Small RNA sequencing reveals the role of pearl millet miRNAs and their targets in salinity stress responses

Abstract Background Sophora tonkinensis Gagnep is a traditional Chinese medical plant that is mainly cultivated in southern China. Drought stress is one of the major abiotic stresses that negatively impacts S. tonkinensis growth. However, the molecular mechanisms governing the responses to drought stress in S. tonkinensis at the transcriptional and posttranscriptional levels are not well understood. Results To identify genes and miRNAs involved in drought stress responses in S. tonkinensis, both mRNA and small RNA sequencing was performed in root samples under control, mild drought, and severe drought conditions. mRNA sequencing revealed 66,476 unigenes, and the differentially expressed unigenes (DEGs) were associated with several key pathways, including phenylpropanoid biosynthesis, sugar metabolism, and quinolizidine alkaloid biosynthesis pathways. A total of 10 and 30 transcription factors (TFs) were identified among the DEGs under mild and severe drought stress, respectively. Moreover, small RNA sequencing revealed a total of 368 miRNAs, including 255 known miRNAs and 113 novel miRNAs. The differentially expressed miRNAs and their target genes were involved in the regulation of plant hormone signal transduction, the spliceosome, and ribosomes. Analysis of the regulatory network involved in the response to drought stress revealed 37 differentially expressed miRNA-mRNA pairs. Conclusion This is the first study to simultaneously profile the expression patterns of mRNAs and miRNAs on a genome-wide scale to elucidate the molecular mechanisms of the drought stress responses of S. tonkinensis. Our results suggest that S. tonkinensis implements diverse mechanisms to modulate its responses to drought stress.

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Dissecting The Mechanism and Functional Role Of Silenced Microrna-125a In Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v122.21.3803.3803 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3803-3803

Author(s):

Melanie L Ufkin ◽

Maria Trissal ◽

Heather Driscoll ◽

Christine W Duarte ◽

Daniel C Link ◽

...

Keyword(s):

Rna Sequencing ◽

Small Rna ◽

Functional Role ◽

Cell Cycle Progression ◽

Small Rna Sequencing ◽

Nb4 Cells ◽

Hematopoietic Malignancies ◽

Rna Expression Profiling ◽

Altered Cell

Abstract Background Acute myeloid leukemia (AML) is a heterogeneous disease marked by a highly variable clinical course and response to therapy. microRNA's (miR) have entered the spotlight for being involved in pathogenesis of numerous diseases, including hematopoietic malignancies. miR-125a-5p previously was identified to be decreased in AML, however its functional role leading to the pathogenesis of AML is unknown leading us to dissect its functional role in AML within these studies. Results Small RNA sequencing illustrated that miR-125a is decreased in many AML FAB subtypes at time of diagnosis and relapse (p<0.05) beyond the previously reported cytogenetically normal AML (Figure 1A&B). miR-125a expression in comparison to clinical aspects (167 AML patients) was conducted using data from the National Cancer Institute the Cancer Genome Atlas (TCGA) Data Portal to support small RNA sequencing findings. The majority of patients had low miR-125a expression (1-250 RPM), which is significantly decreased if compared to the small RNA sequencing results for healthy CD34+ cells (∼5000RPM). Interestingly, there is no difference between gender and miR-125a expression. Though not significant, there was a trend of low miR-125a expression towards low survival rate. To dissect the pathways affected when miR-125a expression is restored in AML, acute promyelocytic leukemia (APL) NB4 cells containing t (15; 17), which had the most significantly decreased expression when AML lines were screened, were utilized for studies. Previously we reported that miR-125a was epigenetically silenced in AML and caused altered cell proliferation, cell cycle progression, and apoptosis, however through RNA expression profiling we now know the potential players altered leading to these altered biological pathways. Among the significant decreased genes were FLT1, MMP-9, IL-32Rα and HIP-1 while several increased genes were of interest such as cathepsin-G, EPX, and SPARC. Though all of these were interesting due to their previous implications within cancer or AML, they are not predicted targets of miR-125a. Therefore, we focused our analysis on identifying a potential target of miR-125a within AML. From profiling results, Trib2 was significantly decreased (p=0.0003, Figure 2) when miR-125a expression was restored in NB4 cells and is a predicted target. RT-qPCR and 3'UTR luciferase confirmed that Trib2 was a target of miR-125a. Trib2 has been implicated in AML in several contexts including inhibition of C/EBPα causing decreased cell differentiation and its ability to interact with HoxA9 to aid in the progression of AML. Although Trib2 has been implicated in cancer, inhibitors are not developed currently but a necessity has been demonstrated. Therefore, we focused our studies on identifying a pathway with known inhibitors. Several reports demonstrate enhanced ErbB2 expression when miR-125a is decreased leading us to test Mubritinib, which selectively inhibits ErbB2 phosphorylation. Previously we demonstrated the profound affect on inhibition of cell cycle progression and altered cell proliferation, differentiation, and apoptosis in NB4 cells. Most strikingly was the lack of affect of the inhibitor on HL60 cells, which do not have decreased miR-125a expression like NB4 cells suggesting that inhibition of the ErbB pathway would be specific for low miR-125a AML. Excitingly, ANOVA pathway analysis revealed the ErbB pathway was significantly altered, such as decreased ErbB receptors (ErbB1 and ErbB3) and downstream effectors (PI3K, AKT, and Stat5). After establishing the role of the ErbB pathway in NB4 cells we tested the effect of Mubritinib on retinoic acid resistant NB4 cells (NB4-LR1), which is indicating that Mubritinib could potentially be utilized for a new therapeutic treatment in NB4-LR1 cells in addition to NB4 cells. Conclusion Decreased miR-125a within AML may give leukemic blasts an advantage in multiple AML subtypes. Through RNA expression profiling, Trib2 was identified as a target of miR-125a within AML. By utilizing pathway analysis in we have identified a potential new therapeutic, Mubritinib, for miR-125a low AML, which is now being tested in vivo. From our strong preliminary work ErbB inhibitors currently being utilized for treating ErbB overexpressing epithelial cancers could be tested in hematopoietic malignancies in addition to identifying a potential role of Trib2 in miR-125a low AML. Disclosures: No relevant conflicts of interest to declare.

