Significant Differences In miRNA Expression Research Articles

MiR-9-5p is Downregulated in Serum Extracellular Vesicles of Patients Treated with Biperiden After Traumatic Brain Injury.

Traumatic brain injury (TBI) is a prevalent and debilitating condition, which often leads to the development of post-traumatic epilepsy (PTE), a condition that yet lacks preventive strategies. Biperiden, an anticholinergic drug, is a promising candidate that has shown efficacy in murine models of PTE. MicroRNAs (miRNAs), small regulatory RNAs, can help in understanding the biological basis of PTE and act as TBI- and PTE-relevant biomarkers that can be detected peripherally, as they are present in extracellular vesicles (EVs) that cross the blood-brain barrier. This study aimed to investigate miRNAs in serum EVs from patients with TBI, and their association with biperiden treatment and PTE. Blood samples of 37 TBI patients were collected 10days after trauma and treatment initiation in a double-blind clinical trial. A total of 18 patients received biperiden, with three subjects developing PTE, and 19 received placebo, with two developing PTE. Serum EVs were characterized by size distribution and protein profiling, followed by high-throughput sequencing of the EV miRNome. Differential expression analysis revealed no significant differences in miRNA expression between TBI patients with and without PTE. Interestingly, miR-9-5p displayed decreased expression in biperiden-treated patients compared to the placebo group. This miRNA regulates genes enriched in stress response pathways, including axonogenesis and neuronal death, relevant to both PTE and TBI. These findings indicate that biperiden may alter miR-9-5p expression in serum EVs, which may play a role in TBI resolution.

Long term effect of INTRABEAM single irradiation on the expression of miRNAs inMCF-7 cells

Intraoperative radiation (IORT) has been increasingly popular in recent years. INTRABEAM has been utilized to treat cancers in various regions of the body as a small intraoperative radiation device. Flat plate, surface, and needle applicators have also been developed by INTRABEAM to meet various irradiation parameters. However, there are still a few basic research on intraoperative radiation in INTRABEAM. The goal of this study was to see how single irradiation affected the expression of miRNAs in breast cancer cells over time. INTRABEAM, an intraoperative radiotherapy device, was used to irradiate MCF-7 cells with a single 6 Gy radiation dosage. Four weeks after the irradiations, high-throughput sequencing of miRNAs was performed. High-throughput sequencing revealed a change in miRNA expression profile four weeks after irradiation. hsa-miR-4485–3p, hsa-miR-215–5p, and hsa-miR-2276–5p were the top three up-regulated miRNAs. hsa-miR-6886–5p, miR-500-y, and miR-6544-x were the top three down-regulated miRNAs. After a single-dose irradiation of 6 Gy with the INTRABEAM device, significant differences in miRNA expression in MCF-7 cells could continue for a long period. INTRABEAM radiotherapy may have therapeutic effect on breast cancer by regulating mRNA expression.

P-239 Embryonic quality assessment from microRNA profiling

Abstract Study question Does the microRNA expression profile in blastocysts change with different embryonic quality parameters? Summary answer MicroRNA expression showed significant changes when aneuploidy was present, in day-6 compared to day-5 blastocysts and with different trophectoderm (TE) morphology grades. What is known already MicroRNA (miRNA) is a gene expression regulatory mechanism used as a diagnostic marker for some conditions. Despite the great magnitude of studies investigating the miRNA role in implantation and pregnancy complications, limited work has been done to evaluate it as a potential marker for preimplantation embryo quality. With the accelerated changes in gene expression at early embryos, the miRNA profile is a potential indicator of embryo quality. This study investigates the blastocyst miRNA profile in relation to three quality parameters: aneuploidy, the day of blastocyst formation and morphology. Study design, size, duration We collected 122 surplus embryos donated for research by 46 consenting couples from Centre for Reproductive and Genetic Health, London. All samples were ICSI derived blastocysts (day5/6) with PGT-A results. For aneuploidy status comparison, samples were categorized according to the number of chromosomes affected (single/multiple) and type of aneuploidy (gain/loss/partial), compared to euploid. The day of blastocyst development has (day5/day6) groups, and finally samples were compared based on TE morphology (excellent/good/fair). Participants/materials, setting, methods Frozen human embryos were individually thawed and transferred to a lysis buffer. The miRNAs were extracted from each sample then sequenced by next generation sequencing (NGS) using miRNA Library Kit (QIAGEN) with NextSeqTM 500 (illumina). The differential expression analysis was conducted using DESeq2 via GeneGlobe (QIAGEN). We set the significance cut-offs to (-2> foldchange >2) and (<0.05 FDR p-value). For gene ontology and pathway annotation, we used miRTargetLink 2.0 and miRPathDB 2.0 platforms. Main results and the role of chance Compared to the euploid blastocysts, all aneuploid categories, except the single chromosome aneuploidy, had significant differences in miRNA expression. Nine miRNAs were altered in the group with multiple aneuploidies, and one was highly upregulated in embryos with partial losses or gains. The majority of differentially expressed (DE) miRNAs in samples with whole chromosomal gain or loss were downregulated. Conversely, four miRNAs showed a significant increase in day-6 blastocysts compared to day-5. Morphology grade comparisons showed no differences between excellent and good TE; however, six miRNAs were significantly changed in fair TE compared to good. The frequently altered miRNAs in these comparisons were ( hsa-let-7c-5p, hsa-miR-206, hsa-miR-184 and hsa-miR-203a-3p ). Unsurprisingly, pathway annotation of the DE miRNAs showed an involvement of key biological processes, like apoptosis, cell-cycle and differentiation, signalling and gene expression regulation, in all groups. The pathways of note in aneuploid blastocysts included DNA damage response, intrinsic and extrinsic apoptotic signalling, and regulation of cell-cycle G1/S associated events. In blastocyst day5/day6 comparison, embryonic and organ development were the most involved pathways, while in TE morphology, kinase activity and phosphorylation along with apoptosis and DNA damage response were significant. Limitations, reasons for caution Differential expression analysis based on inner cell mass (ICM) morphology and blastocyst expansion were not applicable due to the inadequate number of embryos per group; as all the samples collected were in excellent or good quality. Wider implications of the findings Gene expression profiling of miRNAs in human blastocysts will potentially identify biomarkers predictive of implantation success. Trial registration number not applicable

