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Refractory ascites is characterized by severe circulatory dysfunction and increased sensitivity to renal impairment by non‐selective beta‐blockers (NSBB). A novel serum microRNA signature, identified in the secretome of human cirrhotic hepatocytes, allows for the molecular characterization of patients with decompensated cirrhosis more prone to develop significant cardiac impairment after NSBB, as well as those more likely to respond to NSBB in terms of HVPG reduction., Noninvasive staging of decompensated cirrhosis is an unmet clinical need. The aims of this study were to characterize and validate a novel microRNA (miRNA) signature to stage decompensated cirrhosis and predict the portal pressure and systolic cardiac response to nonselective beta‐blockers (NSBBs). Serum samples from patients with decompensated cirrhosis (n = 36) and healthy controls (n = 36) were tested for a novel signature of five miRNAs (miR‐452‐5p, miR‐429, miR‐885‐5p, miR‐181b‐5p, and miR‐122‐5p) identified in the secretome of primary human hepatocytes and for three miRNAs (miR‐192‐5p, miR‐34a‐5p, and miR‐29a‐5p) previously discovered as biomarkers of chronic liver disease. All patients had ascites, which was refractory in 18 (50%), and were placed on NSBBs for variceal bleeding prophylaxis. In all patients, serum miRNAs, hepatic venous pressure gradient, and an echocardiogram study were performed before and 1 month after NSBBs. Patients with cirrhosis had lower serum levels of miR‐429, miR‐885‐5p, miR‐181b‐5p, miR‐122‐5p, miR‐192‐5p, and miR‐29a‐5p (P

MicroRNAs (miRNAs) are small non-coding endogenous RNAs, which are attracting a growing interest as therapeutic molecules due to their central role in major diseases. However, the transformation of these biomolecules into drugs is limited due to their unstability in the bloodstream, caused by nucleases abundantly present in the blood, and poor capacity to enter cells. The conjugation of miRNAs to nanoparticles (NPs) could be an effective strategy for their clinical delivery. Herein, the engineering of non-liposomal lipid nanovesicles, named quatsomes (QS), for the delivery of miRNAs and other small RNAs into the cytosol of tumor cells, triggering a tumor-suppressive response is reported. The engineered pH-sensitive nanovesicles have controlled structure (unilamellar), size (24 weeks), and are prepared by a green, GMP compliant, and scalable one-step procedure, which are all unavoidable requirements for the arrival to the clinical practice of NP based miRNA therapeutics. Furthermore, QS protect miRNAs from RNAses and when injected intravenously, deliver them into liver, lung, and neuroblastoma xenografts tumors. These stable nanovesicles with tunable pH sensitiveness constitute an attractive platform for the efficient delivery of miRNAs and other small RNAs with therapeutic activity and their exploitation in the clinics., The funding was received by Ministerio de Educación, Cultura y Deporte (Grant no. FPU16/01099), Ministerio de Economía, Industria y Competividad (Grants MAT2016-80820-R, MAT2016-80826-R and SAF2016-75241-R), the Ministry of Science and Innovation (MINECO) of Spain through grant PID2019-105622RB-I00, from Instituto de Salud Carlos III (Grant no. CP16/00006, PI17/00564, PI20/00530, DTS20/00018) (Co-funded by European Regional Development Fund/European Social Fund) “Investing in your future”), from the EuroNanoMed II platform through the NanoVax project, from CIBER-BBN through grant TAG-SMARTLY, Joan Petit Foundation, Asociación Matem Lo Bitxo and Asociación Española Contra el Cáncer (Grant no. LABAE18009SEGU), as well as, Generalitat de Catalunya through the Centres de Recerca de Catalunya (CERCA) programme and grant no. 2017-SGR-918, and from Agency for Management of University and Research Grants (AGAUR) (Grant no 2018LLAV0064 and SIFECAT IU68-010017). Furthermore, ICMAB-CSIC acknowledges support from the MINECO through the Severo Ochoa Programme for Centres of Excellence in R&D (SEV-2015-0496 and CEX2019-000917-S). Quatsome production and their physicochemical characterization was performed by the ICTS “NANBIOSIS,” more specifically in the Biomaterial Processing and Nanostructuring Unit (U6), Unit of the CIBER in Bioengineering, Biomaterials & Nanomedicne (CIBER-BBN) located at the Institute of Materials Science of Barcelona (ICMAB-CSIC). The authors thank the UAB Microscopy service for their help in recording cryo-TEM images. The authors also thank Mr. Adolfo de Hoyos-Limon for pKa measurements, Ms. Patricia Pérez for her help in in vitro experiments, the members of Laboratory Animal Service Unit of Vall d'Hebron Research Institute for their help in the in vivo experiment. The authors thank Editage (www.editage.com) and Ms. Christine O'Hara for English language correction. The authors acknowledge Biorender.com for allowing the adaptation of their templates. Retrieved from https://app.biorender.com/biorender-templates., With funding from the Spanish government through the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000917-S).

Dementia with Lewy bodies (DLB) is one of the most common causes of degenerative dementia, after Alzheimer’s disease (AD), and presents pathological and clinical overlap with both AD and Parkinson’s disease (PD). Consequently, only one in three DLB cases is diagnosed correctly. Platelets, previously related to neurodegeneration, contain microRNAs (miRNAs) whose analysis may provide disease biomarkers. Here, we profiled the whole platelet miRNA transcriptome from DLB patients and healthy controls. Differentially expressed miRNAs were further validated in three consecutive studies from 2017 to 2019 enrolling 162 individuals, including DLB, AD, and PD patients, and healthy controls. Results comprised a seven-miRNA biosignature, showing the highest diagnostic potential for the differentiation between DLB and AD. Additionally, compared to controls, two miRNAs were down-regulated in DLB, four miRNAs were up-regulated in AD, and two miRNAs were down-regulated in PD. Predictive target analysis identified three disease-specific clusters of pathways as a result of platelet-miRNA deregulation. Our cross-sectional study assesses the identification of a novel, highly specific and sensitive platelet-associated miRNA-based biosignature, which distinguishes DLB from AD.

Diabetic kidney disease (DKD) is the most common diabetic complication and is a leading cause of end-stage kidney disease. Increasing evidence shows that DKD is regulated not only by many classical signaling pathways but also by epigenetic mechanisms involving chromatin histone modifications, DNA methylation, and non-coding RNA (ncRNAs). In this review, we focus on our current understanding of the role and mechanisms of ncRNAs, including microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) in the pathogenesis of DKD. Of them, the regulatory role of TGF-β/Smad3-dependent miRNAs and lncRNAs in DKD is highlighted. Importantly, miRNAs and lncRNAs as biomarkers and therapeutic targets for DKD are also described, and the perspective of ncRNAs as a novel therapeutic approach for combating diabetic nephropathy is also discussed.

WOS: 000537582600001 PubMed: 32496656 There are many unknown aspects of the pathogenesis of renal cell carcinoma (RCC). The aim of the current study was to define new RCC-related genes and measure their associations with RCC and clinical parameters, especially platelet/lymphocyte ratio which may be an independent predictor of prognosis in patients with RCC and other forms of cancer. Via in silico analysis upon RCC-specific deleted genes in chromosome 3, four possible ceRNAs (ATXN3, ABI2, GOLGB1 and SMAD2) were identified. Then, the expression levels of these genes in tumour and adjacent healthy kidney tissues of 19 RCC patients were determined by real-time PCR. ATXN3 and GOLGB1 gene expression levels increased but ABI2 gene expression level decreased in tumour kidney tissues when compared to normal ones. ATXN3, ABI2 and GOLGB1 gene expression levels were significantly higher in Fuhrman grade 4 than other grades (P < .001). ABI2 gene expression levels were significantly associated with higher platelet/lymphocyte ratio of the patients with RCC (P < .05). ATXN3, ABI2 and GOLGB1 may predict higher RCC grades. Also, ABI2 may regulate platelet/lymphocyte ratio which may be an independent predictor of RCC and other forms of cancer. Scientific Research Projects Commission of Ondokuz Mayis UniversityOndokuz Mayis University [TIP.1904.18.002] This study has been financed by Scientific Research Projects Commission of Ondokuz Mayis University (project number: PYO. TIP.1904.18.002)

The most common scoring system for critically ill patients is the Sequential Organ Failure Assessment (SOFA) score. Little is known about specific molecular signaling networks underlying the SOFA criteria. We characterized these networks and identified specific key regulatory molecules. We prospectively studied seven patients with sepsis and six controls with high-throughput RNA sequencing (RNAseq). Quantitative reverse transcription PCR (RT-qPCR) confirmation was performed in a second independent cohort. Differentially and significantly expressed miRNAs and their target mRNA transcripts were filtered for admission SOFA criteria and marker RNAs for the respective criteria identified. We bioinformatically constructed molecular signaling networks specifically reflecting these criteria followed by RT-qPCR confirmation of RNAs with important regulatory functions in the networks in the second cohort. RNAseq identified 82 miRNAs (45% upregulated) and 3254 mRNAs (50% upregulated) differentially expressed between sepsis patients and controls. Bioinformatic analysis characterized 6 miRNAs and 76 mRNA target transcripts specific for the SOFA criteria. RT-qPCR validated miRNA and mRNAs included IGFBP2 (respiratory system), MMP9 and PDE4B (nervous system), PPARG (cardiovascular system), AKR1B1, ANXA1, and LNC2/NGAL (acute kidney injury), GFER/ALR (liver), and miR-30c-3p (coagulopathy). There are specific canonical networks underlying the SOFA score. Key regulatory miRNA and mRNA transcripts support its biologic validity.

