Oxidative Medicine and Cellular Longevity Changes in the Small RNA Expression in Endothelial Cells in Response to Inflammatory Stimulation

Objective. Endothelial cell inflammation is a common pathophysiological process in many cardiovascular and cerebrovascular diseases. Small RNA is a kind of short nonprotein coding RNA molecule. Changes in the small RNA expression in endothelial cells have been linked to the development of cardiovascular and cerebrovascular diseases. We investigated and verified differentially expressed small RNAs in endothelial cells in response to inflammatory stimulation. Methods. Primary rat endothelial cells were obtained from Sprague-Dawley rats and treated with 10 ng/ml TNF-α for 24 hours. Small RNA sequencing was used to generate extensive small RNA data. Significantly differentially expressed small RNAs identified in the analysis were further confirmed by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Then, we investigated the tissue-specific small RNA expression after RNA extraction from different tissues. Results. Small RNA sequencing demonstrated that 17 miRNAs, 1 piRNA, 10 snoRNAs, and 7 snRNAs were significantly differentially expressed. qRT-PCR identified 3 miRNAs, 2 snoRNAs, and 2 snRNAs with significantly different expression. Analysis of the tissue-specific expression showed that rno-miR-126a-5p was predominantly expressed in the lung, rno-miR-146a-5p in the intestines, and rno-novel-178 in the heart. Rno-piR-017330 was mainly expressed in the muscle. snoR-8966.1 was predominantly expressed in the bone. snoR-6253.1 was mostly expressed in the vessels and bone. snR-29469.1 was mainly expressed in the bone, and snR-85806.1 was predominantly expressed in the vessels and bone. Conclusions. We report for the first time the expression of small RNAs in endothelial cells under inflammatory conditions. TNF-α can regulate the expression of small RNAs in endothelial cells, and their expression is tissue-specific.

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Small RNA sequencing evaluation of renal microRNA biomarkers in dogs with X-linked hereditary nephropathy

Scientific Reports ◽

10.1038/s41598-021-96870-y ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Candice P. Chu ◽

Shiguang Liu ◽

Wenping Song ◽

Ethan Y. Xu ◽

Mary B. Nabity

Keyword(s):

Small Rna ◽

Target Genes ◽

Differentially Expressed ◽

Small Rna Sequencing ◽

Rna Seq ◽

Ckd Progression ◽

Critical Pathways ◽

Qrt Pcr ◽

Hereditary Nephropathy ◽

Differentially Expressed Mirnas

AbstractDogs with X-linked hereditary nephropathy (XLHN) are an animal model for Alport syndrome in humans and progressive chronic kidney disease (CKD). Using mRNA sequencing (mRNA-seq), we have characterized the gene expression profile affecting the progression of XLHN; however, the microRNA (miRNA, miR) expression remains unknown. With small RNA-seq and quantitative RT-PCR (qRT-PCR), we used 3 small RNA-seq analysis tools (QIAGEN OmicSoft Studio, miRDeep2, and CPSS 2.0) to profile differentially expressed renal miRNAs, top-ranked miRNA target genes, and enriched biological processes and pathways in CKD progression. Twenty-three kidney biopsies were collected from 5 dogs with XLHN and 4 age-matched, unaffected littermates at 3 clinical time points (T1: onset of proteinuria, T2: onset of azotemia, and T3: advanced azotemia). We identified up to 23 differentially expressed miRNAs at each clinical time point. Five miRNAs (miR-21, miR-146b, miR-802, miR-142, miR-147) were consistently upregulated in affected dogs. We identified miR-186 and miR-26b as effective reference miRNAs for qRT-PCR. This study applied small RNA-seq to identify differentially expressed miRNAs that might regulate critical pathways contributing to CKD progression in dogs with XLHN.

