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

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

Oxidative Medicine and Cellular Longevity ◽

10.1155/2021/8845520 ◽

2021 ◽

Vol 2021 ◽

pp. 1-9

Author(s):

Peixi Liu ◽

Liuxun Hu ◽

Yuan Shi ◽

Yingjun Liu ◽

Guo Yu ◽

...

Keyword(s):

Endothelial Cells ◽

Small Rna ◽

Small Rnas ◽

Cerebrovascular Diseases ◽

Differentially Expressed ◽

Small Rna Sequencing ◽

Rna Expression ◽

Tissue Specific ◽

Qrt Pcr ◽

Tnf Α

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 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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Genome-Wide Analysis of mRNA and Long Noncoding RNA Profiles in Chronic Actinic Dermatitis

BioMed Research International ◽

10.1155/2017/7479523 ◽

2017 ◽

Vol 2017 ◽

pp. 1-15 ◽

Cited By ~ 2

Author(s):

Dongyun Lei ◽

Lechun Lv ◽

Li Yang ◽

Wenjuan Wu ◽

Yong Liu ◽

...

Keyword(s):

Noncoding Rna ◽

Differentially Expressed ◽

Pathogenetic Mechanism ◽

Bioinformatics Analyses ◽

Inflammatory Lesions ◽

Qrt Pcr ◽

Exposed Skin ◽

Genome Wide ◽

Chronic Actinic Dermatitis ◽

Solid Foundation

Chronic actinic dermatitis (CAD), a photosensitive dermatosis, is characterized by inflammatory lesions, especially on sun-exposed skin. However, its pathogenesis remains unclear. In this study, second-generation RNA sequencing and comprehensive bioinformatics analyses of mRNAs and long noncoding RNAs (lncRNAs) were performed to determine the transcriptome profiles of patients with CAD. A total 6889 annotated lncRNAs, 341 novel lncRNAs, and 65091 mRNAs were identified. Interestingly, patients with CAD and healthy controls showed distinct transcriptome profiles. Indeed, 198 annotated (81.48%) and 45 novel (18.52%) lncRNAs were differentially expressed between the two groups. GO, KEGG, and RGSEA analyses of lncRNAs showed that inflammatory and immune response related pathways played crucial roles in the pathogenetic mechanism of CAD. In addition, we unveiled key differentially expressed lncRNAs, including lncRNA RP11-356I2.4 which plays a role probably by regulating TNFAIP3 and inflammation. qRT-PCR data validated the differentially expressed genes. The newly identified lncRNAs may have potential roles in the development of CAD; these findings lay a solid foundation for subsequent functional exploration of lncRNAs and mRNAs as therapeutic targets for CAD.

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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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Profiling of Differentially Expressed MicroRNAs in Saliva of Parkinson's Disease Patients

Frontiers in Neurology ◽

10.3389/fneur.2021.738530 ◽

2021 ◽

Vol 12 ◽

Author(s):

Yanyan Jiang ◽

Jing Chen ◽

Yunchuang Sun ◽

Fan Li ◽

Luhua Wei ◽

...

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Microarray Analysis ◽

Target Genes ◽

Characteristic Curve ◽

Differentially Expressed ◽

Diagnostic Efficacy ◽

Mirna Microarray ◽

Candidate Mirnas ◽

Diagnostic Potential

Objective: This study aims to identify differentially expressed salivary miRNAs and validate the diagnostic potential for idiopathic Parkinson's disease (PD). Also, the disease specificity of candidate miRNAs was evaluated between PD, multiple system atrophy (MSA), and essential tremor (ET).Methods: We collected salivary samples from 50 PD, 20 ET, and 20 MSA patients, as well as 30 healthy controls (HCs). In the discovery phase, salivary miRNA microarray analysis was performed. In-silico analysis was used to investigate the target genes of differentially expressed miRNAs and clustered pathways. In validation phase, RT-qPCR was performed with samples from 30 PD patients and 30 HCs. Subsequently, we investigated candidate miRNAs in all recruited subjects. Receiver operating characteristic curve and Spearman correlation analysis was performed to determine diagnostic usefulness.Results: We identified 43 miRNAs that were differentially expressed between 5 PD patients and 5 HCs by miRNA microarray analysis. Computational analysis revealed the target genes were clustered in the pathways associated with ubiquitin protein ligase activity. The result of RT-qPCR showed that the miR-29a-3p and miR-29c-3p were found to be significantly downregulated (p = 0.004, p = 0.027), whereas the miR-6756-5p was significantly upregulated in 30 PD patients compared with 30 HCs (p = 0.032). The miR-29a-3p expression level in PD patients was significantly lower than ET patients (p = 0.035), but higher than MSA patients (p < 0.0001). The diagnostic efficacy reached a little higher when the combination of miR-29a-3p and miR-29c-3p.Conclusion: The miRNA combination of salivary miR-29a-3p and miR-29c-3p has potential to be a diagnostic biomarker for idiopathic PD.