// Julia Alejandra Pezuk 1, * , Thiago Luiz Araujo Miller 1, 2, * , Jose Luiz Barbosa Bevilacqua 1 , Alfredo Carlos Simoes Dornellas de Barros 1 , Felipe Eduardo Martins de Andrade 1 , Luiza Freire de Andrade e Macedo 1 , Vera Aguilar 1 , Amanda Natasha Menardo Claro 1, 3 , Anamaria Aranha Camargo 1 , Pedro Alexandre Favoretto Galante 1 and Luiz F. L. Reis 1 1 Hospital Sirio-Libanes, Sao Paulo, Brazil 2 Departamento de Bioquimica, Instituto de Quimica, Universidade de Sao Paulo, Sao Paulo, Brazil 3 Current address: SulAmerica, Sao Paulo, Brazil * These authors contributed equally to this work Correspondence to: Pedro Alexandre Favoretto Galante, email: pgalante@mochsl.org.br Luiz F. L. Reis, email: luiz.reis@hsl.org.br Keywords: micro RNAs, breast cancer, BI-RADS, blood plasma, machine learning Received: June 17, 2017 Accepted: July 25, 2017 Published: September 11, 2017 ABSTRACT A BI-RADS category of 4 from a mammogram indicates suspicious breast lesions, which require core biopsies for diagnosis and have an approximately one third chance of being malignant. Human plasma contains many circulating microRNAs, and variations in their circulating levels have been associated with pathologies, including cancer. Here, we present a novel methodology to identify malignant breast lesions in women with BI-RADS 4 mammography. First, we used the miRNome array and qRT-PCR to define circulating microRNAs that were differentially represented in blood samples from women with breast tumor (BI-RADS 5 or 6) in comparison to controls (BI-RADS 1 or 2). Next, we used qRT-PCR to quantify the level of this circulating microRNAs in patients with mammograms presenting with BI-RADS category 4. Finally, we developed a machine learning method (Artificial Neural Network - ANN) that receives circulating microRNA levels and automatically classifies BI-RADS 4 breast lesions as malignant or benign. We identified a minimum set of three circulating miRNAs (miR-15a, miR-101 and miR-144) with altered levels in patients with breast cancer. These three miRNAs were quantified in plasma from 60 patients presenting biopsy-proven BI-RADS 4 lesions. Finally, we constructed a very efficient ANN that could correctly classify BI-RADS 4 lesions as malignant or benign with approximately 92.5% accuracy, 95% specificity and 88% sensibility. We believe that our strategy of using circulating microRNA and a machine learning method to classify BI-RADS 4 breast lesions is a non-invasive, non-stressful and valuable complementary approach to core biopsy in women with BI-RADS 4 lesions.

Graphical abstract Image 1, Prevalence of Kaposi sarcoma-associated herpesvirus (KSHV/HHV-8) varies greatly in different populations. We hypothesized that the actual prevalence of KSHV/HHV8 infection in humans is underestimated by the currently available serological tests. We analyzed four independent patient cohorts with post-surgical or post-chemotherapy sepsis, chronic lymphocytic leukemia and post-surgical patients with abdominal surgical interventions. Levels of specific KSHV-encoded miRNAs were measured by reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and KSHV/HHV-8 IgG were measured by immunoassay. We also measured specific miRNAs from Epstein Barr Virus (EBV), a virus closely related to KSHV/HHV-8, and determined the EBV serological status by ELISA for Epstein-Barr nuclear antigen 1 (EBNA-1) IgG. Finally, we identified the viral miRNAs by in situ hybridization (ISH) in bone marrow cells. In training/validation settings using independent multi-institutional cohorts of 300 plasma samples, we identified in 78.50% of the samples detectable expression of at least one of the three tested KSHV-miRNAs by RT-qPCR, while only 27.57% of samples were found to be seropositive for KSHV/HHV-8 IgG (P, Highlights • There is no agreement on a standard assay to detect the true prevalence of Kaposi sarcoma-associated herpesvirus (KSHV) infection. • Measurement of the viral miRNAs in plasma by RT-qPCR allows a direct and accurate assessment of viral infection. • Measurement of the viral miRNAs in plasma by RT-qPCR shows prevalence of KSHV infection in immuno-depressed patients. • Measurement of plasma viral miRNAs for viral infection assessment has the potential to become a “gold” standard method in the clinical practice. Chronic viral infections represent risk factors for diseases and development of infection-related complications. There is no agreement on a standard assay to detect the true prevalence of Kaposi sarcoma-associated herpesvirus (KSHV) infection. The current method used in the clinical practice (ELISA-test) identifies a great geographic variation in KSHV seroprevalence and may underestimate the true-prevalence of KSHV infection. Here we showed that detection of plasma viral miRNAs levels for the identification of viral infection (e.g., KSHV, Epstein-Bar virus or EBV) is more accurate than the current method for detection of virus-derived antigen, especially in patients with low number of immune cells.

Duchenne muscular dystrophy (DMD) is characterized by progressive skeletal muscle degeneration. No treatments are currently available to prevent the disease. While the muscle enriched microRNA, miR-133b, has been implicated in muscle biogenesis, its role in DMD remains unknown. To assess miR-133b function in DMD-affected skeletal muscles, we genetically ablated miR-133b in the mdx mouse model of DMD. In the absence of miR-133b, the tibialis anterior muscle of P30 mdx mice is smaller in size and exhibits a thickened interstitial space containing more mononucleated cells. Additional analysis revealed that miR-133b deletion influences muscle fiber regeneration, satellite cell proliferation and differentiation, and induces widespread transcriptomic changes in mdx muscle. These include known miR-133b targets as well as genes involved in cell proliferation and fibrosis. Altogether, our data demonstrate that skeletal muscles utilize miR-133b to mitigate the deleterious effects of DMD.

MicroRNAs from the miR-200 family are commonly associated with the inhibition of the metastatic potential of cancer cells, following inhibition of ZEB transcription factors expression and epithelial-to-mesenchymal transition. However, previous studies performed in pancreatic adenocarcinoma revealed a more complex picture challenging this canonical model. To gain better insights into the role of miR-200 family members in this disease, we analyzed the expression of miR-200a, miR-200b, miR-200c, miR-141, miR-429, and miR-205, and ZEB1, ZEB2, and CDH1 in pancreatic tumors and matching normal adjacent parenchyma and patient-derived xenografts. We found that miR-200a, miR-429, and miR-205 are frequently overexpressed in pancreatic tumors, whereas CDH1 is downregulated, and ZEB1 and ZEB2 levels remain unchanged. Furthermore, we measured a positive correlation between miR-200 family members and CDH1 expression, and a negative correlation between ZEB1 and miR-200c, miR-141, and miR-205 expression, respectively. Interestingly, we identified significant changes in expression of epithelial-to-mesenchymal transition regulators and miR-200 members in patient-derived xenografts. Lastly, functional studies revealed that miR-141 and miR-429 inhibit the tumorigenic potential of pancreatic cancer cells. Taken together, this comprehensive analysis strongly suggests that miRNAs from the miR-200 family, and in particular miR-429, may act as a tumor suppressor gene in pancreatic cancer. Keywords: pancreatic cancer, patient-derived xenograft, miR-200 family, miR-429

[Background] MicroRNAs are small regulatory RNAs with important roles in carcinogenesis. Genetic variants in these regulatory molecules may contribute to disease. We aim to identify allelic variants in microRNAs as susceptibility factors to gastric cancer using association studies and functional approaches., [Methods] Twenty-one single nucleotide variants potentially functional, because of their location in either the seed, mature or precursor region of 22 microRNAs, were selected for association studies. Genetic association with gastric cancer in 365 cases and 1,284 matched controls (European Prospective Investigation into Cancer and Nutrition Cohort) was analysed using logistic regression. MicroRNA overexpression, transcriptome analysis, and target gene validation experiments were performed for functional studies., [Results] rs3746444:T>C, in the seed of MIR499A and mature MIR499B, associated with the cardia adenocarcinoma location; rs12416605:C>T, in the seed of MIR938, associated with the diffuse subtype; and rs2114358:T>C, in the precursor MIR1206, associated with the noncardia phenotype. In all cases, the association was inverse, indicating a protective affect against gastric cancer of the three minor allelic variants. MIR499 rs3746444:T>C and MIR1206 rs2114358:T>C are reported to affect the expression of these miRNAs, but the effect of MIR938 rs12416605:C>T is unknown yet. Functional approaches showed that the expression of MIR938 is affected by rs12416605:C>T and revealed that MIR938 could regulate a subset of cancer-related genes in an allele-specific fashion. Furthermore, we demonstrated that CXCL12, a chemokine participating in gastric cancer metastasis, is specifically regulated by only one of the rs12416605:C>T alleles., [Conclusion] rs12416605 appears to be involved in gastric cancer by affecting the regulatory function of MIR938 on genes related to this cancer type, particularly on CXCL12 posttranscriptional regulation., >la Caixa> Foundation. Grant Number: BM06-130-0; European Regional Development Fund. Grant Number: PI070130 and PI12/01187; Ministerio de Ciencia e Innovación. Grant Number: BFU2010-18477; Fondo de Fomento al Desarrollo Científico y Tecnológico. Grant Number: FONDECYT 1170446; Instituto de Salud Carlos III.

MicroRNA-210-3p is the most prominent hypoxia regulated microRNA, and it has been found significantly overexpressed in different human cancers. We performed the expression analysis of miR-210-3p in a retrospective cohort of breast cancer patients with a median follow-up of 76 months (n = 283). An association between higher levels of miR-210-3p and risk of disease progression (HR: 2.13, 95%CI: 1.33-3.39, P = 0.002) was found in the subgroup of patients treated with Epirubicin and Cyclophosphamide followed by Docetaxel. Moreover, a cut off value of 20.966 established by ROC curve analyses allowed to discriminate patients who developed distant metastases with an accuracy of 85% at 3- (AUC: 0.870, 95%CI: 0.690-1.000) and 83% at 5-years follow up (AUC: 0.832, 95%CI: 0.656–1.000). Whereas the accuracy in discriminating patients who died for the disease was of 79.6% at both 5- (AUC: 0.804, 95%CI: 0.517–1.000) and 10-years (AUC: 0.804. 95%CI: 0.517–1.000) follow-up. In silico analysis of miR-210-3p and Docetaxel targets provided evidence for a putative molecular cross-talk involving microtubule regulation, drug efflux metabolism and oxidative stress response. Overall, our data point to the miR-210-3p involvement in the response to therapeutic regimens including Docetaxel in sequential therapy with anthracyclines, suggesting it may represent a predictive biomarker in breast cancer patients.