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MicroRNA changes in the mouse placenta due to bisphenol A exposure

Epigenomics ◽

10.2217/epi-2021-0339 ◽

2021 ◽

Author(s):

Jiude Mao ◽

Jessica A Kinkade ◽

Nathan J Bivens ◽

Cheryl S Rosenfeld

Keyword(s):

Bisphenol A ◽

Small Rna ◽

Small Rnas ◽

Fetal Brain ◽

Small Rna Sequencing ◽

Rna Expression ◽

Pathway Enrichment ◽

Target Mrnas ◽

Mouse Placenta ◽

Fetal Brain Development

The aim of this study was to determine small RNA expression changes in mouse placenta induced by bisphenol A (BPA) exposure. The methods included exposing female mice to BPA two weeks prior to conception through gestational day 12.5; whereupon fetal placentas were collected, frozen in liquid nitrogen and stored at -80°C. Small RNAs were isolated and used for small RNA-sequencing. The results showed that 43 small RNAs were differentially expressed. Target mRNAs were closely aligned to those expressed by thymus and brain, and pathway enrichment analyses indicated that such target mRNAs regulate neurogenesis and associated neurodevelopment processes. The major conclusions are that BPA induces several small RNAs in mouse placenta that might provide biomarkers for BPA exposure. Further, the placenta might affect fetal brain development through the secretion of miRs.

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Effect of the Diabetic Environment On the Expression of MiRNAs in Endothelial Cells: Mir-149-5p Restoration Ameliorates the High Glucose-Induced Expression of TNF-α and ER Stress Markers

Cellular Physiology and Biochemistry ◽

10.1159/000480330 ◽

2017 ◽

Vol 43 (1) ◽

pp. 120-135 ◽

Cited By ~ 14

Author(s):

Jing Yuan ◽

Minghao Chen ◽

Qingyun Xu ◽

Jianxin Liang ◽

Ruixue Chen ◽

...

Keyword(s):

Nitric Oxide ◽

Endothelial Cells ◽

Vascular Injury ◽

High Glucose ◽

Target Genes ◽

Differentially Expressed ◽

Qrt Pcr ◽

Tnf Α ◽

Mrna And Protein Expression

Background/Aims: This study aimed to screen microRNAs and their corresponding target genes that are associated with vascular injury in type two diabetes mellitus (T2DM), investigate the effects of differentially expressed miRNAs and their target genes on high glucose-induced vascular injury and establish the mechanism underlying these effects. Methods: A high-throughput digital gene expression (DGE) sequencing was performed to sequence microRNAs (miRNAs) and messenger RNAs (mRNAs) and determine their differential expression in human umbilical vein endothelial cells (HUVECs) incubated with serum samples from patients with T2DM and healthy volunteers. The HUVECs were transfected with si-TNF-α (tumor necrosis factor α) and a miR-149-5p inhibitor or mimic in vitro and then treated with normal or high glucose. The relative content of nitric oxide (NO) in the cells was detected using the Griess Reagent System. The mRNA and protein expression of endothelial nitric oxide synthase (eNOS) were determined by qRT-PCR and Western blotting. The content of endothelin-1 (ET-1), von Willebrand factor (vWF), and intercellular adhesion molecular-1 (ICAM-1) were detected using an enzyme-linked immunosorbent assay (ELISA) kit. Apoptosis was determined by flow cytometry using the Annexin V/PI apoptosis detection kit. The mRNA and protein expression levels of ER stress (ERS) markers were determined by qRT-PCR and Western blotting. Results: Based on the high-energy sequencing and in vitro pre-experiment studies, we determined that miR-149-5p and TNF-α were a differentially expressed mRNA/miRNA pair in T2DM with vascular injury. The luciferase reporter assay results demonstrated that miR-149-5p could directly target TNF-α. The upregulation of miR-149-5p reduced the high glucose-induced dysfunction in the HUVECs by significantly decreasing the levels of ET-1, vWF, and ICAM-1 and increasing the level of NO and the expression of eNOS. Additionally, we found that miR-149-5p can improve cell injury and reduce apoptosis by restoring the ameliorated high glucose-induced expression of ERS markers. Conclusion: TNF-α and miR-149-5p were differentially expressed in T2DM vascular endothelial injury. The over-expression of miR-149-5p ameliorates the high glucose-induced injury in the HUVECs by regulating the expression of TNF-α and ERS markers.

