Putative miRNA Research Articles

Circular RNA Expression Profile and Analysis of Their Potential Function in Psoriasis

Background/Aims: Circular RNAs (circRNAs) are evolutionary conserved circular non-coding RNAs that play a role in several diseases by sequestering (sponging) microRNAs (miRNAs). However, their role in psoriasis remains unclear. In the present study, we investigated the expression of circRNAs and analyzed their potential functions in psoriasis. Methods: The SBC human ceRNA array V1.0 was used to analyze circRNA expression in psoriatic lesions and normal healthy skin tissues. Functional analyses were performed using Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Putative miRNA response elements (MREs) were identified using miRNA target prediction software. Six upregulated circRNAs were verified by quantitative real-time reverse transcription polymerase chain reaction in psoriatic lesions and healthy skin tissues. Results: A total of 4956 circRNAs (3016 upregulated and 1940 downregulated; fold change ≥2 and p< 0.05) were identified as differentially expressed in psoriasis. Furthermore, 4405 MREs were identified among the differentially expressed circRNAs. hsa_circ_0061012 was upregulated in psoriatic lesions compared with normal healthy skin tissues. The top five MREs of hsa_circ_0061012 were hsa-miR-7157-5p, hsa-miR-4769-3p, hsa-miR-6817-5p, hsa-miR-4310, and hsa-miR-6882-3p. GO analysis was carried out to investigate the biological functions enriched among the upregulated targets of five miRNAs in psoriasis. The GO analysis identified that most of top 30 of GO enrichment are related to psoriasis. Conclusion: hsa_circ_0061012 might be a candidate biomarker for psoriasis. The results provide a new perspective for a better understanding of ceRNA-mediated gene regulation in psoriasis, and provide a novel theoretical basis for further studies on the function of circRNA in psoriasis.

Might miRNAs Be Related to Mother-to-Child Transmission of HIV-1? A Short Review on Putative Viral miRNAs Encoded by HIV-1

AbstractMicroRNAs (miRNAs) are single-stranded small noncoding RNA molecules that are 22 to 25 nucleotides in length. They are implicated in the regulation of the immune response by modulating differentiation and proliferation of immune cells, production of cytokine types, and activation of the intracellular signaling pathways through posttranscriptional mechanisms. Although their actual functions are not yet fully understood, viral miRNAs are thought to help viruses to replicate and evade host immune response important in infectiousness. The determinants affecting the infectiousness of human immunodeficiency virus-1 (HIV-1) and progression to the disease state vary according to several host and viral factors. Interestingly, mother-to-child transmission rates are as low as 5 to 15%, even when the mother is not receiving antiretroviral therapy. Higher HIV-1 viral loads, and recent maternal infection, are associated with higher transmission rates. Also, cellular tropism is a well-known phenomenon in HIV-1 pathogenesis. Further, cellular and viral miRNAs seem to be involved in the pathogenesis and infectiousness of HIV-1. The aim of this review is to outline the history of the discovery of HIV-1-viral miRNAs and the evidence for their role in pathogenesis.

Identification of Reference Genes for Analysis of microRNA Expression Patterns in Equine Chorioallantoic Membrane and Serum.

MicroRNAs (miRNAs) have important posttranscriptional regulatory abilities, and there is considerable interest in evaluating their expression patterns in different pathophysiological states. The most common method of quantifying miRNA expression is quantitative reverse transcription PCR; however, the identification of tissue-specific and species-specific reference miRNA is a prerequisite for miRNA expression analysis. Currently, no reference genes have been described for evaluating miRNA expression in equine serum and chorioallantoic membrane (CAM) during pregnancy. The aim of the present study was to characterize reference genes for normalization of miRNA expression in CAM and serum in the pregnant equine. To identify the most stable miRNAs in serum, expression of potential candidates was evaluated in serum samples from diestrous mares, pregnant mares and geldings. To identify the most stable miRNAs in CAM, expression of potential candidates was evaluated in CAM, collected from mares at 4, 6 and 10months of pregnancy and immediately postpartum. From a previously generated miRNA sequencing dataset, two separate lists of potential reference miRNAs were identified (serum and CAM) using the NormFinder program, in addition to the commonly used small RNA normalizers, 5S rRNA and U6 snRNA. The putative reference miRNAs were selected using geNorm and NormFinder. In case of a nonsignificant correlation between the results of ranking and stability value between these two programs, ranking from BestKeeper was also included. NormFinder and geNorm consistently identified eca-miR-21-5p, eca-let-7a-5p and eca-miR-10a-5p as the three most stable reference genes for the normalization of serum miRNAs. Within CAM samples, the average ranking obtained from the ranking ofNormFinder, geNorm and BestKeeper identified eca-miR-8908a-1-5p, eca-miR-369-5p and eca-miR-106a-5p as the three most stable miRNAs. These observations provide information about equine-specific reference genes that can be used for normalizing miRNAs expression patterns in CAM and serum during the equine pregnancy.

