endpoint was to identify prognostic (overall survival, OS) miRNA's. Patient samples were analyzed with Agilent human miRNA 8x60K microarrays, each glass slide formatted with eight high-definition 60K arrays. Each array contained 40 probes targeting each of the 1347 miRNA. Data preprocessing included quantile normalization using robust multi-array average (RMA) algorithm. Prognostic and predictive miRNA expression profiles were identified by Spearman's rank correlation test (percentage tumor shrinkage) or log-rank testing (for time-to-event endpoints). RESULTS Data preprocessing kept 49 patients and 424 miRNA for further analysis. Ten miRNA's were significantly associated with OS, with hsa-miR-29a being the strongest prognostic marker (HR=6.44, 95%-CI 2.39-17.33). Patients with high has-miR-29a expression had a significantly lower survival at 10 months compared to patients with a low expression (54% versus 83%). Six out of the 10 miRNA's (hsa-miRN-29a, hsa-miR-542-5p, hsa-miR-502-3p, hsa-miR-376a, hsa-miR-500a, hsa-miR-424) were insensitive to perturbations according to jackknife cross-validation on their HR for OS. The respective principal component analysis (PCA) defined a meta-miRNA signature including the same 6 miRNA's, resulting in a HR of 0.66 (95%-CI 0.53-0.82). CONCLUSION Cell-free circulating miRNA-profiling successfully identified a highly prognostic 6-gene signature in patients with advanced non-squamous NSCLC. Circulating miRNA profiling should further be validated in external cohorts for the selection and monitoring of systemic treatment in patients with advanced NSCLC.

How Do Gastroenterologists Assess Overall Activity of Eosinophilic Esophagitis in Adult Patients?

OBJECTIVES:There is no "gold standard" for assessing disease activity in patients with eosinophilic esophagitis (EoE). We aimed to compare physicians' judgment of EoE activity with patients' judgment of symptom severity. We also aimed to examine the relative contribution of symptoms as well as endoscopic and histologic findings in shaping physicians' judgment of EoE activity.METHODS:Six gastroenterologists (all EoE experts) assessed EoE-associated symptoms in adult patients. Patients completed a symptom instrument and provided global assessment of EoE symptom severity (PatGA) (Likert scale: 0 (inactive) to 10 (most active)). Following esophagogastroduodenoscopy with biopsy sampling, gastroenterologists provided a global assessment of EoE activity (PhysGA) (Likert scale from 0 to 10) based on patient history and endoscopic and histologic findings. Linear regression and analysis of variance was used to quantify the extent to which variations in severity of EoE symptoms and endoscopic and histologic findings explain variations in PhysGA.RESULTS:A total of 149 EoE patients were prospectively included (71.8% male, median age at inclusion 38 years, 71.8% with concomitant allergies). A moderate positive correlation between PhysGA and PatGA (rho=0.442, P<0.001) was observed and the mean difference in the Bland-Altman plot was 1.77. Variations in severity of endoscopic findings, symptoms, and histologic findings alone explained 53%, 49%, and 30%, of the variability in PhysGA, respectively. Together, these findings explained 75% of variability in PhysGA.CONCLUSIONS:Gastroenterologists rate EoE activity mainly on the basis of endoscopic findings and symptoms and, to a lesser extent, on histologic findings.Am J Gastroenterol advance online publication, 3 March 2015; doi:10.1038/ajg.2015.32.

The H19/let-7 double-negative feedback loop contributes to glucose metabolism in muscle cells

The H19 lncRNA has been implicated in development and growth control and is associated with human genetic disorders and cancer. Acting as a molecular sponge, H19 inhibits microRNA (miRNA) let-7. Here we report that H19 is significantly decreased in muscle of human subjects with type-2 diabetes and insulin resistant rodents. This decrease leads to increased bioavailability of let-7, causing diminished expression of let-7 targets, which is recapitulated in vitro where H19 depletion results in impaired insulin signaling and decreased glucose uptake. Furthermore, acute hyperinsulinemia downregulates H19, a phenomenon that occurs through PI3K/AKT-dependent phosphorylation of the miRNA processing factor KSRP, which promotes biogenesis of let-7 and its mediated H19 destabilization. Our results reveal a previously undescribed double-negative feedback loop between sponge lncRNA and target miRNA that contributes to glucose regulation in muscle cells.

Low DICER1 expression is associated with attenuated neutrophil differentiation and autophagy of NB4 APL cells

Successful myeloid differentiation depends on the expression of a series of miRNAs. Thus, it is hardly surprising that miRNAs are globally repressed in AML, a disease mainly characterized by a block in cellular myeloid differentiation. Studies investigating the mechanisms for low miRNA expression in AML has mostly focused on altered transcriptional regulation or deletions, whereas defective miRNA processing has received less attention. In this study, we report that the expression of the key miRNA processing enzyme DICER1 is down-regulated in primary AML patient samples and healthy CD34(+) progenitor cells as compared with granulocytes. In line with these findings, Dicer1 expression was induced significantly in AML cell lines upon neutrophil differentiation. The knocking down of DICER1 in AML cells significantly attenuated neutrophil differentiation, which was paralleled by decreased expression of miRNAs involved in this process. Moreover, we found that inhibiting DICER1 attenuated the activation of autophagy, a cellular recycling process that is needed for proper neutrophil differentiation of AML cells. Our results clearly indicate that DICER1 plays a novel role in neutrophil differentiation as well as in myeloid autophagy of AML cells.