Ovarian cancer is the most lethal gynecological malignancy due to the silent nature on its early onset and the rapid acquisition of drug resistance. Histologically heterogeneous, it includes several subtypes with different mutational landscapes, hampering the development of effective targeted therapies. Non-coding RNAs are emerging as potential new therapeutic targets in cancer. To search for a microRNA signature related to ovarian carcinomas and study its potential as effective targeted therapy, we examined the expression of 768 miRNA in a large collection of tumor samples and found miR-654-5p to be infraexpressed in ovarian serous carcinomas, the most common and aggressive type. Restoration of miR-654-5p levels reduced tumor cell viability in vitro and in vivo and impaired sphere formation capacity and viability of ovarian cancer patient-derived ascitic cells ex vivo. CDCP1 and PLAGL2 oncogenes were found to be the most relevant direct miR-654-5p targets and both genes convey in a molecular signature associated with key cancer pathways relevant to ovarian tumorigenesis, such as MYC, WNT and AKT pathways. Together, we unveiled the tumor suppressor function of miR-654-5p, suggesting that its restoration or co-targeting of CDCP1 and PLAGL2 may be an effective therapeutic approach for ovarian cancer., This work was supported in part by grants from Instituto de la Mujer Dexeus (DEXEUS-B29/012), CIBER (CB16/12/00328), SGR (2017 SGR 1661), the Ministerio de Economia y Competitividad and Fondos FEDER (RTC-2015-3821-1), Instituto Carlos III (PI15/00238 to A.S. and PI17/00564 to M.F.S) and the Miguel Servet Program (CP13/00158 and CPII18/00027 to AS. and CPII16/00006 to MFS). AP and LS were supported by predoctoral VHIR fellowships and CJ by an AGAUR predoctoral fellowship (VHIR: PRED-VHIR-2014-11 and PRED-VHIR-2017; AGAUR: 2017FI_B_00095, respectively).

OBJECTIVES: Pancreatic ductal adenocarcinoma (PDAC) presents the lowest survival rate of all cancers because only 6% of patients reach five-year survival. Alterations in the expression of several microRNAs (miRNAs) occur in the tumor of PDAC and in preneoplastic lesions as the called intraductal papillary mucinous neoplasm (IPMN). Here, we aimed at identifying which miRNAs are significantly altered in liquid biopsies from patients with PDAC and IPMN to find new noninvasive biomarkers for early detection of PDAC. METHODS: We analyzed by real-time quantitative reverse transcription-PCR (qRT-PCR) the expression of 17 circulating miRNAs, previously found to be significantly overexpressed in tissue pancreatic neoplasms, in a set of 182 plasma samples (94 PDAC, 19 IPMN, 18 chronic pancreatitis, and 51 disease-free controls). Then, we analyzed CA19.9 levels in the same plasma set, and we assessed the diagnostic values of differentially expressed miRNAs, CA19.9, and all possible combinations. RESULTS: Of note, 16, 14, and 9 miRNAs were significantly increased in PDAC, IPMN, and chronic pancreatitis, respectively, compared with control plasmas. miR-21-5p, miR-33a-3p, miR-320a, and miR-93-5p showed the highest discriminating capacity for pancreatic neoplasia (PDAC or IPMN) with an area under the receiver operating characteristic curve (AUC) of 0.86, 0.85, 0.85, and 0.80, respectively. 2-miRNA combinations improved these performances reaching AUC = 0.90 for “miR-33a-3p+miR-320a.” Addition of CA19.9 increased the diagnostic potential of miRNA signatures even further achieving an AUC of 0.95 (93% sensitivity and 85% specificity) for the combination of “miR-33a-3p+miR-320a+CA19.9.” CONCLUSIONS: Novel signatures combining miRNAs and CA19.9 could be used as noninvasive biomarkers for early detection of PDAC.

Hormone-refractory prostate cancer frequently relapses from therapy and inevitably progresses to a bone-metastatic status with no cure. Understanding of the molecular mechanisms conferring resistance to androgen deprivation therapy has the potential to lead to the discovery of novel therapeutic targets for type of prostate cancer with poor prognosis. Progression to castration-resistant prostate cancer (CRPC) is characterized by aberrant androgen receptor (AR) expression and persistent AR signaling activity. Alterations in metabolic activity regulated by oncogenic pathways, such as c-Myc, were found to promote prostate cancer growth during the development of CRPC. Non-coding RNAs represent a diverse family of regulatory transcripts that drive tumorigenesis of prostate cancer and various other cancers by their hyperactivity or diminished function. A number of studies have examined differentially expressed non-coding RNAs in each stage of prostate cancer. Herein, we highlight the emerging impacts of microRNAs and long non-coding RNAs linked to reactivation of the AR signaling axis and reprogramming of the cellular metabolism in prostate cancer. The translational implications of non-coding RNA research for developing new biomarkers and therapeutic strategies for CRPC are also discussed.

Altres ajuts: This work was supported by the Marató TV3 grant 1405/10. Altres ajuts: MSCBS/PI12-1702 Altres ajuts: MSCBS/PI15-216 Parkinson disease (PD) and dementia with Lewy bodies (DLB) are Lewy body diseases characterized by abnormal alpha-synuclein deposits and overlapping pathological features in the brain. Several studies have shown that glucocerebrosidase (GBA) deficiency is involved in the development of LB diseases. Here, we aimed to find out if this deficiency starts at the transcriptional level, also involves alternative splicing, and if GBA expression changes in brain are also detectable in blood of patients with LB diseases. The expression of three GBA transcript variants (GBAtv1, GBAtv2 and GBAtv5) was analyzed in samples from 20 DLB, 25 PD and 17 control brains and in blood of 20 DLB, 26 PD patients and 17 unaffected individuals. Relative mRNA expression was determined by real-time PCR. Expression changes were evaluated by the ΔΔCt method. In brain, specific expression profiles were identified in the temporal cortex of DLB and in the caudate nucleus of PD. In blood, significant GBA mRNA diminution was found in both DLB and PD patients. Early PD and early-onset DLB patients showed lowest GBA levels which were normal in PD patients with advanced disease and DLB patients who developed disease after 70 years of age. In conclusion, disease group specific GBA expression profiles were found in mostly affected areas of LBD. In blood, GBA expression was diminished in LB diseases, especially in patients with early onset DLB and in patients with early PD. Age of disease onset exerts an opposite effect on GBA expression in DLB and PD.

The successful treatment of various hematologic diseases with allogeneic hematopoietic cell transplantation is often limited by the occurrence of graft-versus-host disease (GvHD). Several microRNAs (miRs) have recently been shown to impact the biology of GvHD by regulating pro- as well as anti-inflammatory target genes. There is increasing evidence that a single miR can have different effects by preferentially targeting certain genes depending on the cell type that the miR is analyzed in. This review will focus on the role of miRs in myeloid cells during the development of acute and chronic GvHD and autoimmune diseases. Because miRs act on the expression of multiple target genes and may thereby influence the immune system at different functional levels, they are potentially attractive targets for the modification of allogeneic immune responses using miR mimics and inhibitors.

Increasing evidence indicates that microRNAs (miRNAs) are contributing factors to neurodegeneration. Alterations in miRNA signatures have been reported in several neurodegenerative dementias, but data in prion diseases are restricted to ex vivo and animal models. The present study identified significant miRNA expression pattern alterations in the frontal cortex and cerebellum of sporadic Creutzfeldt-Jakob disease (sCJD) patients. These changes display a highly regional and disease subtype-dependent regulation that correlates with brain pathology. We demonstrate that selected miRNAs are enriched in sCJD isolated Argonaute(Ago)-binding complexes in disease, indicating their incorporation into RNA-induced silencing complexes, and further suggesting their contribution to disease-associated gene expression changes. Alterations in the miRNA-mRNA regulatory machinery and perturbed levels of miRNA biogenesis key components in sCJD brain samples reported here further implicate miRNAs in sCJD gene expression (de)regulation. We also show that a subset of sCJD-altered miRNAs are commonly changed in Alzheimer’s disease, dementia with Lewy bodies and fatal familial insomnia, suggesting potential common mechanisms underlying these neurodegenerative processes. Additionally, we report no correlation between brain and cerebrospinal fluid (CSF) miRNA-profiles in sCJD, indicating that CSF-miRNA profiles do not faithfully mirror miRNA alterations detected in brain tissue of human prion diseases. Finally, utilizing a sCJD MM1 mouse model, we analyzed the miRNA deregulation patterns observed in sCJD in a temporal manner. While fourteen sCJD-related miRNAs were validated at clinical stages, only two of those were changed at early symptomatic phase, suggesting that the miRNAs altered in sCJD may contribute to later pathogenic processes. Altogether, the present work identifies alterations in the miRNA network, biogenesis and miRNA-mRNA silencing machinery in sCJD, whereby contributions to disease mechanisms deserve further investigation., Author summary miRNAs are small non-coding RNAs that regulate gene expression through complementary binding to their mRNA targets. Specific miRNA signatures have been proposed for several neurodegenerative diseases supporting the idea that miRNA deregulation is a common disease hallmark. Here we present the comprehensive miRNA signature in sporadic Creutzfeldt-Jakob disease (sCJD). Our study unravels the complex network of regional and disease-subtype miRNA alterations, and the presence of a disturbed miRNA biogenesis pathway and miRNA-mRNA silencing machinery. We also highlight the existence of time-dependent miRNA profiles and identify commonly regulated miRNAs between several dementias with cortical pathology sharing a partial clinical overlap and pathological involvement with sCJD. The present data shed light on the potential role of miRNAs as a contributing factor of pathogenic molecular traits associated with sCJD.