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Comprehensive MicroRNA Expression Profile of the Mammary Gland in Lactating Dairy Cows With Extremely Different Milk Protein and Fat Percentages

Frontiers in Genetics ◽

10.3389/fgene.2020.548268 ◽

2020 ◽

Vol 11 ◽

Author(s):

Xiaogang Cui ◽

Shengli Zhang ◽

Qin Zhang ◽

Xiangyu Guo ◽

Changxin Wu ◽

...

Keyword(s):

Mammary Gland ◽

Rna Sequencing ◽

Differentially Expressed Genes ◽

Small Rna ◽

Milk Protein ◽

Milk Composition ◽

Differentially Expressed ◽

Small Rna Sequencing ◽

Rna Seq

A total of 31 differentially expressed genes in the mammary glands were identified in our previous study using RNA sequencing (RNA-Seq), for lactating cows with extremely high and low milk protein and fat percentages. To determine the regulation of milk composition traits, we herein investigated the expression profiles of microRNA (miRNA) using small RNA sequencing based on the same samples as in the previous RNA-Seq experiment. A total of 497 known miRNAs (miRBase, release 22.1) and 49 novel miRNAs among the reads were identified. Among these miRNAs, 71 were found differentially expressed between the high and low groups (p < 0.05, q < 0.05). Furthermore, 21 of the differentially expressed genes reported in our previous RNA-Seq study were predicted as target genes for some of the 71 miRNAs. Gene ontology and KEGG pathway analyses showed that these targets were enriched for functions such as metabolism of protein and fat, and development of mammary gland, which indicating the critical role of these miRNAs in regulating the formation of milk protein and fat. With dual luciferase report assay, we further validated the regulatory role of 7 differentially expressed miRNAs through interaction with the specific sequences in 3′UTR of the targets. In conclusion, the current study investigated the complexity of the mammary gland transcriptome in dairy cattle using small RNA-seq. Comprehensive analysis of differential miRNAs expression and the data from previous study RNA-seq provided the opportunity to identify the key candidate genes for milk composition traits.