Pan-cancer analysis of microRNA expression profiles highlights microRNAs enriched in normal body cells as effective suppressors of multiple tumor types: A study based on TCGA database

BackgroundMicroRNAs (miRNAs) are frequently deregulated in various types of cancer. While antisense oligonucleotides are used to block oncomiRs, delivery of tumour-suppressive miRNAs holds great potential as a potent anti-cancer strategy. Here, we aim to determine, and functionally analyse, miRNAs that are lowly expressed in various types of tumour but abundantly expressed in multiple normal tissues.MethodsThe miRNA sequencing data of 14 cancer types were downloaded from the TCGA dataset. Significant differences in miRNA expression between tumor and normal samples were calculated using limma package (R programming). An adjusted p value < 0.05 was used to compare normal versus tumor miRNA expression profiles. The predicted gene targets were obtained using TargetScan, miRanda, and miRDB and then subjected to gene ontology analysis using Enrichr. Only GO terms with an adjusted p < 0.05 were considered statistically significant. All data from wet-lab experiments (cell viability assays and flow cytometry) were expressed as means ± SEM, and their differences were analyzed using GraphPad Prism software (Student’s t test, p < 0.05).ResultsBy compiling all publicly available miRNA profiling data from The Cancer Genome Atlas (TCGA) Pan-Cancer Project, we reveal a small set of tumour-suppressing miRNAs (which we designate as ’normomiRs’) that are highly expressed in 14 types of normal tissues but poorly expressed in corresponding tumour tissues. Interestingly, muscle-enriched miRNAs (e.g. miR-133a/b and miR-206) and miRNAs from DLK1-DIO3 locus (e.g. miR-381 and miR-411) constitute a large fraction of the normomiRs. Moreover, we define that the CCCGU motif is absent in the oncomiRs’ seed sequences but present in a fraction of tumour-suppressive miRNAs. Finally, the gain of function of candidate normomiRs across several cancer cell types indicates that miR-206 and miR-381 exert the most potent inhibition on multiple cancer types in vitro.ConclusionOur results reveal a pan-cancer set of tumour-suppressing miRNAs and highlight the potential of miRNA-replacement therapies for targeting multiple types of tumour.

MicroRNA Expression Analysis of Mice Retinas with Oxygen-Induced Retinopathy by RNA Sequencing.

Purpose To characterize the microRNA (miRNA) expression profiles in the retinas of mice with oxygen-induced retinopathy by RNA sequencing and to ascertain miRNAs associated with retinal neovascularization. Methods Retina samples were obtained from 3 groups (6 retinas/group) of OIR mice and normal mice at P17. RNA was isolated from 24 retina samples and then detected on an Illumina HiSeq. Twelve retina samples were used for quantitative polymerase chain reaction to validate the RNA sequencing. Bioinformatics analyses were performed. Result The RNA sequence showed that 565 miRNAs were detected in the retina of OIR mice and 583 miRNAs in the retina of normal control mice. A total of 553 miRNAs were expressed in both groups. Thirty-eight miRNAs showed altered expression in both groups (p ≤ 0.05). Compared with the control group, 2 miRNAs were significantly upregulated in the OIR group, while 36 miRNAs were significantly downregulated. Meanwhile, 2 candidate miRNAs (miR-181a-5p and miR-21a-5p) with significant differences in miRNA expression (p < 0.01) were selected for validation. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to confirm the relative expression of the two miRNAs. Bioinformatics analyses showed that pathways involved in ischemic retinopathy (such as TGF-β, Ras, Hippo, PI3K-Akt, VEGF, and HIF-1 signaling pathways) were enriched. Conclusions Our study provided an overall view of miRNA profiling in the OIR retina. These miRNA profiles provide a valuable framework for the potential therapy of retinal angiogenesis.