Cristina Esquinas,1,2 Sabina Janciauskiene,3 Ricardo Gonzalo,4 Gemma Mas de Xaxars,4 Beata Olejnicka,5 Irene Belmonte,6 Miriam Barrecheguren,1 Esther Rodriguez,1 Alexa Nuñez,1 Francisco Rodriguez-Frias,6 Marc Miravitlles1 1Pneumology Department, University Hospital Vall d’Hebron, CIBER de Enfermedades Respiratorias (CIBERES), Barcelona, 2Public Health, Mental, Maternal and Child Health Nursing Department, Faculty of Medicine and Health Sciences, University of Barcelona, Barcelona, Spain; 3Department of Respiratory Medicine, Hannover Medical School, BREATH, German Center for Lung Research (DZL), Hannover, Germany; 4Statistics and Bioinformatics Unit (UEB), Vall d’Hebron Research Institute (VHIR), Barcelona, Spain; 5Department of Experimental Medical Science, Lund University, Lund, Sweden; 6Biochemistry Department, University Hospital Vall d’Hebron, Barcelona, Spain Introduction: COPD has complex etiologies involving both genetic and environmental determinants. Among genetic determinants, the most recognized is a severe PiZZ (Glu342Lys) inherited alpha1-antitrypsin deficiency (AATD). Nonetheless, AATD patients present a heterogeneous clinical evolution, which has not been completely explained by sociodemographic or clinical factors. Here we performed the gene expression profiling of blood cells collected from mild and severe COPD patients with PiZZ AATD. Our aim was to identify differences in messenger RNA (mRNA) and microRNA (miRNA) expressions that may be associated with disease severity. Materials and methods: Peripheral blood mononuclear cells from 12 COPD patients with PiZZ AATD (6 with severe disease and 6 with mild disease) were used in this pilot, high-throughput microarray study. We compared the cellular expression levels of RNA and miRNA of the 2 groups, and performed functional and enrichment analyses using the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene-ontology (GO) terms. We also integrated the miRNA and the differentially expressed putative target mRNA. For data analyses, we used the R statistical language R Studio (version 3.2.5). Results: The severe and mild COPD–AATD groups were similar in terms of age, gender, exacerbations, comorbidities, and use of augmentation therapy. In severe COPD–AATD patients, we found 205 differentially expressed genes (DEGs) (114 upregulated and 91 downregulated) and 28 miRNA (20 upregulated and 8 downregulated) compared to patients with mild COPD–AATD disease. Of these, hsa-miR-335-5p was downregulated and 12 target genes were involved in cytokine signaling, MAPK/mk2, JNK signaling cascades, and angiogenesis were much more highly expressed in severe compared with mild patients. Conclusions: Despite the small sample size, we identified downregulated miRNA (hsa-miR-335) and the activation of pathways related to inflammation and angiogenesis on comparing patients with severe vs mild COPD–AATD. Nonetheless, our findings warrant further validation in large studies. Keywords: alpha1-antitrypsin deficiency, COPD, gene expression, miRNAs, integrative analysis

MicroRNAs usually regulate gene expression negatively, and aberrant expression has been involved in the development of several types of cancers. Microarray profiling of microRNA expression was performed to define a microRNA signature in a series of mycosis fungoides tumor stage (MFt, n=21) and CD30+ primary cutaneous anaplastic large cell lymphoma (CD30+ cALCL, n=11) samples in comparison with inflammatory dermatoses (ID, n=5). Supervised clustering confirmed a distinctive microRNA profile for cutaneous T-cell lymphoma (CTCL) with respect to ID. A 40 microRNA signature was found in MFt including upregulated onco-microRNAs (miR-146a, miR-142-3p/5p, miR-21, miR-181a/b, and miR-155) and downregulated tumor-suppressor microRNAs (miR-200ab/429 cluster, miR-10b, miR-193b, miR-141/200c, and miR-23b/27b). Regarding CD30+ cALCL, 39 differentially expressed microRNAs were identified. Particularly, overexpression of miR-155, miR-21, or miR-142-3p/5p and downregulation of the miR-141/200c clusters were observed. DNA methylation in microRNA gene promoters, as expression regulatory mechanism for deregulated microRNAs, was analyzed using Infinium 450K array and approximately one-third of the differentially expressed microRNAs showed significant DNA methylation differences. Two different microRNA methylation signatures for MFt and CD30+ cALCL were found. Correlation analysis showed an inverse relationship for microRNA promoter methylation and microRNA expression. These results reveal a subgroup-specific epigenetically regulated microRNA signatures for MFt and CD30+ cALCL patients.

Background: Contemporary challenges of prostate cancer (PCa) include overdiagnosis and overtreatment, entailing the need for novel clinical tools to improve risk stratification and therapy selection. PCa diagnosis and prognostication might be perfected using epigenetic biomarkers, among which aberrant DNA methylation of microRNA promoters has not been systematically explored. Herein, we identified aberrantly methylated microRNAs promoters in PCa and assessed its diagnostic and prognostic biomarker potential. Methods: Using HumanMethylation450 BeadChip-based analysis differentially methylated CpGs in microRNA promoters were identified. Promoter methylation of six microRNAs (miR-34b/c, miR-129-2, miR-152, miR-193b, miR-663a and miR-1258) was analyzed by qMSP in three sets (180 prostatectomies, 95 urine sediments and 74 prostate biopsies). Biomarkers’ diagnostic (validity estimates) and prognostic [disease-free (DFS) and disease-specific survival (DSS)] performance was assessed. Results: Significantly higher promoter methylation levels in PCa were confirmed for six candidate microRNAs. Except for miR-152, all displayed AUC values higher than 0.90, with miR-1258 and miR-193b disclosing the best performance (AUC = 0.99 and AUC = 0.96, respectively). In urine samples, miR-193b showed the best performance (91.6% sensitivity, 95.7% specificity, AUC = 0.96). Moreover, higher miR-129-2 independently predicted for shorter DSS and miR−34b/c methylation levels independently predicted for shorter DFS and DSS. Conclusions: Quantitative miR-193b, miR-129-2 and miR-34b/c promoter methylation might be clinically useful PCa biomarkers for non-invasive detection/diagnosis and prognostication, both in tissue and urine samples. The authors would like to acknowledge funding attributed to this study, namely research grants from Research Center of Portuguese Oncology Institute of Porto (CI-IPOP 4–2012; CI-IPOP 19–2016)) and by Federal funds through Programa Operacional Temático Factores de Competitividade (COMPETE) with co-participation from the European Community Fund (FEDER) and by national funds through Fundação para a Ciência e Tecnología (FCT) under the projects EXPL/BIM-ONC/0556/2012. JRC was supported by a FCT-Fundação para a Ciência e a Tecnologia fellowship (SFRH/BD/71293/2010).

MicroRNAs are critical regulators of gene networks in normal and abnormal biological processes. Focusing on invasive ductal breast cancer (IDC), we have found dysregulated expression in tumor samples of several microRNAs, including the miR200 family, along progression from primary tumors to distant metastases, further reflected in higher blood levels of miR-200b and miR-7 in IDC patients with regional or distant metastases relative to patients with primary node-negative tumors. Forced expression of miR-200s in MCF10CA1h mammary cells induced an enhanced epithelial program, aldehyde dehydrogenase (ALDH) activity, mammosphere growth and ability to form branched tubuloalveolar structures while promoting orthotopic tumor growth and lung colonization in vivo. MiR-200s also induced the constitutive activation of the PI3K-Akt signaling through downregulation of PTEN, and the enhanced mammosphere growth and ALDH activity induced in MCF10CA1h cells by miR-200s required the activation of this signaling pathway. Interestingly, the morphology of tumors formed in vivo by cells expressing miR-200s was reminiscent of metaplastic breast cancer (MBC). Indeed, the epithelial components of MBC samples expressed significantly higher levels of miR-200s than their mesenchymal components and displayed a marker profile compatible with luminal progenitor cells. We propose that microRNAs of the miR-200 family promote traits of highly proliferative breast luminal progenitor cells, thereby exacerbating the growth and metastatic properties of transformed mammary epithelial cells, This work was supported by grants to MICINN (FIS-PI080274), MINECO (SAF2012-40017-C02-02) and RTICC (RD12/0036/0036) to PLF; MICINN (SAF2011-24686), MINECO (SAF2012-40017-C02-01 and SAF2015-66984-C2-1-R), Catalan Agència d’Ajuts Universitaris i de Recerca (AGAUR; 2009SGR1482), and Xarxa de Referència en Biotecnologia to TMT; CIBERBBN and Fundación ECO to TMT, MM, PLF and JB; and RTICC (RD12/0037/0016) and AGAUR (2014SGR660) to MFS. The Hospital Clínic-IDIBAPS Biobank is part of the Xarxa de Bancs de Tumors de Catalunya (XBTC), financed by the Pla Director d’Oncologia de Catalunya, and the Red Nacional de Biobancos (RNBB, ReTBioH), funded by the ISCIII (RETICS RD09-0076/0038).