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Small RNA sequencing of extracellular vesicles identifies circulating miRNAs related to inflammation and oxidative stress in HIV patients

BMC Immunology ◽

10.1186/s12865-020-00386-5 ◽

2020 ◽

Vol 21 (1) ◽

Author(s):

Sukrutha Chettimada ◽

David R. Lorenz ◽

Vikas Misra ◽

Steven M. Wolinsky ◽

Dana Gabuzda

Keyword(s):

Oxidative Stress ◽

Rna Sequencing ◽

Extracellular Vesicles ◽

Small Rna ◽

Stress Responses ◽

Hiv Positive ◽

Small Rna Sequencing ◽

And Oxidative Stress ◽

Potential Biomarkers ◽

Hiv Negative

Abstract Background Extracellular vesicles (EVs) are nano-sized particles secreted by most cells. EVs carry nucleic acids that hold promise as potential biomarkers in various diseases. Human immunodeficiency virus type 1 (HIV) infects CD4+ T cells and induces immune dysfunction, inflammation, and EV secretion, but little is known about EV small RNA cargo in relation to immune dysregulation in HIV-infected individuals. Here, we characterize small RNA carried by circulating EVs in HIV-positive subjects on antiretroviral therapy (ART) relative to uninfected controls by next-generation RNA sequencing. Results Plasma EVs isolated from HIV-positive and HIV-negative subjects in test (n = 24) and validation (n = 16) cohorts were characterized by electron microscopy, nanoparticle tracking analysis, and immunoblotting for exosome markers. EVs were more abundant in plasma from HIV-positive compared to HIV-negative subjects. Small RNA sequencing of plasma EVs in the test cohort identified diverse small RNA species including miRNA, piRNA, snRNA, snoRNA, tRNA, and rRNA, with miRNA being the most abundant. A total of 351 different miRNAs were detected in plasma EVs, with the top 50 miRNAs accounting for 90% of all miRNA reads. miR-26a-5p was the most abundant miRNA, followed by miR-21-5p and miR-148-3p. qRT-PCR analysis showed that six miRNAs (miR-10a-5p, − 21-5p, −27b-3p, − 122-5p, −146a-5p, − 423-5p) were significantly increased in plasma EVs from HIV-positive compared to HIV-negative subjects in the validation cohort. Furthermore, miR-21-5p, −27b-3p, −146a-5p, and − 423-5p correlated positively with metabolite markers of oxidative stress and negatively with anti-inflammatory polyunsaturated fatty acids. Over-representation and pathway enrichment analyses of miRNAs and their target genes predicted functional association with oxidative stress responses, interferon gamma signaling, Toll-like receptor signaling, TGF beta signaling, and Notch signaling. Conclusions HIV-positive individuals on ART have increased abundance of circulating EVs carrying diverse small RNAs, with miRNAs being the most abundant. Several miRNAs associated with inflammation and oxidative stress are increased in circulating EVs of HIV-positive individuals, representing potential biomarkers of targetable pathways that contribute to disease pathogenesis.

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Role of miRNA in Micro-Environment Mediated Drug Resistance in Acute Promyelocytic Leukemia to Arsenic Trioxide

Blood ◽

10.1182/blood.v128.22.5125.5125 ◽

2016 ◽

Vol 128 (22) ◽

pp. 5125-5125 ◽

Cited By ~ 1

Author(s):

Saravanan Ganesan ◽

Vairavan Lakshmanan ◽

Hamenth Kumar Palani ◽

Nithya Balasundaram ◽

Ansu Abu Alex ◽

...

Keyword(s):