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Finding differentially expressed sRNA-Seq regions with srnadiff

PLoS ONE ◽

10.1371/journal.pone.0256196 ◽

2021 ◽

Vol 16 (8) ◽

pp. e0256196

Author(s):

Matthias Zytnicki ◽

Ignacio González

Keyword(s):

Gene Regulation ◽

Small Rna ◽

Small Rnas ◽

Differentially Expressed ◽

External Information ◽

Small Rna Sequencing ◽

Small Interfering Rnas ◽

Genomic Annotation ◽

Wide Range ◽

Precursor Rna

Small RNAs (sRNAs) encompass a great variety of molecules of different kinds, such as microRNAs, small interfering RNAs, Piwi-associated RNA, among others. These sRNAs have a wide range of activities, which include gene regulation, protection against virus, transposable element silencing, and have been identified as a key actor in determining the development of the cell. Small RNA sequencing is thus routinely used to assess the expression of the diversity of sRNAs, usually in the context of differentially expression, where two conditions are compared. Tools that detect differentially expressed microRNAs are numerous, because microRNAs are well documented, and the associated genes are well defined. However, tools are lacking to detect other types of sRNAs, which are less studied, and whose precursor RNA is not well characterized. We present here a new method, called srnadiff, which finds all kinds of differentially expressed sRNAs. To the extent of our knowledge, srnadiff is the first tool that detects differentially expressed sRNAs without the use of external information, such as genomic annotation or additional sequences of sRNAs.

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Small Non-coding RNAome of ageing chondrocytes

10.1101/2020.06.17.156927 ◽

2020 ◽

Author(s):

Panagiotis Balaskas ◽

Jonathan A. Green ◽

Tariq M. Haqqi ◽

Philip Dyer ◽

Yalda A. Kharaz ◽

...

Keyword(s):

Rna Sequencing ◽

Small Rna ◽

Differentially Expressed ◽

Cartilage Tissue ◽

Small Rna Sequencing ◽

High Grade ◽

Human Cartilage ◽

Fragment Analysis ◽

Qrt Pcr ◽

Non Coding Rnas

ABSTRACTBackgroundAgeing is one of the leading risk factors predisposing cartilage to musculoskeletal diseases, including osteoarthritis. Cumulative evidence suggests that small non-coding RNAs play a role in cartilage-related pathological changes. However, little research has been conducted on the effect of ageing on the expression of small non-coding RNAs in cartilage. By using small RNA sequencing, we investigated changes in the expression of small non-coding RNAs between young and old equine chondrocytes.MethodsChondrocytes were extracted from five young (4±1 years) and five old (17.4±1.9 years) macroscopically normal equine metacarpophalangeal joints. Following RNA extraction cDNA libraries were prepared and subjected to small RNA sequencing using the Illumina MiSeq platform. Differential expression analysis was performed in R using package DESeq2. For tRNA fragment analysis, tRNA reads were aligned to horse tRNA sequences using Bowtie2 version 2.2.5. Selected microRNA and small nucleolar RNA findings were validated using qRT-PCR in an extended cohort of equine chondrocytes. tRNA fragments were further investigated in low and high grade OA human cartilage tissue.ResultsIn total, 83 sncRNAs were differentially expressed between young and old equine chondrocytes, including microRNAs, snoRNAs, snRNAs and tRNAs. Of these, 34 were expressed higher and 49 were expressed lower in old chondrocytes compared to young. qRT-PCR analysis confirmed findings in an extended cohort of equine chondrocytes. Ingenuity Pathway Analysis of differentially expressed microRNAs and their predicted target genes linked them to cartilage and OA-related pathways and diseases. tRNA fragment analysis revealed that tiRNA-5035-GluCTC and tiRNA-5031-GluCTC-1 were reduced in both high grade OA human cartilage and old equine chondrocytes.ConclusionFor the first time, we have measured the effect of ageing on the expression of small non-coding RNAs in equine chondrocytes. Changes were detected in a number of different sncRNA species, including microRNAs, small nucleolar RNAs and tRNA fragments. This study supports a role for small non-coding RNAs in ageing cartilage and their potential involvement in age-related cartilage diseases.

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Seminal Plasma piRNA Array Analysis Identifies Possible Biomarker piRNAs for the Diagnosis of Asthenozoospermia

10.21203/rs.3.rs-149605/v1 ◽

2021 ◽

Author(s):

ling he ◽

Xing wu ◽

rong wu ◽

ping guo ◽

Wan sun ◽

...