Identification and characterization of Prunus persica miRNAs in response to UVB radiation in greenhouse through high-throughput sequencing

BackgroundMicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression of target mRNAs involved in plant growth, development, and abiotic stress. As one of the most important model plants, peach (Prunus persica) has high agricultural significance and nutritional values. It is well adapted to be cultivated in greenhouse in which some auxiliary conditions like temperature, humidity, and UVB etc. are needed to ensure the fruit quality. However, little is known about the genomic information of P. persica under UVB supplement. Transcriptome and expression profiling data for this species are therefore important resources to better understand the biological mechanism of seed development, formation and plant adaptation to environmental change. Using a high-throughput miRNA sequencing, followed by qRT-PCR tests and physiological properties determination, we identified the responsive-miRNAs under low-dose UVB treatment and described the expression pattern and putative function of related miRNAs and target genes in chlorophyll and carbohydrate metabolism.ResultsA total of 164 known peach miRNAs belonging to 59 miRNA families and 109 putative novel miRNAs were identified. Some of these miRNAs were highly conserved in at least four other plant species. In total, 1794 and 1983 target genes for known and novel miRNAs were predicted, respectively. The differential expression profiles of miRNAs between the control and UVB-supplement group showed that UVB-responsive miRNAs were mainly involved in carbohydrate metabolism and signal transduction. UVB supplement stimulated peach to synthesize more chlorophyll and sugars, which was verified by qRT-PCR tests of related target genes and metabolites’ content measurement.ConclusionThe high-throughput sequencing data provided the most comprehensive miRNAs resource available for peach study. Our results identified a series of differentially expressed miRNAs/target genes that were predicted to be low-dose UVB-responsive. The correlation between transcriptional profiles and metabolites contents in UVB supplement groups gave novel clues for the regulatory mechanism of miRNAs in Prunus. Low-dose UVB supplement could increase the chlorophyll and sugar (sorbitol) contents via miRNA-target genes and therefore improve the fruit quality in protected cultivation of peaches.

Identification of circular RNAs and their targets during tomato fruit ripening

Circular RNAs (circRNAs) have been found to be highly abundant and evolutionarily conserved in both animals and plants. Tomato is a widely characterized model plant, but very little information is available about the feature and functions of circRNAs in this species. In the present study, to invest whether circRNAs are involved in fruit ripening process, we used deep sequencing to monitor the changes in circRNAs between mature green tomato fruit and red ripening ones. A total of 705 circRNAs were detected in these two samples, of which 340 were differentially regulated. CircRNAs were reported to function as cis-regulator and miRNA sponge to regulate physiological and biochemical metabolism in animals. In this study, 19 differentially expressed circRNAs were found to function as putative miRNA sponges to regulate the expression of 94 target mRNAs. Furthermore, Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation of both parent genes and target mRNAs were performed to reveal the functions of differentially expressed circRNAs during ripening. The results revealed that circRNAs may be involved in regulating fruit ripening by mediating metabolism adaptation, hormone balance, and photosynthesis related pathways. Besides, circRNAs were predicted to be involved in transcription regulation through targeting transcription factors such as ethylene-responsive transcription factor (ERF), squamosa promoter binding-like protein (SBP) and myeloblastosis (MYB) proto-oncogene protein. These results shed light on the role of circRNAs in fruit ripening and help to identify novel circRNAs in tomato.