Drug Resistance ◽

Arsenic Trioxide ◽

Rna Sequencing ◽

Acute Promyelocytic Leukemia ◽

Small Rna ◽

Stromal Cells ◽

Promyelocytic Leukemia ◽

Small Rna Sequencing ◽

Nb4 Cells

Abstract Role of stromal microenvironment in drug resistance has been extensively reported for several cancers. We have demonstrated earlier that there is significant micro-environment mediated drug resistance (EM-DR) to arsenic trioxide (ATO) in acute promyelocytic leukemia (APL) and that this was predominantly driven by upregulation of the NF-ⱪB pathway in the malignant cell. In our current study we have probed the molecular mechanism of ATO resistance in further detail. The role of microRNA (miRNA) in mediating this cross talk, if any, has not been reported on. We undertook a study to evaluate the potential role played by miRNA in EM-DR to ATO in APL. Using NGS based small RNA sequencing we identified two miRNA's that were differentially regulated in NB4 cells upon co-culture with HS-5 stromal cells (FDR corrected p values < 0.05). The two miRNAs were hsa-miR-23a-5p (downregulated) and hsa-miR-125a-3p (upregulated)](Fig 1a). These miRNAs have also been previously reported to be involved in NF-kB regulation, specifically miR125a-3p has been reported to be involved in activation of the NF-kB pathway and miR-23a- 5p can be repressed by the same pathway. These results were consistent with our earlier reported observations that NF-kB pathway is dysregulated and enhances drug resistance to ATO. We also observed miR-23a-5p mimics were able to restore the sensitivity of NB4 cells to ATO even in the presence of stromal cells (Fig 1b). Consistent with the above small RNA sequencing and our previously reported microarray data, using quantitative proteomics approach we have identified that both NFkB signaling and metallothionein 2A (MT-2A) levels are upregulated in leukemic cells upon stromal co-culture. MT 2A is a known target for hsa-mir-23a-5p. MT's are known to sequester heavy metals such as arsenic and could potentially reduce their cytotoxic effect. The role of metallothionein in ATO resistance in APL and possibly other leukemia's needs further evaluation. This data along with that reported earlier by us illustrates multiple levels of regulation of the NF-kB pathway and resistance to ATO by stromal cell co-culture. Disclosures No relevant conflicts of interest to declare.

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Role of Novel Micro-RNA Identified by Small RNA Sequencing in the Disease Progression of Hepatitis C Infection

Journal of Clinical and Experimental Hepatology ◽

10.1016/j.jceh.2017.05.060 ◽

2017 ◽

Vol 7 ◽

pp. S28-S29

Author(s):

Suchandrima Ghosh ◽

Joyeeta Chakraborty ◽

Amit Ghosh ◽

Priyanka Kumari ◽

Susree Roy ◽

...

Keyword(s):

Hepatitis C ◽

Disease Progression ◽

Rna Sequencing ◽

Small Rna ◽

Micro Rna ◽

Small Rna Sequencing ◽

Hepatitis C Infection

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Faculty Opinions recommendation of Identification of nutrient-responsive Arabidopsis and rapeseed microRNAs by comprehensive real-time polymerase chain reaction profiling and small RNA sequencing.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1161246.621733 ◽

2009 ◽

Author(s):

Alain Gojon

Keyword(s):

Polymerase Chain Reaction ◽

Real Time ◽

Rna Sequencing ◽

Small Rna ◽

Small Rna Sequencing ◽

Chain Reaction ◽

Polymerase Chain

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Small RNA Sequencing to Discover Circulating MicroRNA Biomarkers of Testicular Toxicity in Dogs

International Journal of Toxicology ◽

10.1177/1091581820961515 ◽

2020 ◽

pp. 109158182096151

Author(s):

Jennifer C. Shing ◽

Kai Schaefer ◽

Shaun E. Grosskurth ◽

Andy H. Vo ◽

Tatiana Sharapova ◽

...

Keyword(s):

Rna Sequencing ◽

Small Rna ◽

Exploratory Study ◽

Quantitative Polymerase Chain Reaction ◽

Small Rna Sequencing ◽

Circulating Microrna ◽

Testicular Toxicity ◽

Potential Candidate ◽

Drug Induced ◽

Organ Systems

Predictive indicators of testicular toxicity could improve drug development by allowing early in-life screening for this adverse effect before it becomes severe. We hypothesized that circulating microRNAs (miRNAs) could serve as testicular toxicity biomarkers in dogs. Herein, we describe the results of an exploratory study conducted to discover biomarkers of drug-induced testicular injury. Following a dose-selection study using the testicular toxicant ethylene glycol monomethyl ether (EGME), we chose a dose of 50 mg/kg/d EGME to avoid systemic toxicity and treated 2 groups of dogs (castrated, non-castrated) for 14 to 28 days. Castrated animals were used as negative controls to identify biomarkers specific for testicular toxicity because EGME can cause toxicity to organ systems in addition to the testis. Blood was collected daily during the dosing period, followed by recovery for 29 to 43 days with less frequent sampling. Dosing was well tolerated, resulting in mild-to-moderate degeneration in testes and epididymides. Global profiling of serum miRNAs at selected dosing and recovery time points was completed by small RNA sequencing. Bioinformatics data analysis using linear modeling demonstrated several circulating miRNAs that were differentially abundant during the dosing period compared with baseline and/or castrated control samples. Confirmatory reverse transcription quantitative polymerase chain reaction data in these animals was unable to detect sustained alterations of miRNAs in serum, except for 1 potential candidate cfa-miR-146b. Taken together, we report the results of a comprehensive exploratory study and suggest future directions for follow-up research to address the challenge of developing diagnostic biomarkers of testicular toxicity.