Colorectal cancer (CRC) is one of the leading causes of death in the United States. Chemotherapy and radiotherapy are some of the most commonly used treatments, but are often associated with severe side effects, and are not entirely curative. It is therefore important to consider other preventative treatment options. Phytochemicals are naturally occurring bioactive compounds which have been shown to play a role in cancer prevention and treatment, especially in regards to a person’s lifestyle and diet. Recent evidence has shown that phytochemicals may exert their chemopreventative effects by targeting micro RNAs (miRNAs), which regulate the downstream expression of tumor suppressors and oncogenes. MiRNAs are small, endogenous, noncoding RNAs that regulate several biological processes through post-translational regulation. The dysregulation of miRNA expression has been shown to be associated with colorectal cancer. In this review, we will summarize and discuss several phytochemicals, which have been shown to exert chemopreventative effects in colorectal cancer by the modulation of miRNA expression.

Histone modification is associated with resistance to anti-cancer drugs. Epigenetic modifications of histones can regulate resistance to anti-cancer drugs. It has been reported that histone deacetylase 3 (HDAC3) regulates responses to anti-cancer drugs, angiogenic potential, and tumorigenic potential of cancer cells in association with cancer-associated genes (CAGE), and in particular, a cancer/testis antigen gene. In this paper, we report the roles of microRNAs that regulate the expression of HDAC3 and CAGE involved in resistance to anti-cancer drugs and associated mechanisms. In this review, roles of HDAC3-miRNAs-CAGE molecular networks in resistance to anti-cancer drugs, and the relevance of HDAC3 as a target for developing anti-cancer drugs are discussed.

International audience; The female mammary gland is a very dynamic organ that undergoes continuous tissue remodeling during adulthood. Although it is well established that the number of menstrual cycles and pregnancy (in this case transiently) increase the risk of breast cancer, the reasons are unclear. Growing clinical and experimental evidence indicates that improper involution plays a role in the development of this malignancy. Recently, we described the miR-424(322)/503 cluster as an important regulator of mammary epithelial involution after pregnancy. Here, through the analysis of ∼3000 primary tumors, we show that miR-424(322)/503 is commonly lost in a subset of aggressive breast cancers and describe the genetic aberrations that inactivate its expression. Furthermore, through the use of a knockout mouse model, we demonstrate for the first time that loss of miR-424(322)/503 promotes breast tumorigenesis in vivo. Remarkably, we found that loss of miR-424(322)/503 promotes chemoresistance due to the up-regulation of two of its targets: BCL-2 and insulin-like growth factor-1 receptor (IGF1R). Importantly, targeted therapies blocking the aberrant activity of these targets restore sensitivity to chemotherapy. Overall, our studies reveal miR-424(322)/ 503 as a tumor suppressor in breast cancer and provide a link between mammary epithelial involution, tumori-genesis, and the phenomenon of chemoresistance.

Inflammation is the body's response to noxious stimuli such as infectious, physiological or chemical agents, it releases various inflammatory mediators via immune cells such as neutrophils, macrophages, and lymphocytes. These inflammatory mediators are growth factors, chemokines, and cytokines. Reactive oxygen species (ROS) and nitrogen species (RNS) activate transcriptional factors (NF-KB, STAT-3) and bring about cellular proliferation, genomic instability, angiogenesis, resistance to apoptosis, invasion, and metastasis. The presence of inflammatory mediators in the tumour microenvironment inhibits or promotes inflammation-induced cancer, depending on various stages of immune surveillance of the tumor i.e. by immunoediting, immunoprocessing, and immunoevasion. Myeloid derived suppressor cells are immature myeloid progenitor cells. They are the major immune-suppressor cells in the tumour inflammatory microenvironment that activate transcriptional factor NF-KB, STAT-3 to bring about tumour progression. Another gene which the micro RNA's are noncoding RNA molecules is found to have a link with inflammation and cancer. This article discusses the roles of inflammatory mediators involved in antitumour or protumour activity within the context of the tumour microenvironment.

Symptoms caused by bacterial, viral and malarial infections usually overlap and aetiologic diagnosis is difficult. Patient management in low-resource countries with limited laboratory services has been based predominantly on clinical evaluation and syndromic approaches. However, such clinical assessment has limited accuracy both for identifying the likely aetiological cause and for the early recognition of patients who will progress to serious or fatal disease. Plasma-detectable biomarkers that rapidly and accurately diagnose severe infectious diseases could reduce morbidity and decrease the unnecessary use of usually scarce therapeutic drugs. The discovery of microRNAs (miRNAs) has opened exciting new avenues to identify blood biomarkers of organ-specific injury. This review assesses current knowledge on the relationship between malaria disease and miRNAs, and evaluates how future research might lead to the use of these small molecules for identifying patients with severe malaria disease and facilitate treatment decisions. This work was supported by ISCIII-Subdirección General de Evaluación Plan Nacional I+D+I 2013-2016 (grant PI13/01478 cofunded by the Fondo Europeo de Desarrollo Regional [FEDER] and CES10/021-I3SNS to AM), the Departament d’Universitats i Recerca de la Generalitat de Catalunya (Agencia de Gestión de Ayudas Universitarias y de Investigación (AGAUR); grant 2014SGR263) and the CERCA Institutes Integration Program (SUMA 2013; Secretaría de Universidades e Investigación del Departamento de Economía y Conocimiento de la Generalidad de Cataluña, AGAUR and Fundación Institució dels Centres de Recerca de Catalunya). QB has a fellowship from the programme Miguel Servet of the ISCIII (Plan Nacional de I+D+I 2008-2011, grant number: CP11/00269). XE is supported by the Generalitat de Catalunya AGAUR 2014 SGR-1138 and the Spanish Ministry of Economy and Competitiveness, ‘Centro de Excelencia Severo Ochoa 2013-2017′, SEV-2012-0208.

Recently, microRNAs (miRNA) have been proposed as regulators in the different processes involved in alcohol intake, and differences have been found in the miRNA expression profile in alcoholics. However, no study has focused on analyzing polymorphisms in genes encoding miRNAs and daily alcohol consumption at the population level. Our aim was to investigate the association between a functional polymorphism in the pre-miR-27a (rs895819 A>G) gene and alcohol consumption in an elderly population. We undertook a cross-sectional study of PREvención con DIeta MEDiterránea (PREDIMED)-Valencia participants (n = 1007, including men and women aged 67 7 years) and measured their alcohol consumption (total and alcoholic beverages) through a validated questionnaire. We found a strong association between the pre-miR-27a polymorphism and total alcohol intake, this being higher in GG subjects (5.2 0.4 in AA, 5.9 0.5 in AG and 9.1 1.8 g/day in GG; padjusted = 0.019). We also found a statistically-significant association of the pre-miR-27a polymorphism with the risk of having a high alcohol intake (>2 drinks/day in men and >1 in women): 5.9% in AA versus 17.5% in GG; padjusted < 0.001. In the sensitivity analysis, this association was homogeneous for sex, obesity and Mediterranean diet adherence. In conclusion, we report for the first time a significant association between a miRNA polymorphism (rs895819) and daily alcohol consumption. This study was funded, by the Spanish Ministry of Health (Instituto de Salud Carlos III) and the Ministerio de Economía y Competitividad-Fondo Europeo de Desarrollo Regional (Projects CNIC-06/2007, RTIC G03/140, CIBER 06/03, PI06-1326, PI07-0954, PI11/02505, SAF2009-12304, AGL2010-22319-C03-03 and PRX14/00527), by the University Jaume I (Project P1-1B2013-54), by Contracts 53-K06-5-10 and 58-1950-9-001 from the U.S. Department of Agriculture Research Service, USA, by the Generalitat Valenciana (ACOMP2010-181, AP111/10, AP-042/11, ACOM2011/145, ACOMP/2012/190, ACOMP/2013/159 and ACOMP/213/165), and with the collaboration of the Real Colegio Complutense at Harvard University, Cambridge. MA, USA. Rocío Barragán’s contract is funded by the Ayudas para la contratación de personal investigador en formación de caracter predoctoral, Programa “VALencia Investigación más Desarrollo” (VALi+d). Conselleria d’Educació, Investigació, Cultura i Esport. Generalitat Valenciana, Spain (ACIF/2013/168).

// Giuseppina Roscigno 1, 2 , Cristina Quintavalle 1, 2 , Elvira Donnarumma 3 , Ilaria Puoti 1 , Angel Diaz-Lagares 4 , Margherita Iaboni 1 , Danilo Fiore 1 , Valentina Russo 1 , Matilde Todaro 5 , Giulia Romano 6 , Renato Thomas 7 , Giuseppina Cortino 7 , Miriam Gaggianesi 5 , Manel Esteller 4 , Carlo M. Croce 6 , Gerolama Condorelli 1, 2 1 Department of Molecular Medicine and Medical Biotechnology, “Federico II” University of Naples, Naples, Italy 2 IEOS-CNR, Naples, Italy 3 IRCCS-SDN, Naples, Italy 4 Epigenetic and Cancer Biology Program (PEBC) IDIBELL, Hospital Duran I Reynals, Barcelona, Spain 5 Department of Surgical and Oncological Sciences, Cellular and Molecular Pathophysiology Laboratory, University of Palermo, Palermo, Italy 6 Department of Molecular Virology, Immunology and Medical Genetics, Human Cancer Genetics Program, Comprehensive Cancer Center, The Ohio State University, Columbus, OH, USA 7 Department of Surgical and Oncology, Clinica Mediterranea, Naples, Italy Correspondence to: Gerolama Condorelli, e-mail: gecondor@unina.it Keywords: microRNAs, breast cancer, cancer stem cells, DNMT Received: June 15, 2015 Accepted: October 09, 2015 Published: October 19, 2015 ABSTRACT Cancer stem cells (CSCs) are a small part of the heterogeneous tumor cell population possessing self-renewal and multilineage differentiation potential as well as a great ability to sustain tumorigenesis. The molecular pathways underlying CSC phenotype are not yet well characterized. MicroRNAs (miRs) are small noncoding RNAs that play a powerful role in biological processes. Early studies have linked miRs to the control of self-renewal and differentiation in normal and cancer stem cells. We aimed to study the functional role of miRs in human breast cancer stem cells (BCSCs), also named mammospheres. We found that miR-221 was upregulated in BCSCs compared to their differentiated counterpart. Similarly, mammospheres from T47D cells had an increased level of miR-221 compared to differentiated cells. Transfection of miR-221 in T47D cells increased the number of mammospheres and the expression of stem cell markers. Among miR-221’s targets, we identified DNMT3b. Furthermore, in BCSCs we found that DNMT3b repressed the expression of various stemness genes, such as Nanog and Oct 3/4 , acting on the methylation of their promoters, partially reverting the effect of miR-221 on stemness. We hypothesize that miR-221 contributes to breast cancer tumorigenicity by regulating stemness, at least in part through the control of DNMT3b expression.