Dysregulation of microRNAs as the risk factor of lymph node metastasis in papillary thyroid carcinoma: systematic review.

Papillary thyroid carcinoma (PTC) has an excellent prognosis with a relatively low mortality rate, but a small portion of PTC patients suffer from an aggressive form of the disease. In such cases early detection of lymph node metastasis (LNM) is as paramount as it is problematic. The routine use of central neck lymph node dissection is not recommended. New methods to detect LNM are needed. MicroRNAs are a potential biomarker for diagnosis and prognosis of PTC. In this review we summarise the current knowledge regarding dysregulated miRNAs and their association with LNM in PTS patients. The PubMed and EBSCO databases were searched using terms for "microRNA", "thyroid carcinoma", and "prognosis" by using Boolean operators. Based on eligibility and exclusion criteria, articles were screened and reviewed in full, methodological data of included studies were extracted, and risk of bias analysis performed. In total, 446 unique studies were extracted from the mentioned databases, and based on inclusion and exclusion criteria 27 studies were included in this review. Of them 17 analysed tissue microRNAs, 5 analysed circulating microRNAs, and 5 studies analysed both tissue and circulating samples. MiRNA-146B, miRNA-221, miRNA-222, miRNA-21, miRNA-204, miRNA-451, miRNA-199a-3p, and miRNA-30a-3p were dysregulated in at least 2 separate studies. A sizable portion of studies failed to show statistically significant differences in miRNA expression between LNM-positive and -negative patients. Different methodologies and disparities of patient populations could explain these discrepancies.\ This research supports the statement that specific up- and downregulated miRNAs are associated with LNM in PTC patients. However, the prognostic value of these miRNAs is limited. Additional targeted cohort studies are required to elucidate the role of miRNAs in defining individualised treatment strategies for thyroid cancer patients.

Expression characteristics of pineal miRNAs at ovine different reproductive stages and the identification of miRNAs targeting the AANAT gene

BackgroundMany recent studies have shown that miRNAs play important roles in the regulation of animal reproduction, including seasonal reproduction. The pineal gland is a crucial hub in the regulation of seasonal reproduction. However, little is known about the expression characteristics of pineal miRNAs in different reproductive seasons (anestrus and breeding season). Therefore, the expression profiles and regulatory roles of ovine pineal miRNAs were investigated during different reproductive stages using Solexa sequencing technology and dual luciferase reporter assays.ResultsA total of 427 miRNAs were identified in the sheep pineal gland. Significant differences in miRNA expression were demonstrated between anestrus and the breeding season in terms of the frequency distributions of miRNA lengths, number of expressed miRNAs, and specifically and highly expressed miRNAs in each reproductive stage. KEGG analysis of the differentially expressed (DE) miRNAs between anestrus and the breeding season indicated that they are significantly enriched in pathways related to protein synthesis, secretion and uptake. Furthermore, transcriptome analysis revealed that many target genes of DE miRNAs in the ribosome pathway showed relatively low expression in the breeding season. On the other hand, analyses combining miRNA-gene expression data with target relationship validation in vitro implied that miR-89 may participate in the negative regulation of aralkylamine N-acetyltransferase (AANAT) mRNA expression by targeting its 3’UTR at a unique binding site.ConclusionsOur results provide new insights into the expression characteristics of sheep pineal miRNAs at different reproductive stages and into the negative regulatory effects of pineal miRNAs on AANAT mRNA expression.

MicroRNA Expression Profiling of Cutaneous Squamous Cell Carcinomas Arising in Different Sites.

To examine the microRNA (miRNA) expression profile of cutaneous squamous cell carcinoma (cSCC) tumors from aggressive head and neck locations compared with nonaggressive anatomic sites and normal controls. Retrospective analysis of miRNA expression. Tertiary care center. Tissue samples were collected from 3 anatomic regions: aggressive head and neck sites (ie, ears/lip), nonaggressive locations (ie, extremities/trunk), and adjacent normal skin. RNA was isolated from tissue cores of 45 samples (18 aggressive sites, 15 nonaggressive sites, and 12 normal-adjacent skin). miRNA expression analysis was completed for approximately 800 miRNAs using the NanoString nCounter panel. Five candidate miRNAs were selected for validation. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed on the original samples plus 30 additional tissue samples (7 aggressive sites, 14 nonaggressive sites, and 9 normal-adjacent skin). Five candidate miRNAs with significant differences in miRNA expression (P < 0 ≤ .001) from discovery samples were selected: miR-21, miR-31, let-7g, miR-93, and miR-22. Relative expression for these miRNAs using qRT-PCR in the new sample set did not reveal any significant differences using 1-way analysis of variance. When sets were combined, miR-21 showed increased expression in aggressive tumors relative to nonaggressive tumors (P = .009), but no others reached statistical significance. cSCC behaves more aggressively when originating from specific anatomical subsites of the head and neck. Of 5 miRNAs evaluated, only miR-21 showed significantly higher expression in tumors from aggressive sites relative to nonaggressive sites. Larger sample sizes are needed to evaluate other miRNAs.