Turmeric (Curcuma longa): miRNAs and their regulating targets are involved in development and secondary metabolite pathways

Turmeric has been used as a therapeutic herb over centuries in traditional medicinal systems due to the presence of several secondary metabolite compounds. microRNAs are known to regulate gene expression at the post-transcriptional level by transcriptional cleavage or translation repression. miRNAs have been demonstrated to play an active role in secondary metabolism regulation. The present work was focused on the identification of the miRNAs involved in the regulation of secondary metabolite and development process of turmeric. Eighteen miRNA families were identified for turmeric. Sixteen miRNA families were observed to regulate 238 target transcripts. LncRNAs targets of the putative miRNA candidates were also predicted. Our results indicated their role in binding, reproduction, stress, and other developmental processes. Gene annotation and pathway analysis illustrated the biological function of the targets regulated by the putative miRNAs. The miRNA-mediated gene regulatory network also revealed co-regulated targets that were regulated by two or more miRNA families. miR156 and miR5015 were observed to be involved in rhizome development. miR5021 showed regulation for terpenoid backbone biosynthesis and isoquinoline alkaloid biosynthesis pathways. The flavonoid biosynthesis pathway was observed to be regulated by miR2919. The analysis revealed the probable involvement of three miRNAs (miR1168.2, miR156b and miR1858) in curcumin biosynthesis. Other miRNAs were found to be involved in the growth and developmental process of turmeric. Phylogenetic analysis of selective miRNAs was also performed.

Multiple microRNAs function as self-protective modules in acetaminophen-induced hepatotoxicity in humans.

Acetaminophen (APAP) overdose is the leading cause of acute liver failure. Yet the mechanisms underlying adaptive tolerance toward APAP-induced liver injury are not fully understood. To better understand molecular mechanisms contributing to adaptive tolerance to APAP is an underpinning foundation for APAP-related precision medicine. In the current study, the mRNA and microRNA (miRNA) expression profiles derived from next generation sequencing data for APAP-treated (5 and 10mM) HepaRG cells and controls were analyzed systematically. Putative miRNAs targeting key dysregulated genes involved in APAP hepatotoxicity were selected using in silico prediction algorithms, un-biased gene ontology, and network analyses. Luciferase reporter assays, RNA electrophoresis mobility shift assays, and miRNA pull-down assays were performed to investigate the role of miRNAs affecting the expression of dysregulated genes. Levels of selected miRNAs were measured in serum samples obtained from children with APAP overdose (58.6-559.4mg/kg) and from healthy controls. As results, 2758 differentially expressed genes and 47 miRNAs were identified. Four of these miRNAs (hsa-miR-224-5p, hsa-miR-320a, hsa-miR-449a, and hsa-miR-877-5p) suppressed drug metabolizing enzyme (DME) levels involved in APAP-induced liver injury by downregulating HNF1A, HNF4A and NR1I2 expression. Exogenous transfection of these miRNAs into HepaRG cells effectively rescued them from APAP toxicity, as indicated by decreased alanine aminotransferase levels. Importantly, hsa-miR-320a and hsa-miR-877-5p levels were significantly elevated in serum samples obtained from children with APAP overdose compared to health controls. Collectively, these data indicate that hsa-miR-224-5p, hsa-miR-320a, hsa-miR-449a, and hsa-miR-877-5p suppress DME expression involved in APAP-induced hepatotoxicity and they contribute to an adaptive response in hepatocytes.

Computational deciphering of biotic stress associated genes in tomato (Solanum lycopersicum)

Tomato (Solanum lycopersicum) is one of the major vegetable plant and a model system for fruit development. Its global importance is due to its lycopene pigment which has anti-oxidative and anti-cancerous properties. Though >1.5M biotic stress associated ESTs of tomato are available but cumulative analysis to predict genes is warranted. Availability of whole genome de novo assembly can advantageously be used to map them over different chromosome. Further, available 0.14M catalogued markers can be used to introgress specific desirable genes in varietal improvement program. We report here 57 novel genes associated with biotic stress of tomato along with 50 genes having physical location over different chromosomes. We also report 52 cis-regulating elements and 69 putative miRNAs which are involved in regulation of these biotic stresses associated genes. These putative candidate genes associated with biotic stress can be used in molecular breeding in the endeavor of tomato productivity along with its sustainable germplasm management.