OBJECTIVE: To evaluate the risk of progression of diabetic retinopathy (DR) using new strategies to obtain genetic information in type 2 diabetes (T2D) based on interfering ribonucleic acid (RNA). MATERIAL AND METHODS: A prospective multicentre case-control study of 132 participants was distributed into: T2D with (+DR) or without (-DR) (T2DG; n=77), and a control group (CG; n=55). After an eye examination and personal interview, tears were collected for molecular analysis (expression of microRNAs [miRNAs] (miRCURY ARN Isolation Kit, Qiagen)]. Libraries, 137 vs. 140bp (GeneMapper, Applied Biosystems), were obtained in 18 samples (T2DG+DR=6; T2DG-DR=6; CG=6) by performing next-generation sequencing (NGS). SPSS 15.0 statistical program was used to perform data analysis. RESULTS: The mean age was 67+/-12 years in the T2DG vs. 55+/-21 years in the CG. Distribution men/women: 25/30 in T2DG vs. 51/28 in CG. A family history of DM, diet compliance, smoking, drinking and exercise, showed significant differences between groups (P

// Alfons Navarro 1 , Rut Tejero 1 , Nuria Vinolas 2 , Anna Cordeiro 1 , Ramon M. Marrades 3 , Dolors Fuster 1 , Oriol Caritg 1 , Jorge Moises 3 , Carmen Munoz 1 , Laureano Molins 5 , Josep Ramirez 4 , Mariano Monzo 1 1 Molecular Oncology and Embryology Laboratory, Human Anatomy Unit, School of Medicine, University of Barcelona, IDIBAPS, Barcelona, Spain 2 Department of Medical Oncology, Institut Clinic Malalties Hemato-Oncologiques (ICMHO), Hospital Clinic de Barcelona, University of Barcelona, IDIBAPS, Barcelona, Spain 3 Department of Pneumology, Institut Clinic del Torax (ICT), Hospital Clinic de Barcelona, University of Barcelona, IDIBAPS, CIBER de Enfermedades Respiratorias (CIBERES), Barcelona, Spain 4 Department of Pathology, Centro de Diagnostico Biomedico (CDB), Hospital Clinic de Barcelona, University of Barcelona, IDIBAPS, CIBERES, Barcelona, Spain 5 Department of Thoracic Surgery, Institut Clinic del Torax (ICT), Hospital Clinic de Barcelona, University of Barcelona, Barcelona, Spain Correspondence to: Alfons Navarro, e-mail: anavarroponz@ub.edu Keywords: PIWI proteins, piwiRNAs, PIWIL1, PIWIL4, NSCLC Received: November 14, 2014 Accepted: December 21, 2014 Published: January 23, 2015 ABSTRACT The expression of Piwi-interacting RNAs, small RNAs that bind to PIWI proteins, was until recently believed to be limited to germinal stem cells. We have studied the expression of PIWI genes during human lung embryogenesis and in paired tumor and normal tissue prospectively collected from 71 resected non-small-cell lung cancer patients. The mRNA expression analysis showed that PIWIL1 was highly expressed in 7-week embryos and downregulated during the subsequent weeks of development. PIWIL1 was expressed in 11 of the tumor samples but in none of the normal tissue samples. These results were validated by immunohistochemistry, showing faint cytoplasmic reactivity in the PIWIL1 -positive samples. Interestingly, the patients expressing PIWIL1 had a shorter time to relapse (TTR) ( p = 0.006) and overall survival (OS) ( p = 0.0076) than those without PIWIL1 expression. PIWIL2 and 4 were downregulated in tumor tissue in comparison to the normal tissue ( p < 0.001) and the patients with lower levels of PIWIL4 had shorter TTR ( p = 0.048) and OS ( p = 0.033). In the multivariate analysis, PIWIL1 expression emerged as an independent prognostic marker. Using 5-Aza-dC treatment and bisulfite sequencing, we observed that PIWIL1 expression could be regulated in part by methylation. Finally, an in silico study identified a stem-cell expression signature associated with PIWIL1 expression.

Long non-coding RNAs (lncRNAs) are deregulated in several tumors, although their role in acute myeloid leukemia (AML) is mostly unknown. We have examined the expression of the lncRNA HOX antisense intergenic RNA myeloid 1 (HOTAIRM1) in 241 AML patients. We have correlated HOTAIRM1 expression with a miRNA expression profile. We have also analyzed the prognostic value of HOTAIRM1 expression in 215 intermediate-risk AML (IR-AML) patients. The lowest expression level was observed in acute promyelocytic leukemia (P < 0.001) and the highest in t(6; 9) AML (P = 0.005). In 215 IR-AML patients, high HOTAIRM1 expression was independently associated with shorter overall survival OR: 2.04; P = 0.001), shorter leukemia-free survival (OR: 2.56; P < 0.001) and a higher cumulative incidence of relapse (OR: 1.67; P = 0.046). Moreover, HOTAIRM1 maintained its independent prognostic value within the favorable molecular subgroup (OR: 3.43; P = 0.009). Interestingly, HOTAIRM1 was overexpressed in NPM1-mutated AML (P < 0.001) and within this group retained its prognostic value (OR: 2.21; P = 0.01). Moreover, HOTAIRM1 expression was associated with a specific 33- microRNA signature that included miR-196b (P < 0.001). miR-196b is located in the HOX genomic region and has previously been reported to have an independent prognostic value in AML. miR-196b and HOTAIRM1 in combination as a prognostic factor can classify patients as high-, intermediate-, or low-risk (5-year OS: 24% vs 42% vs 70%; P = 0.004). Determination of HOTAIRM1 level at diagnosis provided relevant prognostic information in IR-AML and allowed refinement of risk stratification based on common molecular markers. The prognostic information provided by HOTAIRM1 was strengthened when combined with miR-196b expression. Furthermore, HOTAIRM1 correlated with a 33-miRNA signature., Marina Diaz-Beya is supported by ISCII (Rio Hortega CM13/00205). This research was in part supported by Fundacion Espanola de Hematologia Hemoterapia (beca de investigacion MDB). This research is also supported by grants from Fondo de Investigaciones Sanitarias/Instituto de Salud Carlos III PI13/00999 (IP: Dr. Jordi Esteve), RETICS RD12/0036/0010 (JE; MDB) and SDCSD from School of Medicine, University of Barcelona, AECC-Catalunya 2013 (AN) (sponsored by Mat Holding). Anna Cordeiro is an APIF fellow of the University of Barcelona

The aim of this study was to assess the cellular miRNA expression behaviour in testes with spermatogenic failure (SpF). We performed a high-throughput screen of 623 mature miRNAs by a quantitative RT-qPCR-based approach in histologically well-defined testicular samples with spermatogenic disruption at different germ-cell stages, which revealed altered patterns of miRNA expression. We focussed on the differentially expressed miRNAs whose expression correlated with the number of testicular mature germ-cells and described the combined expression values of a panel of three miRNAs (miR-449a, miR-34c-5p and miR-122) as a predictive test for the presence of mature germ-cells in testicular biopsy. Additionally, we determined decreased cellular miRNA content in developing germ-cells of SpF testis; this was more noticeable the earlier the stage of germ-cell differentiation was affected by maturation failure. Furthermore, we showed that the miRNA expression profile in mature sperm from mild SpF patients was widely altered. Our results suggest that the cellular miRNA content of developed germ-cells depends heavily on the efficacy of the spermatogenic process. What is more, spermatozoa that have fulfilled the differentiation process still retain the dysregulated miRNA pattern observed in the developing SpF germ-cells. This altered miRNA molecular signature may have functional implications for the male gamete.

Acute myeloid leukemia (AML) is a heterogeneous disease whose prognosis is mainly related to the biological risk conferred by cytogenetics and molecular profiling. In elderly patients (>= 60 years) with normal karyotype AML miR-3151 have been identified as a prognostic factor. However, miR-3151 prognostic value has not been examined in younger AML patients. In the present work, we have studied miR-3151 alone and in combination with BAALC, its host gene, in a cohort of 181 younger intermediate-risk AML (IR-AML) patients. Patients with higher expression of miR-3151 had shorter overall survival (P = 0.0025), shorter leukemia-free survival (P = 0.026) and higher cumulative incidence of relapse (P = 0.082). Moreover, in the multivariate analysis miR-3151 emerged as independent prognostic marker in both the overall series and within the unfavorable molecular prognostic category. Interestingly, the combined determination of both miR-3151 and BAALC improved this prognostic stratification, with patients with low levels of both parameters showing a better outcome compared with those patients harboring increased levels of one or both markers (P = 0.003). In addition, we studied the microRNA expression profile associated with miR-3151 identifying a six-microRNA signature. In conclusion, the analysis of miR-3151 and BAALC expression may well contribute to an improved prognostic stratification of younger patients with IR-AML., Marina Diaz-Beya is supported by ISCIII (Rio Hortega CM13/00205). This research was in part supported by Fundacion Espanola de Hematologia y Hemoterapia (beca de investigacion MDB). This research is also supported by grants from Fondo de Investigaciones Sanitarias/Instituto de Salud Carlos III PI13/00999 (PI: JE), RETICS RD12/0036/0010 (JE; MDB) and SDCSD from School of Medicine, University of Barcelona, AECC-Catalunya 2013 (AN) (sponsored by Mat Holding), and grants AGAUR 2014SGR-1281, ISCIII RD12/0036/0071 and PI014/00450 (JS). Anna Cordeiro is an APIF fellow of the University of Barcelona.