Endometrial Liquid Biopsy Provides a miRNA Roadmap of the Secretory Phase of the Human Endometrium.

Endometrial liquid biopsy (ELB) is a minimally invasive alternative for research and diagnosis in endometrial biology. We sought to establish an endometrial micro ribonucleic acid (miRNA) roadmap based on ELB during the secretory phase of the menstrual cycle in both natural and hormonal replacement therapy (HRT) cycles. Human ELB samples (n = 58) were obtained from healthy ovum donors undergoing a natural and an HRT cycle consecutively. miRNA profiles were identified using next-generation sequencing (NGS). For functional analysis, messenger ribonucleic acid targets were chosen among those reported in the endometrial receptivity analysis. The human endometrial secretory phase is characterized by a dynamic miRNA secretion pattern that varies from the prereceptive to the receptive stages. No differences in miRNA profiles were found among natural versus HRT cycles in the same women, reinforcing the similarities in functional and clinical outcomes in natural versus medicated cycles. Bioinformatic analysis revealed 62 validated interactions and 81 predicted interactions of miRNAs differentially expressed in the HRT cycle. Annotation of these genes linked them to 51 different pathways involved in endometrial receptivity. This NGS-based study describes the miRNA signature in human ELB during the secretory phase of natural and HRT cycles. A consistent endometrial miRNA signature was observed in the acquisition of endometrial receptivity. Interestingly, no significant differences in miRNA expression were found in natural versus HRT cycles reinforcing the functional clinical similarities between both approaches.

Circulating Exosomal miRNA Signature in Pregnancies with Gestational Diabetes Mellitus across Gestation

Exosomes are small nanovesicles that carry bioactive molecules (e.g., miRNAs) which can be delivered to other cells and modify their phenotype. Recently, we have established that the number of circulating exosomes is higher in women with gestational diabetes mellitus (GDM); however, the exosomal miRNA profile in GDM across gestation has not been established yet. The aim of this study was to establish the circulating exosomal miRNA profile in women with normal glucose tolerance (NGT) and GDM women (BMI-matched) in a prospective cohort of patients at three time points during pregnancy. Exosomal RNA was sequenced using an Illumina NextSeq 500 platform. Linear mixed modelling was performed on the normalized miRNAs across gestation for normal and GDM pregnancies, using the lme4 package in R. Statistically significant differences in miRNA expression between NGT and GDM women was determined using likelihood ratio tests. In the first trimester of pregnancy, 92% (84 out of 92) of exosomal miRNAs were significantly elevated in plasma of women with GDM when compared to NGT women. Interestingly, in the second trimester of pregnancy the expression of these miRNA decreased in the GDM women with no significance difference observed in the exosomal miRNA profile between GDM and NGT women. Gene ontology analysis revealed that miRNAs which changed expression across gestation for GDM pregnancies are involved in the regulation of insulin. In summary, we have identified a range of exosomal miRNAs which change expression across gestation for GDM and normal pregnancies. Gene ontology analysis revealed that these miRNAs are involved in the regulation of insulin, playing significant role in the pathophysiology of GDM. Disclosure D. Guanzon: None. A. Lai: None. K. Scholz-Romero: None. F. Zuñiga: None. E. Diaz: None. H. McIntyre: Speaker's Bureau; Self; Novo Nordisk Inc.. Research Support; Self; Danish Diabetes Academy. M. Lappas: None. C. Salomon: None.

Peripheral blood microRNA and VEGFAmRNA changes following electroconvulsive therapy: implications for psychotic depression

MicroRNAs are short, non-coding molecules that regulate gene expression. Here, we investigate the role of microRNAs in depression and electroconvulsive therapy (ECT). We performed three studies: a deep sequencing discovery-phase study of miRNA changes in whole blood following ECT (n = 16), followed by a validation study in a separate cohort of patients pre-/post-ECT (n = 37) and matched healthy controls (n = 34). Changes in an experimentally validated gene target (VEGFA) were then analysed in patients pre-/post-ECT (n = 97) and in matched healthy controls (n = 53). In the discovery-phase study, we found no statistically significant differences in miRNA expression from baseline to end of treatment in the group as a whole, but post hoc analysis indicated a difference in patients with psychotic depression (n = 3). In a follow-up validation study, patients with psychotic depression (n = 7) had elevated baseline levels of miR-126-3p (t = 3.015, P = 0.006) and miR-106a-5p (t = 2.598, P = 0.025) compared to healthy controls. Following ECT, these differences disappeared. Baseline VEGFA levels were significantly higher in depressed patients compared to healthy controls (F(1,144) = 27.688, P = <0.001). Following ECT, there was a significant change in VEGFA levels in the psychotic group only (t = 2.915, P = 0.010). Molecular differences (miRNA and VEGFA) may exist between psychotic and non-psychotic depression treated with ECT.