Effect of lipopolysaccharide on the expression of inflammatory mRNAs and microRNAs in the mouse oviduct.

Infection with Gram-negative bacteria is a major cause of aberrant inflammation in the oviduct; consequences can include tubal-based infertility and/or ectopic pregnancy. Understanding the inflammatory response is necessary for the development of novel treatment options that counter inflammation-induced infertility. The aim of the present study was to determine the effect of intraperitoneal (i.p.) administration of Escherichia coli-derived lipopolysaccharide (LPS) on the acute expression of inflammatory mRNAs and microRNAs (miRNAs) in the oviduct. On the day of oestrus, 6- to 8-week-old CD1 mice were injected i.p. with 0, 2 or 10µg LPS in 100μL phosphate-buffered saline. Mice were killed 24h later and the oviducts collected for gene expression analyses. The effect of treatment on the expression of mRNAs and miRNAs was evaluated by one-way analysis of variance (ANOVA), with treatment means of differentially expressed (P<0.05) transcripts separated using Scheffé's test. LPS treatment affected 49 of 179 targeted inflammatory mRNAs and 51 of 578 miRNAs (P<0.05). The identity of differentially expressed miRNAs predicted as regulators of chemokine and interleukin ligand mRNAs was then extracted using the microRNA.org database. The results of the present study indicate that systemic treatment with LPS induces a robust inflammatory response in the oviducts of mice, and identify key mRNAs and putative miRNAs modulating this effect.

MicroRNA regulation of CYP 1A2, CYP3A4 and CYP2E1 expression in acetaminophen toxicity

MicroRNAs (miRNAs) that regulate the cytochrome P-450 isoforms involved in acetaminophen (APAP) toxicity were examined in HepaRG cells treated with APAP (20 mM). In-vitro studies found that APAP protein adducts were increased at 1 h, followed by ALT increases at 12 and 24 h. CYP1A2, CYP3A4 and CYP2E1 mRNA levels were decreased, while miRNAs were increased for miR-122-5p, miR-378a-5p, miR-27b-3p at 6 h and miR-125b-5p at 12 h and miR-27b-3p at 24 h. Putative miRNA binding sites on the 3′UTRs of the CYPs were identified in-silico. Overexpression of miR-122-5p and miR-378a-5p in cells suppressed protein expression of CYP1A2, CYP3A4 and CYP2E1. Luciferase reporter assays confirmed the interaction between miR-122 and the 3′UTR of the CYP1A2 and CYP3A4. Thus, the in-vitro experiments showed that miR-122-5p and miR-378a-5p upregulation were associated with translational repression of CYPs. Serum samples of children with APAP overdose had significant elevation of miR-122-5p, miR-378a-5p, miR-125b-5p and miR-27b-3p, compared to healthy controls and receiver operator curves of the miRNAs had AUCs of 91 to 100%. Collectively, the data suggest that miRNA elevations in APAP toxicity represent a regulatory response to modify CYP1A2, CYP3A4 and CYP2E1 translation due to cellular stress and injury.

Role of MicroRNA-423-5p in posttranscriptional regulation of the intestinal riboflavin transporter-3.