Keyword(s):

Logistic Regression ◽

Small Rna ◽

Metal Ion ◽

Roc Analysis ◽

Characteristic Curve ◽

Differentially Expressed ◽

Small Rna Sequencing ◽

Bioinformatics Analyses ◽

Diagnostic Efficacy ◽

Qrt Pcr

Abstract Asthenozoospermia (AZS) is characterized by reduced sperm motility, and its pathogenesis remains poorly understood. Piwi-interacting RNAs (piRNAs) have been recognized to play important roles in spermatogenesis. However, little is known about the correlation of piRNAs with AZS. In this study, small RNA sequencing was performed on samples from AZS patients and fertile controls. Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to validate the small RNA-seq results. Bioinformatics analyses were performed to predict the functions of differentially expressed piRNAs. Logistic regression models were constructed, and receiver operating characteristic curve (ROC) analysis was used to evaluate their diagnostic performance. A total of 114 upregulated and 169 downregulated piRNAs were detected in AZS patients. GO and KEGG analyses showed that the differentially expressed piRNAs were mainly associated with transcription, signal transduction, cell differentiation, metal ion binding and focal adhesion. These results were verified by qRT-PCR of 8 selected piRNAs. Among the differentially expressed piRNAs tested, piR-hsa-32694, piR-hsa-26591, piR-hsa-18725, piR-hsa-18586 and piR-hsa-2912 produced qRT-PCR results consistent with the sequencing results in AZS compared with controls in the first cohort, whereas the other three genes did not show significant differences in expression. piR-hsa-32694, piR-hsa-26591, piR-hsa-18725, and piR-hsa-18586 were significantly upregulated in AZS. The diagnostic power of the four piRNAs was further analysed using ROC analysis; piR-hsa-26591 exhibited an area under the ROC curve (AUC) of 0.913 (95% CI: 0.795-0.994). Logistic regression modelling and subsequent ROC analysis indicated that the combination of the 4 piRNAs reached good diagnostic efficacy (AUC: 0.935).

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Identification and validation of miRNA reference genes in poplar under pathogen stress

10.21203/rs.3.rs-225734/v1 ◽

2021 ◽

Author(s):

Lichun Zhang ◽

Xiaoqian Yang ◽

Yiyi Yin ◽

Jinxing Wang ◽

Yanwei Wang

Keyword(s):

Gene Expression ◽

Reference Gene ◽

Small Rna ◽

Reference Genes ◽

Expression Profiles ◽

Small Rna Sequencing ◽

Rna Seq ◽

Internal Standards ◽

Qrt Pcr ◽

Canker Pathogen

Abstract Quantitative real time polymerase chain reaction (qRT-PCR) is a common method to analyze gene expression. Due to differences in RNA quantity, quality, and reverse transcription efficiency between qRT-PCR samples, reference genes are used as internal standards to normalize gene expression. However, few universal genes especially miRNAs have been identified as reference so far. Therefore, it is essential to identify reference genes that can be used across various experimental conditions, stress treatments, or tissues. In this study, 14 microRNAs (miRNAs) and 5.8S rRNA were assessed for expression stability in poplar trees infected with canker pathogen. Using three reference gene analysis programs, we found that miR156g and miR156a exhibited stable expression throughout the infection process. miR156g and miR156a were then tested as internal standards to measure the expression of miR1447 and miR171c, and the results were compared to small RNA sequencing (RNA-seq) data. We found that when miR156a was used as the reference gene, the expression of miR1447 and miR171c were consistent with the small RNA-seq expression profiles. Therefore, miR156a was the most stable miRNAs examined in this study, and could be used as a reference gene in poplar under canker pathogen stress, which should enable comprehensive comparisons of miRNAs expression and avoid the bias caused by different lenth between detected miRNAs and traditional referece genes. The present study has expanded the miRNA reference genes available for gene expression studies in trees under biotic stress.