MicroRNA expression changes following synthesis of three full-sib Populus triploid populations with different heterozygosities.

Through high-throughput sequencing, we compared the relative expression levels of miRNA in three full-sib Populus triploid populations with that in their parents and one diploid hybrid population. We found similar numbers of miRNAs differentially expressed between the parents and the four progeny hybrid populations. In addition, unbalanced parental expression level dominance of miRNAs were found in the three allotriploid and interspecific hybrid populations, which may reprogram gene expression networks and contribute to the growth of Populus hybrids. These results indicated that hybridization has a great impact on the miRNA expression variation in the newly synthesized Populus triploid and diploid hybrid populations. However, we also found no significant differences in miRNA expression among one diploid and three triploid hybrid populations, hinting that miRNA abundances do not increase with the genome content. No dosage effect of miRNA expression could lead to dosage-dependent negative effects on target genes and their downstream pathway in polyploids. We speculate that polyploids may gain advantages from the slight decrease in miRNA regulation, suggesting an important molecular mechanism of polyploid advantage. Hybridization with three types of induced 2n gametes transmitted different parental heterozygosities has been proven as an efficient method for Populus triploid production. Several researches have shown that miRNA could be non-additively expressed in allopolyploids. However, it is still unclear whether the non-additively expressed miRNAs result from the effect of hybridization or polyploidization, and whether a dose response to the additional genomic content exists for the expression of miRNA. Toward this end, through high-throughput sequencing, we compared the expression levels of miRNA in three full-sib Populus triploid populations with that in their parents and one interspecific hybrid population. We found similar numbers of miRNAs differentially expressed between the parents and the four progeny hybrid populations. Unbalanced parental expression level dominance of miRNAs were found in the three triploid and diploid hybrid populations, which may reprogram gene expression networks and affect the growth of Populus hybrids. These results indicated that hybridization has a great impact on the miRNA expression variation in the newly synthesized Populus triploid and diploid hybrid populations. However, we also found no significant differences in miRNA expression among the three triploid populations and the diploid hybrid population. No dosage effect of miRNA expression could lead to dosage-dependent negative effects on target genes and their downstream pathway in polyploids. We speculate that polyploids may gain advantages from the decrease in miRNA negative regulation, suggesting an important molecular mechanism of polyploid advantage.

Abstract A11: Profiling of miRNAs expression in pediatric brain tumors

Abstract Introduction: The understanding of pediatric CNS tumors biology is essential in the development of disease stratification and in development of less toxic therapeutic agents as well as finding novel markers for early diagnosis. MicroRNAs are short 18-25 nucleotide small non-coding RNA molecules regulate gene expression. Recent studies showed that miRNAs play a significant role in brain tumor biology; and may up-regulated or down-regulated in malignancies, which referred their oncogenic or tumor suppressor effect. MiRNA expression patterns have been linked to clinical outcomes, tumor regulation; such as tumor progression, cell growth, cell death and metastasis. The identification of tumor- specific miRNA signatures may assist in future in the discovery of new biomarkers with diagnostic and prognostic utility. The main objective of the present study is to detect the expression of different miRNAs in different subtypes of pediatric CNS tumors to distinguish between them in discovering biomarkers for early detection in addition to develop novel therapies. Methods: The expression level of 82 miRNAs were detected in 120 cases of pediatric CNS tumors from fixed formalin paraffin embedded tissues (FFPE), divided into four subtypes including; Low Grade Glioma, High Grade Glioma, Ependymoma, and Medulloblastoma using quantitative real time PCR (qRTPCR). Results: Analysis of qRT-PCR data showed significant differences in miRNA expression between tumor subtypes with P value&amp;lt; 0.05. Low expression of (miR-221, miR-9, and miR-181c/d) and over expression in miR-101, miR-222, miR-139, miR-1827 and miR-34c) in Medulloblastoma patients compared to other subtypes. Low expression of miR-10a and overexpression in (miR-10b, and miR-29a) in Ependymoma patients compared to other subtypes. Low expression of miR-26a and over expression in (miR-19a/b, miR-24, miR-27a, miR- 584 and miR-527) in Low Grade Glioma patients compared to other subtypes. Conclusion: MicroRNAs are differentially expressed between different subtypes of pediatric CNS tumors suggesting that they may play a significant role in diagnosis to distinguish between different subtypes. A greater understanding of aberrant miRNA expression in pediatric brain tumors may support in the development of novel therapies. In addition, the characterization of tumor-specific miRNA signatures may play an important role in the discovery of biomarkers with diagnostic or prognostic utility. Note: This abstract was not presented at the conference. Citation Format: Marwa Tantawy, Mariam Elzayat, Dina Yehia, Hala Taha. Profiling of miRNAs expression in pediatric brain tumors. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr A11.