Riboflavin (RF) is essential for normal cellular functions and health. Humans obtain RF from exogenous sources via intestinal absorption that involves a highly specific carrier-mediated process. We have recently established that the riboflavin transporter-3 (RFVT3) is vital for the normal intestinal RF uptake process and have characterized certain aspects of its transcriptional regulation. Little is known, however, about how this transporter is regulated at the posttranscriptional level. We address this issue by focusing on the role of microRNAs. Using bioinformatics, we identified two potential interacting miRNAs with the human (h) RFVT3-3'-UTR, and showed (using pmirGLO-hRFVT3-3'-UTR) that the hRFVT3-3'-UTR is, indeed, a target for miRNA effect. Of the two putative miRNAs identified, miR-423-5p was found to be highly expressed in intestinal epithelial cells and that its mimic affected luciferase reporter activity of the pmirGLO-hRFVT3-3'-UTR construct, and also led to inhibition in RF uptake by intestinal epithelial Caco-2 and HuTu-80 cells. Furthermore, cells transfected with mutated seed sequences for miR-423-5p showed an abrogation in inhibitory effect of the miR-423-5p mimic on luciferase activity. While miR-423-5p did not affect the level of expression of the hRFVT3 mRNA, it did lead to a significant inhibition in the level of expression of its protein. Similarly, miR-423-5p was found to affect the level of expression of the mouse RFVT3 in cultured intestinal enteroids. These findings demonstrate, for the first time, that the RFVT3 is a target for posttranscriptional regulation by miRNAs in intestinal epithelial cells and that this regulation has functional consequences on intestinal RF uptake.NEW & NOTEWORTHY Our findings show for the first time that RFVT3 is a target for posttranscriptional regulation by miR-423-5p in intestinal epithelial cells, and this regulation has functional consequences on intestinal riboflavin (RF) uptake process.

In Silico Analysis of Small RNAs Suggest Roles for Novel and Conserved miRNAs in the Formation of Epigenetic Memory in Somatic Embryos of Norway Spruce.

Epigenetic memory in Norway spruce affects the timing of bud burst and bud set, vitally important adaptive traits for this long-lived forest species. Epigenetic memory is established in response to the temperature conditions during embryogenesis. Somatic embryogenesis at different epitype inducing (EpI) temperatures closely mimics the natural processes of epigenetic memory formation in seeds, giving rise to epigenetically different clonal plants in a reproducible and predictable manner, with respect to altered bud phenology. MicroRNAs (miRNAs) and other small non-coding RNAs (sRNAs) play an essential role in the regulation of plant gene expression and may affect this epigenetic mechanism. We used NGS sequencing and computational in silico methods to identify and profile conserved and novel miRNAs among small RNAs in embryogenic tissues of Norway spruce at three EpI temperatures (18, 23 and 28°C). We detected three predominant classes of sRNAs related to a length of 24 nt, followed by a 21–22 nt class and a third 31 nt class of sRNAs. More than 2100 different miRNAs within the prevailing length 21–22 nt were identified. Profiling these putative miRNAs allowed identification of 1053 highly expressed miRNAs, including 523 conserved and 530 novels. 654 of these miRNAs were found to be differentially expressed (DEM) depending on EpI temperature. For most DEMs, we defined their putative mRNA targets. The targets represented mostly by transcripts of multiple-repeats proteins, like TIR, NBS-LRR, PPR and TPR repeat, Clathrin/VPS proteins, Myb-like, AP2, etc. Notably, 124 DE miRNAs targeted 203 differentially expressed epigenetic regulators. Developing Norway spruce embryos possess a more complex sRNA structure than that reported for somatic tissues. A variety of the predicted miRNAs showed distinct EpI temperature dependent expression patterns. These putative EpI miRNAs target spruce genes with a wide range of functions, including genes known to be involved in epigenetic regulation, which in turn could provide a feedback process leading to the formation of epigenetic marks. We suggest that TIR, NBS and LRR domain containing proteins could fulfill more general functions for signal transduction from external environmental stimuli and conversion them into molecular response. Fine-tuning of the miRNA production likely participates in both developmental regulation and epigenetic memory formation in Norway spruce.

MicroRNA Profiling Reveals Distinct Profiles for Tissue-Derived and Cultured Endothelial Cells

Endothelial plasticity enables the cells to switch their phenotype according to the surrounding vascular microenvironment. MicroRNAs (miRNAs) are small noncoding RNAs that control endothelial plasticity. The objective of this study was to investigate the differences in miRNA profiles of tissue-derived cells and cultured endothelial cells. To this end, miRNA expression was profiled from freshly isolated tissue-derived human vascular endothelial cells and endothelial cells cultured until cellular senescence using miRNA sequencing. In addition, the data was searched for putative novel endothelial miRNAs and miRNA isoforms. The data analysis revealed a striking change in endothelial miRNA profile as the cells adapted from tissue to cell culture environment and the overall miRNA expression decreased significantly in cultured compared to tissue-derived endothelial cells. In addition to changes in mechanosensitive miRNA expression, alterations in senescence-associated and endothelial-to-mesenchymal-transition-associated miRNAs were observed in aging cells. Collectively, the data illustrates the adaptability of endothelial cell miRNA expression that mirrors prevailing cellular environment.