Purpose: We examined expression profiles of 16 micro RNAs (miRNAs) in triple negative breast cancers to identify their potential as biomarkers for lymph node metastasis. Methods: The expression profiles of miR-9, miR-21, miR-30a, miR-30d, miR-31, miR-34a, miR-34c, miR-100, miR-122, miR-125b, miR-146a, miR-146b, miR-155, miR-181a, miR-200c, and miR-205 were examined by using real-time quantitative reverse transcription polymerase chain reaction in tumor samples and corresponding benign breast tissues. Their associations with histopathological features and prognostic parameters were assessed. Results: When compared with the expression in benign breast tissues, seven of the miRNAs (miR-31, miR-205, miR-34a, miR-146a, miR-125b, miR-34c, and miR-181a) were downregulated more than 1.5-fold in tumor tissues, whereas, only miR-21 was found to be upregulated more than 1.5-fold in tumor tissues. Although miR-200c levels were decreased only 1.12-fold in tumor tissues, the reduced expressions of miR-200c and miR-205 were signifi cantly associated with lymph node metastasis (p= 0.021 and p= 0.016, respectively). Conclusion: Our results demonstrate that miR-205 and miR-200c expression levels may be useful in predicting lymph node metastasis in triple negative breast cancer patients.

PURPOSE: To identify tissue microRNAs predictive of sunitinib activity in patients with metastatic renal-cell-carcinoma (MRCC) and to evaluate in vitro their mechanism of action in sunitinib resistance. METHODS: We screened 673 microRNAs using TaqMan Low-density-Arrays (TLDAs) in tumors from MRCC patients with extreme phenotypes of marked efficacy and resistance to sunitinib, selected from an identification cohort (n = 41). The most relevant differentially expressed microRNAs were selected using bioinformatics-based target prediction analysis and quantified by qRT-PCR in tumors from patients presenting similar phenotypes selected from an independent cohort (n = 101). In vitro experiments were conducted to study the role of miR-942 in sunitinib resistance. RESULTS: TLDAs identified 64 microRNAs differentially expressed in the identification cohort. Seven candidates were quantified by qRT-PCR in the independent series. MiR-942 was the most accurate predictor of sunitinib efficacy (p = 0.0074). High expression of miR-942, miR-628-5p, miR-133a, and miR-484 was significantly associated with decreased time to progression and overall survival. These microRNAs were also overexpressed in the sunitinib resistant cell line Caki-2 in comparison with the sensitive cell line. MiR-942 overexpression in Caki-2 up-regulates MMP-9 and VEGF secretion which, in turn, promote HBMEC endothelial migration and sunitinib resistance. CONCLUSIONS: We identified differentially expressed microRNAs in MRCC patients presenting marked sensitivity or resistance to sunitinib. MiR-942 was the best predictor of efficacy. We describe a novel paracrine mechanism through which high miR-942 levels in MRCC cells up-regulates MMP-9 and VEGF secretion to enhance endothelial migration and sunitinib resistance. Our results support further validation of these miRNA in clinical confirmatory studies.

BACKGROUND: The emerging relationship between microRNAs (miRNA) and viral-control is a topic of interest in the field of HIV. Host-genome might play an important role in the control of viremia. The aim of this study was to assess the specific miRNA profile that could contribute to the control of HIV replication in Elite Controllers. RESULTS: After adequate normalization, expression profile of 286 human miRNAs (hsa-miR) was evaluated in phytohaemagglutinin-stimulated PBMCs from 29 individuals classified in 4 groups: 8 elite controllers (EC; viral load 5000 cp/ml without treatment), 8 patients under antiretroviral treatment (ART; VL

Background: Hemophagocytic lymphohistiocytosis (HLH) is a life threatening hyper inflammatory disease. Micro RNAs (miRNA) are about 22 nucleotide-long, small RNAs encoded with genes, and they have regulatory functions in immune response. Objective: To determine the miRNA expression levels of 11 secondary HLH patients, we evaluated the associations of miRNA levels with pathogenesis, clinical presentation, and prognosis of the disease. Patients and Methods: Patients who were diagnosed with secondary HLH from January 2011 to December 2012 were included in this study. We profiled the expressions of 379 miRNAs in plasma of both HLH patients and healthy controls. Patients were evaluated regarding with age, clinical findings, miRNA expresions, laboratory data, treatment, and prognosis, by using descriptive statistics. Results: A total of 11 secondary HLH patients and 11 healthy children were included in this study. miR-205-5p was expressed in all case and controls and expression level of miR-205-5p was found 6.21 fold higher than control group (p=0.01). We detected the second highest expression percent in miR-194-5p with 81% of cases and controls. Expression level of miR-194-5p was found to have 163 fold higher than controls (p= 0.009). miR-30c-5p showed 77% expression percent in cases and controls together. The expression level of this miRNA was detected 9 fold decreased in HLH patients compared to healthy children (p= 0.031). Conclusion: We showed that miR-205-5p, miR-194-5p and miR-30c-5p could be useful plasma biomarkers for HLH. Further research is needed in larger and homogenous study groups, especially for these miRNAs as biomarkers for HLH.

MicroRNAs (miRNAs) play important roles in diverse biological processes and are emerging as key regulators of tumorigenesis and tumor progression. To explore the dysregulation of miRNAs in breast cancer, a genome-wide expression profiling of 939 miRNAs was performed in 50 breast cancer patients. A total of 35 miRNAs were aberrantly expressed between breast cancer tissue and adjacent normal breast tissue and several novel miRNAs were identified as potential oncogenes or tumor suppressor miRNAs in breast tumorigenesis. miR-125b exhibited the largest decrease in expression. Enforced miR-125b expression in mammary cells decreased cell proliferation by inducing G2/M cell cycle arrest and reduced anchorage-independent cell growth of cells of mammary origin. miR-125b was found to perform its tumor suppressor function via the direct targeting of the 3'-UTRs of ENPEP, CK2-alpha, CCNJ, and MEGF9 mRNAs. Silencing these miR-125b targets mimicked the biological effects of miR-125b overexpression, confirming that they are modulated by miR-125b. Analysis of ENPEP, CK2-alpha, CCNJ, and MEGF9 protein expression in breast cancer patients revealed that they were overexpressed in 56%, 40-56%, 20%, and 32% of the tumors, respectively. The expression of ENPEP and CK2-alpha was inversely correlated with miR-125b expression in breast tumors, indicating the relevance of these potential oncogenic proteins in breast cancer patients. Our results support a prognostic role for CK2-alpha, whose expression may help clinicians predict breast tumor aggressiveness. In particular, our results show that restoration of miR-125b expression or knockdown of ENPEP, CK2-alpha, CCNJ, or MEGF9 may provide novel approaches for the treatment of breast cancer.

Prostate cancer (PCa) is the most frequently diagnosed type of cancer in developed countries. The decisive method of diagnosis is based on the results of biopsies, morphologically evaluated to determine the presence or absence of cancer. Although this approach leads to a confident diagnosis in most cases, it can be improved by using the molecular markers present in the tissue. Both miRNAs and proteins are considered excellent candidates for biomarkers in formalin-fixed paraffin-embedded (FFPE) tissues, due to their stability over long periods of time. In the last few years, a concerted effort has been made to develop the necessary tools for their reliable measurement in these types of samples. Furthermore, the use of these kinds of markers may also help in establishing tumor grade and aggressiveness, as well as predicting the possible outcomes in each particular case for the different treatments available. This would aid clinicians in the decision-making process. In this review, we attempt to summarize and discuss the potential use of microRNA and protein profiles in FFPE tissue samples as markers to better predict PCa diagnosis, progression, and response to therapy.

Glioblastoma multiforme (GBM) is one of the most deadly types of cancer. To date, the best clinical approach for treatment is based on administration of temozolomide (TMZ) in combination with radiotherapy. Much evidence suggests that the intracellular level of the alkylating enzyme O(6)-methylguanine-DNA methyltransferase (MGMT) impacts response to TMZ in GBM patients. MGMT expression is regulated by the methylation of its promoter. However, evidence indicates that this is not the only regulatory mechanism present. Here, we describe a hitherto unknown microRNA-mediated mechanism of MGMT expression regulation. We show that miR-221 and miR-222 are upregulated in GMB patients and that these paralogues target MGMT mRNA, inducing greater TMZ-mediated cell death. However, miR-221/miR-222 also increase DNA damage and, thus, chromosomal rearrangements. Indeed, miR-221 overexpression in glioma cells led to an increase in markers of DNA damage, an effect rescued by re-expression of MGMT. Thus, chronic miR-221/222-mediated MGMT downregulation may render cells unable to repair genetic damage. This, associated also to miR-221/222 oncogenic potential, may poor GBM prognosis.

The hepatic microRNA (miRNA), miR-122, is the most abundant miRNA within the liver, where it accounts for 70% of the total miRNA pool. It is known that miR-122, as an unusual host factor, increases the abundance of hepatitis C virus (HCV) RNA in HCV infection by binding directly to the 5’-UTR of the viral genome. Therefore, it has been suggested as a potential target for the treatment of hepatitis C. However, recent evidence shows that miR-122 decreases HBV replication through the inhibitory effect of p53 on HBV transcription, and consequently it acts as a tumor-suppressor through both a decrease in HBV replication and by directly targeting cyclin G1, as well as Wnt/beta-catenin, and NDRG3 pathways. This paper will briefly discuss the underlying mechanisms for the dual role of miR-122 in viral hepatitis, and explains why therapeutic applications of miR-122 may differ based on the underlying disease.