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Altered Fecal Small RNA Profiles in Colorectal Cancer Reflect Gut Microbiome Composition in Stool Samples

mSystems ◽

10.1128/msystems.00289-19 ◽

2019 ◽

Vol 4 (5) ◽

Cited By ~ 9

Author(s):

Sonia Tarallo ◽

Giulio Ferrero ◽

Gaetano Gallo ◽

Antonio Francavilla ◽

Giuseppe Clerico ◽

...

Keyword(s):

Colorectal Cancer ◽

Gut Microbiota ◽

Small Rna ◽

Gut Microbiome ◽

Small Rnas ◽

Area Under The Curve ◽

Small Rna Sequencing ◽

Predictive Tool ◽

Cross Sectional ◽

Stool Samples

ABSTRACT Dysbiotic configurations of the human gut microbiota have been linked to colorectal cancer (CRC). Human small noncoding RNAs are also implicated in CRC, and recent findings suggest that their release in the gut lumen contributes to shape the gut microbiota. Bacterial small RNAs (bsRNAs) may also play a role in carcinogenesis, but their role has been less extensively explored. Here, we performed small RNA and shotgun sequencing on 80 stool specimens from patients with CRC or with adenomas and from healthy subjects collected in a cross-sectional study to evaluate their combined use as a predictive tool for disease detection. We observed considerable overlap and a correlation between metagenomic and bsRNA quantitative taxonomic profiles obtained from the two approaches. We identified a combined predictive signature composed of 32 features from human and microbial small RNAs and DNA-based microbiome able to accurately classify CRC samples separately from healthy and adenoma samples (area under the curve [AUC] = 0.87). In the present study, we report evidence that host-microbiome dysbiosis in CRC can also be observed by examination of altered small RNA stool profiles. Integrated analyses of the microbiome and small RNAs in the human stool may provide insights for designing more-accurate tools for diagnostic purposes. IMPORTANCE The characteristics of microbial small RNA transcription are largely unknown, while it is of primary importance for a better identification of molecules with functional activities in the gut niche under both healthy and disease conditions. By performing combined analyses of metagenomic and small RNA sequencing (sRNA-Seq) data, we characterized both the human and microbial small RNA contents of stool samples from healthy individuals and from patients with colorectal carcinoma or adenoma. With the integrative analyses of metagenomic and sRNA-Seq data, we identified a human and microbial small RNA signature which can be used to improve diagnosis of the disease. Our analysis of human and gut microbiome small RNA expression is relevant to generation of the first hypotheses about the potential molecular interactions occurring in the gut of CRC patients, and it can be the basis for further mechanistic studies and clinical tests.

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Small RNAs in parasitic nematodes – forms and functions

Parasitology ◽

10.1017/s0031182019001689 ◽

2019 ◽

Vol 147 (8) ◽

pp. 855-864

Author(s):

Collette Britton ◽

Roz Laing ◽

Eileen Devaney

Keyword(s):

Gene Expression ◽

Small Rna ◽

Small Rnas ◽

Target Genes ◽

Parasitic Nematodes ◽

Small Rna Sequencing ◽

Small Interfering Rnas ◽

Rna Sequences ◽

C Elegans

AbstractSmall RNAs are important regulators of gene expression. They were first identified in Caenorhabditis elegans, but it is now apparent that the main small RNA silencing pathways are functionally conserved across diverse organisms. Availability of genome data for an increasing number of parasitic nematodes has enabled bioinformatic identification of small RNA sequences. Expression of these in different lifecycle stages is revealed by small RNA sequencing and microarray analysis. In this review we describe what is known of the three main small RNA classes in parasitic nematodes – microRNAs (miRNAs), Piwi-interacting RNAs (piRNAs) and small interfering RNAs (siRNAs) – and their proposed functions. miRNAs regulate development in C. elegans and the temporal expression of parasitic nematode miRNAs suggest modulation of target gene levels as parasites develop within the host. miRNAs are also present in extracellular vesicles released by nematodes in vitro, and in plasma from infected hosts, suggesting potential regulation of host gene expression. Roles of piRNAs and siRNAs in suppressing target genes, including transposable elements, are also reviewed. Recent successes in RNAi-mediated gene silencing, and application of small RNA inhibitors and mimics will continue to advance understanding of small RNA functions within the parasite and at the host–parasite interface.

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