Haloperidol induces pharmacoepigenetic response by modulating miRNA expression, global DNA methylation and expression profiles of methylation maintenance genes and genes involved in neurotransmission in neuronal cells.

IntroductionHaloperidol has been extensively used in various psychiatric conditions. It has also been reported to induce severe side effects. We aimed to evaluate whether haloperidol can influence host methylome, and if so what are the possible mechanisms for it in neuronal cells. Impact on host methylome and miRNAs can have wide spread alterations in gene expression, which might possibly help in understanding how haloperidol may impact treatment response or induce side effects.MethodsSK-N-SH, a neuroblasoma cell line was treated with haloperidol at 10μm concentration for 24 hours and global DNA methylation was evaluated. Methylation at global level is maintained by methylation maintenance machinery and certain miRNAs. Therefore, the expression of methylation maintenance genes and their putative miRNA expression profiles were assessed. These global methylation alterations could result in gene expression changes. Therefore genes expressions for neurotransmitter receptors, regulators, ion channels and transporters were determined. Subsequently, we were also keen to identify a strong candidate miRNA based on biological and in-silico approach which can reflect on the pharmacoepigenetic trait of haloperidol and can also target the altered neuroscience panel of genes used in the study.ResultsHaloperidol induced increase in global DNA methylation which was found to be associated with corresponding increase in expression of various epigenetic modifiers that include DNMT1, DNMT3A, DNMT3B and MBD2. The expression of miR-29b that is known to putatively regulate the global methylation by modulating the expression of epigenetic modifiers was observed to be down regulated by haloperidol. In addition to miR-29b, miR-22 was also found to be downregulated by haloperidol treatment. Both these miRNA are known to putatively target several genes associated with various epigenetic modifiers, pharmacogenes and neurotransmission. Interestingly some of these putative target genes involved in neurotransmission were observed to be upregulated while CHRM2 gene expression was down regulated.ConclusionsHaloperidol can influence methylation traits thereby inducing a pharmacoepigenomic response, which seems to be regulated by DNMTs and their putative miRNA expression. Increased methylation seems to influence CHRM2 gene expression while microRNA could influence neurotransmission, pharmacogene expression and methylation events. Altered expression of various therapeutically relevant genes and miRNA expression, could account for their role in therapeutic response or side effects.

LncRNAs in Secondary Hair Follicle of Cashmere Goat: Identification, Expression, and Their Regulatory Network in Wnt Signaling Pathway

ABSTRACTLong noncoding RNAs (lncRNAs) are a novel class of eukaryotic transcripts. They are thought to act as a critical regulator of protein-coding gene expression. Herein, we identified and characterized 13 putative lncRNAs from the expressed sequence tags from secondary hair follicle of Cashmere goat. Furthermore, we investigated their transcriptional pattern in secondary hair follicle of Liaoning Cashmere goat during telogen and anagen phases. Also, we generated intracellular regulatory networks of upregulated lncRNAs at anagen in Wnt signaling pathway based on bioinformatics analysis. The relative expression of six putative lncRNAs (lncRNA-599618, -599556, -599554, -599547, -599531, and -599509) at the anagen phase is significantly higher than that at telogen. Compared with anagen, the relative expression of four putative lncRNAs (lncRNA-599528, -599518, -599511, and -599497) was found to be significantly upregulated at telogen phase. The network generated showed that a rich and complex regulatory relationship of the putative lncRNAs and related miRNAs with their target genes in Wnt signaling pathway. Our results from the present study provided a foundation for further elucidating the functional and regulatory mechanisms of these putative lncRNAs in the development of secondary hair follicle and cashmere fiber growth of Cashmere goat.