Comparisons of serum miRNA expression profiles in patients with diabetic retinopathy and type 2 diabetes mellitus

OBJECTIVES:The aim of this study was to compare the expression levels of serum miRNAs in diabetic retinopathy and type 2 diabetes mellitus.METHODS:Serum miRNA expression profiles from diabetic retinopathy cases (type 2 diabetes mellitus patients with diabetic retinopathy) and type 2 diabetes mellitus controls (type 2 diabetes mellitus patients without diabetic retinopathy) were examined by miRNA-specific microarray analysis. Quantitative real-time polymerase chain reaction was used to validate the significantly differentially expressed serum miRNAs from the microarray analysis of 45 diabetic retinopathy cases and 45 age-, sex-, body mass index- and duration-of-diabetes-matched type 2 diabetes mellitus controls. The relative changes in serum miRNA expression levels were analyzed using the 2-ΔΔCt method.RESULTS:A total of 5 diabetic retinopathy cases and 5 type 2 diabetes mellitus controls were included in the miRNA-specific microarray analysis. The serum levels of miR-3939 and miR-1910-3p differed significantly between the two groups in the screening stage; however, quantitative real-time polymerase chain reaction did not reveal significant differences in miRNA expression for 45 diabetic retinopathy cases and their matched type 2 diabetes mellitus controls.CONCLUSION:Our findings indicate that miR-3939 and miR-1910-3p may not play important roles in the development of diabetic retinopathy; however, studies with a larger sample size are needed to confirm our findings.

MicroRNA profiles in colorectal carcinomas, adenomas and normal colonic mucosa: variations in miRNA expression and disease progression.

MiRNAs are small, non-protein-coding RNA molecules that regulate gene expression either by post-transcriptionally suppressing mRNA translation or by mRNA degradation. We examine differentially expressed miRNAs in colorectal carcinomas, adenomas and normal colonic mucosa. Data come from population-based studies of colorectal cancer conducted in Utah and the Kaiser Permanente Medical Care Program. A total of 1893 carcinoma/normal-paired samples and 290 adenoma tissue samples were run on the Agilent Human miRNA Microarray V19.0 which contained 2006 miRNAs. We tested for significant differences in miRNA expression between paired carcinoma/adenoma/normal colonic tissue samples. Fewer than 600 miRNAs were expressed in >80% of people for colonic tissue; of these 86.5% were statistically differentially expressed between carcinoma and normal colonic mucosa using a false discovery rate of 0.05. Roughly half of these differentially expressed miRNAs showed a progression in levels of expression from normal to adenoma to carcinoma tissue. Other miRNAs appeared to be altered at the normal to adenoma stage, while others were only altered at the adenoma to carcinoma stage or only at the normal to carcinoma stage. Evaluation of the Agilent platform showed a high degree of repeatability (r = 0.98) and reasonable agreement with the NanoString platform. Our data suggest that miRNAs are highly dysregulated in colorectal tissue among individuals with colorectal cancer; the pattern of disruption varies by miRNA as tissue progresses from normal to adenoma to carcinoma.

P100: MicroRNA-21, -155, -200c and -205 in carcinogenesis of larynx

The purpose of the study was to access pattern of microRNA expression in tissue of precancerous lesions and larynx cancer. A total of 25 people with a diagnosis of dysplasia II–III (n = 10), control group (dysplasia 0, n = 15) and larynx cancer patients (n = 46) were examined. Fresh frozen biopsies and adjacent normal epithelium were used. The diagnosis was verified by histology. All qRT-PCR data were analyzed using the Pfaffl analysis. Multiplex RT-PCR on miRNA templates were performed as described by Chen et al. (2005). TaqMan miRNA assays (Iyevleva et al., 2012) were used to quantify expression of mature miRNAs of interest (miR-18a, -21, -155, -200a, -200c, -205, -221, -494). Data were analyzed using Welsh t-test with a 5% FDR correction. According to our results, aberrant expression of some miRNAs was showed. There was no significant differences in miRNA expression in a total group (n = 25) although a trend towards overexpression of oncogenic miRNA-21 and -155 according to severity of dysplastic changes in a tissue were present. Detailed analysis showed significant overexpression of that miRNAs as well as miRNA-200c and -205 in a group of dysplasia II- III against control group (15 vs 10, p = 0.019, p = 0.045 and p = 0.020, p = 0.038, respectively) but these results did not meet 5% FDR correction. Data showed overexpression of the same microRNA (-205, -155, -200 and -21) in larynx cancer patients compared to control group (46 vs 15, p = 0.0002, p = 0.008, p = 0.009, p = 0.013, respectively). It should be pointed that microRNA pattern both in larynx cancer and patients with dysplasia II-III was very close showing similarity of these groups at molecular level. Frequency of cases with microRNA-205 overexpression was 2.98 times higher in cancer patients than in those who had no malignant transformation (CI 95%, 1.41–16.26, p = 0.007). Data showed that up regulation of microRNA-205 could be a marker of disease progression (OR = 4.79). Three other microRNAs did not show any promising results as biomarkers of cancer progression but data obtained has interesting fundamental value. These miRs (-21, -155, -205 and -200c) are known to be regulators of processes that play an emergent role in carcinogenesis and our results obtained in vivo highlight and expand knowledge about some aspects of larynx cell transformation. Data suggest that miRNAs changing its expression according to dysplasia progress and could be important players in complex process of carcinogenesis as well as could be potential markers of disease progression.