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Extensive Analysis of miRNA Trimming and Tailing Indicates that AGO1 Has a Complex Role in miRNA Turnover

Plants ◽

10.3390/plants10020267 ◽

2021 ◽

Vol 10 (2) ◽

pp. 267

Author(s):

Axel J. Giudicatti ◽

Ariel H. Tomassi ◽

Pablo A. Manavella ◽

Agustin L. Arce

Keyword(s):

Small Rna ◽

Stress Responses ◽

Cellular Localization ◽

Mirna Biogenesis ◽

Small Rna Sequencing ◽

Sequencing Data ◽

Extensive Analysis ◽

Small Regulatory Rnas ◽

Regulatory Rnas ◽

Argonaute 1

MicroRNAs are small regulatory RNAs involved in several processes in plants ranging from development and stress responses to defense against pathogens. In order to accomplish their molecular functions, miRNAs are methylated and loaded into one ARGONAUTE (AGO) protein, commonly known as AGO1, to stabilize and protect the molecule and to assemble a functional RNA-induced silencing complex (RISC). A specific machinery controls miRNA turnover to ensure the silencing release of targeted-genes in given circumstances. The trimming and tailing of miRNAs are fundamental modifications related to their turnover and, hence, to their action. In order to gain a better understanding of these modifications, we analyzed Arabidopsis thaliana small RNA sequencing data from a diversity of mutants, related to miRNA biogenesis, action, and turnover, and from different cellular fractions and immunoprecipitations. Besides confirming the effects of known players in these pathways, we found increased trimming and tailing in miRNA biogenesis mutants. More importantly, our analysis allowed us to reveal the importance of ARGONAUTE 1 (AGO1) loading, slicing activity, and cellular localization in trimming and tailing of miRNAs.

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Small RNA-Sequencing: Approaches and Considerations for miRNA Analysis

Diagnostics ◽

10.3390/diagnostics11060964 ◽

2021 ◽

Vol 11 (6) ◽

pp. 964

Author(s):

Sarka Benesova ◽

Mikael Kubista ◽

Lukas Valihrach

Keyword(s):

Rna Sequencing ◽

Small Rna ◽

High Sensitivity ◽

Small Rna Sequencing ◽

Rna Seq ◽

Liquid Biopsies ◽

Comprehensive Overview ◽

Rna Molecules ◽

Novel Mirna ◽

The Many

MicroRNAs (miRNAs) are a class of small RNA molecules that have an important regulatory role in multiple physiological and pathological processes. Their disease-specific profiles and presence in biofluids are properties that enable miRNAs to be employed as non-invasive biomarkers. In the past decades, several methods have been developed for miRNA analysis, including small RNA sequencing (RNA-seq). Small RNA-seq enables genome-wide profiling and analysis of known, as well as novel, miRNA variants. Moreover, its high sensitivity allows for profiling of low input samples such as liquid biopsies, which have now found applications in diagnostics and prognostics. Still, due to technical bias and the limited ability to capture the true miRNA representation, its potential remains unfulfilled. The introduction of many new small RNA-seq approaches that tried to minimize this bias, has led to the existence of the many small RNA-seq protocols seen today. Here, we review all current approaches to cDNA library construction used during the small RNA-seq workflow, with particular focus on their implementation in commercially available protocols. We provide an overview of each protocol and discuss their applicability. We also review recent benchmarking studies comparing each protocol’s performance and summarize the major conclusions that can be gathered from their usage. The result documents variable performance of the protocols and highlights their different applications in miRNA research. Taken together, our review provides a comprehensive overview of all the current small RNA-seq approaches, summarizes their strengths and weaknesses, and provides guidelines for their applications in miRNA research.

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