α, γ-Mangostins Induce Autophagy and Show Synergistic Effect with Gemcitabine in Pancreatic Cancer Cell Lines.

Pancreatic cancer is one of the most lethal and aggressive cancers in the world. However, no effective treatment is currently available for pancreatic cancer. The objective of this study was to determine the anti-pancreatic cancer effect of α-mangostin (αM) and γ-mangostin (γM) extracted from the pericarp of Garcinia mangostana L.. Both αM and γM reduced the viability of pancreatic cancer cells MIA PaCa-2 and PANC-1 in a dose-dependent manner. These compounds induced apoptosis by increasing c-PARP and c-Caspase 3 levels. They also induced autophagy by increasing levels of microtubule-associated protein 1A/1B light chain 3B (LC3II) in both cell lines while decreasing sequestosome 1 (p62) in MIA PaCa-2. Both αM and γM induced autophagy through increasing phosphorylation levels of AMP-activated protein kinase (p-AMPK) and p38-mitogen activated protein kinase (p-p38) while decreasing phosphorylation level of mammalian target of rapamycin complex 1 (p-mTOR). Of various microRNAs (miRNA), miR-18a was found to be a putative regulatory miRNA for autophagy induced by αM or γM. In combination with gemcitabine, a compound frequently used in pancreatic cancer treatment, αM and γM showed synergistic anti-cancer effects in MIA PaCa-2. Collectively, these results suggest that αM and γM can induce apoptosis and autophagy in pancreatic cancer cells and that their anti-cancer effect is likely to be associated with miR-18a. In conclusion, αM and γM might be used as a potential new therapy for pancreatic cancer.

The use of high-throughput small RNA sequencing reveals differentially expressed microRNAs in response to aster yellows phytoplasma-infection in Vitis vinifera cv. 'Chardonnay'.

Phytoplasmas are cell wall-less plant pathogenic bacteria responsible for major crop losses throughout the world. In grapevine they cause grapevine yellows, a detrimental disease associated with a variety of symptoms. The high economic impact of this disease has sparked considerable interest among researchers to understand molecular mechanisms related to pathogenesis. Increasing evidence exist that a class of small non-coding endogenous RNAs, known as microRNAs (miRNAs), play an important role in post-transcriptional gene regulation during plant development and responses to biotic and abiotic stresses. Thus, we aimed to dissect complex high-throughput small RNA sequencing data for the genome-wide identification of known and novel differentially expressed miRNAs, using read libraries constructed from healthy and phytoplasma-infected Chardonnay leaf material. Furthermore, we utilised computational resources to predict putative miRNA targets to explore the involvement of possible pathogen response pathways. We identified multiple known miRNA sequence variants (isomiRs), likely generated through post-transcriptional modifications. Sequences of 13 known, canonical miRNAs were shown to be differentially expressed. A total of 175 novel miRNA precursor sequences, each derived from a unique genomic location, were predicted, of which 23 were differentially expressed. A homology search revealed that some of these novel miRNAs shared high sequence similarity with conserved miRNAs from other plant species, as well as known grapevine miRNAs. The relative expression of randomly selected known and novel miRNAs was determined with real-time RT-qPCR analysis, thereby validating the trend of expression seen in the normalised small RNA sequencing read count data. Among the putative miRNA targets, we identified genes involved in plant morphology, hormone signalling, nutrient homeostasis, as well as plant stress. Our results may assist in understanding the role that miRNA pathways play during plant pathogenesis, and may be crucial in understanding disease symptom development in aster yellows phytoplasma-infected grapevines.