Identification of differentially expressed microRNAs involved in non-traumatic osteonecrosis through microRNA expression profiling

Accumulating evidence has recently indicated a vital role of microRNAs (miRNAs) in the development of various bone diseases. However, the biological role of miRNAs in the pathogenesis of non-traumatic osteonecrosis of femoral head (ONFH) has not yet been investigated. The present study aimed to profile the differential miRNA expression between non-traumatic ONFH and femoral neck fracture and to develop further understanding of the molecular mechanisms involved in the pathogenesis of non-traumatic ONFH. Femoral heads from 4 patients with non-traumatic ONFH and 4 with femoral neck fracture were used to analyze the miRNA expression profiles in bone tissue using the Exiqon miRCURY™ LNA Array (v.18.0). The results of miRNA microarray analysis were further confirmed by real-time quantitative polymerase chain reaction (qPCR). The differentially expressed miRNA target genes and signaling pathways involved were predicted by bioinformatics analysis. MiRNA microarray chip analysis revealed that 22 miRNAs were significantly up-regulated and 17 were significantly down-regulated in the non-traumatic ONFH samples compared with the femoral neck fracture samples. The real-time qPCR also confirmed the microarray data. Bioinformatics analysis demonstrated that toll-like receptor (TLR), neurotrophin and NOD-like receptor signaling pathway were most likely to be regulated by these differential miRNAs. This miRNA microarray study reveals significant differences in miRNA expression between patients with non-traumatic ONFH and those with femoral neck fracture. Our data also manifests that the signaling pathways regulated by these differentially expressed miRNAs might be important in the pathogenesis of non-traumatic ONFH.

MicroRNAome in decidua: a new approach to assess the maintenance of pregnancy

To comparatively analyze the human microRNAomes between normal pregnant and miscarriage deciduas by an in-depth sequencing of microRNA (miRNA); and to specifically examine miRNA-199b-5p and serum/glucocorticoid regulated kinase 1 (SGK1) in vivo and in vitro for their possible roles in pregnancy maintenance. Samples of deciduas from 6-8-week spontaneous miscarriages and normal pregnant women were irrespectively collected and comparatively analyzed by miRNA sequencing. The miR-199b-5p and SGK1 expressions were validated in vivo and in vitro. University research and clinical institutes. In this experimental study, samples of deciduas were obtained from October 2011 to April 2012 from 29 women with spontaneous miscarriages and 35 normal pregnant women (control group) who underwent pregnancy termination at 6-8 weeks at our university gynecology unit. Endometrial biopsies, cell transfection, and production of an miR-199b-5p transgenic mouse model. In-depth sequencing of the miRNAome on human deciduas was performed for statistically significant differences in miRNA expression. Expression levels of SGK1 were detected by quantitative polymerase chain reaction and immunoblotting (Western blot) in vitro while miR-199b-5p is overexpressed or knockdown in miR-199b-5p transgenic mice. Expression of the 1,921 known miRNAs was analyzed in the study. In aborted deciduas, 0.57% of the miRNAs were expressed abundantly (>10,000 transcripts per million) and represented 86.38% of all the miRNA reads. Six miRNAs were down-regulated (let-7a-5p, let-7f-5p, let-7g-5p, let-7e-5p, let-7d-5p, and miR-98), whereas miR-199b-5p was significantly up-regulated. Overexpression or knockdown of miR-199b-5p in HEK293T and Ishikawa cells decreased or increased SGK1 expression. Furthermore, overexpression of miR-199b-5p in human endometrial stromal cells or in transgenic mouse decreased SGK1 expression at the mRNA and protein levels, respectively. Among the miRNAomes, the abundant expression of the let-7 members was decreased in aborted samples, whereas miR-199b-5p expression was consistently increased. A significant inverse correlation was found between miR-199b-5p and SGK1 in vivo and in vitro.