Journal article The early detection of oligonucleotide biomarkers of disease, such as microRNAs, has been established as a fundamental factor in cancer diagnosis. As the levels of these small molecules (microRNAs) in blood have recently been found to be significantly affected in cancer patients, they offer a means of early stage detection of cancer. Towards the goal of creating a novel method of DNA hybridization detection, we report the detection of specific sequences of small oligonucleotides in a model experiment carried out in serum. The results shown here display the versatility of the DE-ATRP method in synthesizing a specific polymer structure capable of changing its physical properties in the presence of double stranded DNA. The polymer was labeled and used to detect single-stranded DNA in serum successfully. peer-reviewed

MicroRNAs (miRNAs) play an important role in carcinogenesis through the regulation of their target genes. miRNA-related single nucleotide polymorphisms (miR-SNPs) can affect miRNA biogenesis and target sites and can alter microRNA expression and functions. We examined 11 miR-SNPs, including 5 in microRNA genes, 3 in microRNA binding sites and 3 in microRNA-processing machinery components, and evaluated time to recurrence (TTR) according to miR-SNP genotypes in 175 surgically resected non-small-cell lung cancer (NSCLC) patients. Significant differences in TTR were found according to KRT81 rs3660 (median TTR: 20.3 months for the CC genotype versus 86.8 months for the CG or GG genotype; P = 0.003) and XPO5 rs11077 (median TTR: 24.7 months for the AA genotype versus 73.1 months for the AC or CC genotypes; P = 0.029). Moreover, when patients were divided according to stage, these differences were maintained for stage I patients (P = 0.002 for KRT81 rs3660; P

International audience; BACKGROUND: Micro RNAs are small, non-coding, single-stranded RNAs that negatively regulate gene expression at the post-transcriptional level. Since miR-143 was found to be down-regulated in prostate cancer cells, we wanted to analyze its expression in human prostate cancer, and test the ability of miR-43 to arrest prostate cancer cell growth in vitro and in vivo. RESULTS: Expression of miR-143 was analyzed in human prostate cancers by quantitative PCR, and by in situ hybridization. miR-143 was introduced in cancer cells in vivo by electroporation. Bioinformatics analysis and luciferase-based assays were used to determine miR-143 targets. We show in this study that miR-143 levels are inversely correlated with advanced stages of prostate cancer. Rescue of miR-143 expression in cancer cells results in the arrest of cell proliferation and the abrogation of tumor growth in mice. Furthermore, we show that the effects of miR-143 are mediated, at least in part by the inhibition of extracellular signal-regulated kinase-5 (ERK5) activity. We show here that ERK5 is a miR-143 target in prostate cancer. CONCLUSIONS: miR-143 is as a new target for prostate cancer treatment.

MicroRNAs (miRNAs) are small, noncoding RNAs that can contribute to cancer development and progression by acting as oncogenes or tumor suppressor genes. Recent studies have also linked different sets of miRNAs to metastasis through either the promotion or suppression of this malignant process. Interestingly, epigenetic silencing of miRNAs with tumor suppressor features by CpG island hypermethylation is also emerging as a common hallmark of human tumors. Thus, we wondered whether there was a miRNA hypermethylation profile characteristic of human metastasis. We used a pharmacological and genomic approach to reveal this aberrant epigenetic silencing program by treating lymph node metastatic cancer cells with a DNA demethylating agent followed by hybridization to an expression microarray. Among the miRNAs that were reactivated upon drug treatment, miR-148a, miR-34b/c, and miR-9 were found to undergo specific hypermethylation-associated silencing in cancer cells compared with normal tissues. The reintroduction of miR-148a and miR-34b/c in cancer cells with epigenetic inactivation inhibited their motility, reduced tumor growth, and inhibited metastasis formation in xenograft models, with an associated down-regulation of the miRNA oncogenic target genes, such as C-MYC, E2F3, CDK6, and TGIF2. Most important, the involvement of miR-148a, miR-34b/c, and miR-9 hypermethylation in metastasis formation was also suggested in human primary malignancies ( n = 207) because it was significantly associated with the appearance of lymph node metastasis. Our findings indicate that DNA methylation-associated silencing of tumor suppressor miRNAs contributes to the development of human cancer metastasis.

Learning and memory difficulties observed in patients with fragile X syndrome, as well as in a mouse model for the syndrome, are partially due to impaired translational regulation of neuronal mRNAs encoding key molecules for the synaptic structure and function. There has been intense interest in characterizing the mRNAs that are regulated by the fragile X mental retardation protein (FMRP) in the neuronal cell. A large number of candidate FMRP-interacting mRNAs has been identified over the last few years and three models have been described so far to explain the specificity of these interactions. Here, we report our vision on how they could work in the same and/or in different pathways and suggest that the three mechanisms may not be mutually exclusive. ispartof: Neuroreport vol:15 issue:16 pages:2447-2450 ispartof: location:England status: published

Intermittent hypoxia (IH) during sleep is one of the major abnormalities occurring in patients suffering from obstructive sleep apnea (OSA), a highly prevalent disorder affecting 6–15% of the general population, particularly among obese people. IH has been proposed as a major determinant of oncogenetically-related processes such as tumor growth, invasion and metastasis. During the growth and expansion of tumors, fragmented DNA is released into the bloodstream and enters the circulation. Circulating tumor DNA (cirDNA) conserves the genetic and epigenetic profiles from the tumor of origin and can be isolated from the plasma fraction. Here we report a microarray-based epigenetic profiling of cirDNA isolated from blood samples of mice engrafted with TC1 epithelial lung cancer cells and controls, which were exposed to IH during sleep (XenoIH group, n=3) or control conditions, (i.e., room air (RA); XenoRA group, n=3) conditions. To prepare the targets for microarray hybridization, we applied a previously developed method that enriches the modified fraction of the cirDNA without amplification of genomic DNA. Regions of differential cirDNA modification between the two groups were identified by hybridizing the enriched fractions for each sample to Affymetrix GeneChip Human Promoter Arrays 1.0R. Microarray raw and processed data were deposited in NCBI's Gene Expression Omnibus (GEO) database (accession number: GSE61070).

// Alfons Navarro 1 , Tania Diaz 2 , Natalia Tovar 2 , Fabiola Pedrosa 2 , Rut Tejero 1 , Maria Teresa Cibeira 2 , Laura Magnano 2 , Laura Rosinol 2 , Mariano Monzo 1 , Joan Blade 2 , Carlos Fernandez de Larrea 2 1 Molecular Oncology and Embryology Laboratory, Human Anatomy Unit, School of Medicine, University of Barcelona, 08036 Barcelona, Spain 2 Amyloidosis and Myeloma Unit, Department of Hematology, Hospital Clinic de Barcelona, Institut d’Investigacions Biomediques August Pi I Sunyer (IDIBAPS), University of Barcelona, 08036 Barcelona, Spain Correspondence to: Carlos Fernandez de Larrea, e-mail: cfernan1@clinic.ub.es Keywords: miRNA, serum, myeloma, autologous transplantation, complete remission Received: October 26, 2014 Accepted: November 17, 2014 Published: January 22, 2015 ABSTRACT We have examined serum microRNA expression in multiple myeloma (MM) patients at diagnosis and at complete response (CR) after autologous stem-cell transplantation (ASCT), in patients with stable monoclonal gammopathy of undetermined significance, and in healthy controls. MicroRNAs were first profiled using TaqMan Human MicroRNA Arrays. Differentially expressed microRNAs were then validated by individual TaqMan MicroRNA assays and correlated with CR and progression-free survival (PFS) after ASCT. Supervised analysis identified a differentially expressed 14-microRNA signature. The differential expression of miR-16 ( P = 0.028), miR-17 ( P = 0.016), miR-19b ( P = 0.009), miR-20a ( P = 0.017) and miR-660 ( P = 0.048) at diagnosis and CR was then confirmed by individual assays. In addition, high levels of miR-25 were related to the presence of oligoclonal bands ( P = 0.002). Longer PFS after ASCT was observed in patients with high levels of miR-19b (6 vs. 1.8 years; P < 0.001) or miR-331 (8.6 vs. 2.9 years; P = 0.001). Low expression of both miR-19b and miR-331 in combination was a marker of shorter PFS (HR 5.3; P = 0.033). We have identified a serum microRNA signature with potential as a diagnostic and prognostic tool in MM.

Unveiling the regulatory pathways maintaining hepatic stellate cells (HSC) in a quiescent (q) phenotype is essential to develop new therapeutic strategies to treat fibrogenic diseases. To uncover the miRNA-mRNA regulatory interactions in qHSCs, HSCs were FACS-sorted from healthy livers and activated HSCs (aHSCs) were generated in vitro. MiRNA Taqman array analysis showed HSCs expressed a low number of miRNAs (n = 259), from which 47 were down-regulated and 212 up-regulated upon activation. Computational integration of miRNA and gene expression profiles revealed that 66% of qHSC-associated miRNAs correlated with more than 6 altered target mRNAs (17,28 ± 10,7 targets/miRNA) whereas aHSC-associated miRNAs had an average of 1,49 targeted genes. Interestingly, interaction networks generated by miRNA-targeted genes in qHSCs were associated with key HSC activation processes. Next, selected miRNAs were validated in healthy and cirrhotic human livers and miR-192 was chosen for functional analysis. Down-regulation of miR-192 in HSCs was found to be an early event during fibrosis progression in mouse models of liver injury. Moreover, mimic assays for miR-192 in HSCs revealed its role in HSC activation, proliferation and migration. Together, these results uncover the importance of miRNAs in the maintenance of the qHSC phenotype and form the basis for understanding the regulatory networks in HSCs.