Biocomputational identification and validation of novel microRNAs predicted from bubaline whole genome shotgun sequences

MicroRNAs (miRNAs) are small (19–25 base long), non-coding RNAs that regulate post-transcriptional gene expression by cleaving targeted mRNAs in several eukaryotes. The miRNAs play vital roles in multiple biological and metabolic processes, including developmental timing, signal transduction, cell maintenance and differentiation, diseases and cancers. Experimental identification of microRNAs is expensive and lab-intensive. Alternatively, computational approaches for predicting putative miRNAs from genomic or exomic sequences rely on features of miRNAs viz. secondary structures, sequence conservation, minimum free energy index (MFEI) etc. To date, not a single miRNA has been identified in bubaline (Bubalus bubalis), which is an economically important livestock. The present study aims at predicting the putative miRNAs of buffalo using comparative computational approach from buffalo whole genome shotgun sequencing data (INSDC: AWWX00000000.1). The sequences were blasted against the known mammalian miRNA. The obtained miRNAs were then passed through a series of filtration criteria to obtain the set of predicted (putative and novel) bubaline miRNA. Eight miRNAs were selected based on lowest E-value and validated by real time PCR (SYBR green chemistry) using RNU6 as endogenous control. The results from different trails of real time PCR shows that out of selected 8 miRNAs, only 2 (hsa-miR-1277-5p; bta-miR-2285b) are not expressed in bubaline PBMCs. The potential target genes based on their sequence complementarities were then predicted using miRanda. This work is the first report on prediction of bubaline miRNA from whole genome sequencing data followed by experimental validation. The finding could pave the way to future studies in economically important traits in buffalo.

Genome-wide identification of soybean microRNA responsive to soybean cyst nematodes infection by deep sequencing

BackgroundThe soybean cyst nematode (SCN), Heterodera glycines, is one of the most devastating diseases limiting soybean production worldwide. It is known that small RNAs, including microRNAs (miRNAs) and small interfering RNAs (siRNAs), play important roles in regulating plant growth and development, defense against pathogens, and responses to environmental changes.ResultsIn order to understand the role of soybean miRNAs during SCN infection, we analyzed 24 small RNA libraries including three biological replicates from two soybean cultivars (SCN susceptible KS4607, and SCN HG Type 7 resistant KS4313N) that were grown under SCN-infested and -noninfested soil at two different time points (SCN feeding establishment and egg production). In total, 537 known and 70 putative novel miRNAs in soybean were identified from a total of 0.3 billion reads (average about 13.5 million reads for each sample) with the programs of Bowtie and miRDeep2 mapper. Differential expression analyses were carried out using edgeR to identify miRNAs involved in the soybean-SCN interaction. Comparative analysis of miRNA profiling indicated a total of 60 miRNAs belonging to 25 families that might be specifically related to cultivar responses to SCN. Quantitative RT-PCR validated similar miRNA interaction patterns as sequencing results.ConclusionThese findings suggest that miRNAs are likely to play key roles in soybean response to SCN. The present work could provide a framework for miRNA functional identification and the development of novel approaches for improving soybean SCN resistance in future studies.

MicroRNA dependent and independent deregulation of long non-coding RNAs by an oncogenic herpesvirus.

Kaposi’s sarcoma (KS) is a highly prevalent cancer in AIDS patients, especially in sub-Saharan Africa. Kaposi’s sarcoma-associated herpesvirus (KSHV) is the etiological agent of KS and other cancers like Primary Effusion Lymphoma (PEL). In KS and PEL, all tumors harbor latent KSHV episomes and express latency-associated viral proteins and microRNAs (miRNAs). The exact molecular mechanisms by which latent KSHV drives tumorigenesis are not completely understood. Recent developments have highlighted the importance of aberrant long non-coding RNA (lncRNA) expression in cancer. Deregulation of lncRNAs by miRNAs is a newly described phenomenon. We hypothesized that KSHV-encoded miRNAs deregulate human lncRNAs to drive tumorigenesis. We performed lncRNA expression profiling of endothelial cells infected with wt and miRNA-deleted KSHV and identified 126 lncRNAs as putative viral miRNA targets. Here we show that KSHV deregulates host lncRNAs in both a miRNA-dependent fashion by direct interaction and in a miRNA-independent fashion through latency-associated proteins. Several lncRNAs that were previously implicated in cancer, including MEG3, ANRIL and UCA1, are deregulated by KSHV. Our results also demonstrate that KSHV-mediated UCA1 deregulation contributes to increased proliferation and migration of endothelial cells.