The MicroRNAome of Pregnancy: Deciphering miRNA Networks at the Maternal-Fetal Interface

MicroRNAs (miRNAs) post-transcriptionally regulate a vast network of genes by inhibiting mRNA translation. Aberrant miRNA expression profiles have been implicated in pathologies and physiological processes including pregnancy and angiogenesis. Using our established model of implantation failure and spontaneous fetal loss in pigs (Sus scrofa), 236 miRNAs were profiled and compared between 1) non-pregnant and pregnant endometrium, 2) maternal and fetal tissues, and 3) viable and growth-arrested conceptus attachment sites by microarray and Real-Time PCR. Many significant differences in miRNA expression were observed between each of the aforementioned comparisons, and several were validated by PCR. Results indicated which miRNAs were important during pregnancy, which were elevated on the maternal or fetal side of the maternal-fetal interface, and they implicated the maternal expression of miR-10a, 27a, 29c, 323, 331-5p, 339-3p, 374b-5p, and 935 in the spontaneous loss observed in pigs. Several putative mRNA targets of the miRNAs (elevated in endometrium associated with arresting conceptuses) were assessed by quantitative Real-Time PCR and were depressed, supporting their regulation by miRNAs. Finally, targets were clustered by function to obtain ranked lists of gene networks that indicated which pathways/physiological processes might be important in non-pregnant (extracellular matrix factors) versus pregnant endometrium (nuclear transcription factor regulation), maternal (blood vessel development) versus fetal (neuronal differentiation) tissue, and healthy (extracellular matrix factors) versus arresting (GRAM domain) conceptus attachment sites. Overall, we demonstrate the presence of miRNAs on both sides of the maternal-fetal interface, implicate them in spontaneous fetal loss, and present a unique glimpse into the vast microRNAome of pregnancy.

Molecular Characterization By miRNA Expression Profiling in Acute Promyelocytic Leukemia (APL): deregulation Of Mirnas May Identify Patients At Risk Of Early Death

BackgroundAPL is a highly curable malignancy with large co-operative group trials showing mortality under 10%. Despite aggressive care 30% of patients die early at presentation or shortly after initiation of treatment. Understanding the biology of the disease may help us identify the causes for these poor outcomes and allow for even better outcomes. Recently, a class of noncoding RNAs called miRNAs has emerged as critical gene regulators in cell growth, disease, and development. miRNAs are 18–24 nucleotide long noncoding RNAs, which regulate gene expression by pairing with 3' untranslated region (UTR) of target mRNA and inhibiting protein translation and/or inducing mRNA degradation. miRNAs modulate critical cell processes including cell growth, development, and differentiation. Our study investigates the differences in expression of miRs in APL patients with early mortality versus surviving patients. DesignThe miRNA expression profile of APL survived (n=7), vs APL died (n=6) were evaluated using the Affymetrix miRNA microarray platform on GeneChip miRNA 3.0 array in paraffin-embedded samples. Following hybridization and data acquisition, we used Partek Genomics Suite ® software for RMA normalization and to determine statistically significant differences in miRNA expression between experimental groups by ANOVA and pairwise comparisons (two-sided α=0.05). ResultsThe miRNA expression profiles of dead versus alive patients shows statistically significant (1.5 to 14X, p<0.05) downregulation of a set of 30 miRNAs, and upregulation of 6 miRNAs (p<0.05). Although the role of these miRNAs has not been studied in APL, they have been described in other tumors, having a role in tumor suppression, differentiation, cell proliferation, chemoresistance and inflammation. The miRNA (mir-100), which plays a key role in the myeloid differentiation, was amongst the highest miRNAs that were downregulated in our dead patients (14X). SignificanceTo the best of our knowledge, the current study represents the first global miRNA profiling exploration in understanding the early death in APL patients. Previous studies have demonstrated that miR-100 regulates G1/S transition and S-phase entry and blocks the terminal differentiation by targeting RBSP3 (a phosphatase-like tumor suppressor). Although it is overexpressed in APL patients compared to normal cells, in patients who died it shows a significant down regulation (14X). Other tumor cell lines have shown that a down regulation in miR-100 imparts chemotherapy resistance. Combined with down regulation in miR-27a (3.5 X), which has been shown to impart chemoresistance in APL cell lines, and dysregulation of miR 30c (down 5 fold) and miR-663 (up 2.3 fold), we predict that these patients may be less sensitive to treatment. Reduction in tumor suppressor function is another way that tumors can be more aggressive. Multiple miRNAs that have been implicated as tumor suppressive were down regulated in dead patients. miRs-100, -125b, -150 (down 14X, 10X and 9X respectively) have been shown to have tumor suppressive function in non-APL studies. This finding in combination with other downregulated miRNAs (a set of 15 miRs that have confirmed tumor suppressive roles), and two upregulated miRs may lead to a collective decrease in tumor suppression leading to more aggressive disease and poor outcome. miR-125b and 27a have been implicated in pro-inflammatory pathways and their dysregulation may lead to a sustained pro-inflammatory state that is peculiar to this disease and may contribute to the early morbidity and mortality observed in this group. ConclusionsWe hypothesize that dysregulation of microRNAs leads to a sustained pro-inflammatory state and might impart decreased sensitivity to treatment, and that eventually may lead to the poor outcomes observed. Further studies are needed to validate these results in a larger patient population. If confirmed, our findings can lead to targeted approaches to improve APL outcomes. Disclosures:No relevant conflicts of interest to declare.