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Evaluation of keratoconus progression

British Journal of Ophthalmology ◽

10.1136/bjophthalmol-2017-311651 ◽

2018 ◽

Vol 103 (4) ◽

pp. 551-557 ◽

Cited By ~ 6

Author(s):

Mehdi Shajari ◽

Gernot Steinwender ◽

Kim Herrmann ◽

Kate Barbara Kubiak ◽

Ivana Pavlovic ◽

...

Keyword(s):

Logistic Regression ◽

Roc Analysis ◽

Operating Characteristic ◽

Characteristic Curve ◽

Youden Index ◽

Back Surface ◽

Corneal Surface ◽

Maximum Elevation ◽

Index Index ◽

Variance Index

AimTo define variables for the evaluation of keratoconus progression and to determine cut-off values.MethodsIn this retrospective cohort study (2010–2016), 265 eyes of 165 patients diagnosed with keratoconus underwent two Scheimpflug measurements (Pentacam) that took place 1 year apart ±3 months. Variables used for keratoconus detection were evaluated for progression and a correlation analysis was performed. By logistic regression analysis, a keratoconus progression index (KPI) was defined. Receiver-operating characteristic curve (ROC) analysis was performed and Youden Index calculated to determine cut-off values.ResultsVariables used for keratoconus detection showed a weak correlation with each other (eg, correlation r=0.245 between RPImin and Kmax, p<0.001). Therefore, we used parameters that took several variables into consideration (eg, D-index, index of surface variance, index for height asymmetry, KPI). KPI was defined by logistic regression and consisted of a Pachymin coefficient of −0.78 (p=0.001), a maximum elevation of back surface coefficient of 0.27 and coefficient of corneal curvature at the zone 3 mm away from the thinnest point on the posterior corneal surface of −12.44 (both p<0.001). The two variables with the highest Youden Index in the ROC analysis were D-index and KPI: D-index had a cut-off of 0.4175 (70.6% sensitivity) and Youden Index of 0.606. Cut-off for KPI was −0.78196 (84.7% sensitivity) and a Youden Index of 0.747; both 90% specificity.ConclusionsKeratoconus progression should be defined by evaluating parameters that consider several corneal changes; we suggest D-index and KPI to detect progression.

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Small RNA Sequencing Uncovers New miRNAs and moRNAs Differentially Expressed in Normal and Primary Myelofibrosis CD34+ Cells

PLoS ONE ◽

10.1371/journal.pone.0140445 ◽

2015 ◽

Vol 10 (10) ◽

pp. e0140445 ◽

Cited By ~ 17

Author(s):

Paola Guglielmelli ◽

Andrea Bisognin ◽

Claudia Saccoman ◽

Carmela Mannarelli ◽

Alessandro Coppe ◽

...

Keyword(s):

Rna Sequencing ◽

Small Rna ◽

Primary Myelofibrosis ◽

Differentially Expressed ◽

Small Rna Sequencing ◽

Cd34 Cells

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Small RNA Sequencing Reveals Differentially Expressed miRNAs in Necrotizing Enterocolitis in Rats

BioMed Research International ◽

10.1155/2020/5150869 ◽

2020 ◽

Vol 2020 ◽

pp. 1-9

Author(s):

Ren-qiang Yu ◽

Min Wang ◽

Shan-yu Jiang ◽

Ying-hui Zhang ◽

Xiao-yu Zhou ◽

...

Keyword(s):

Wnt Signaling ◽

Signaling Pathway ◽

Rna Sequencing ◽

Necrotizing Enterocolitis ◽

Small Rna ◽

Mirna Expression ◽

Expression Patterns ◽

Wnt Signaling Pathway ◽

Differentially Expressed ◽

Small Rna Sequencing

Necrotizing enterocolitis (NEC) is the leading cause of death due to gastrointestinal disease in preterm infants. The role of miRNAs in NEC is still unknown. The objective of this study was to identify differentially expressed (DE) miRNAs in rats with NEC and analyze their possible roles. In this study, a NEC rat model was established using Sprague-Dawley rat pups. Small RNA sequencing was used to analyze the miRNA expression profiles in the NEC and control rats. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were carried out to identify target mRNAs for the DE miRNAs and to explore their potential roles. The DE miRNAs were verified by real-time quantitative PCR (RT-qPCR). The status of intestinal injury and the elevated levels of inflammatory cytokines in the NEC group confirmed that the NEC model was successfully established. The 16 miRNAs were found to be differentially expressed between the NEC group and the control group of rats. Bioinformatics analysis indicated that the parental genes of the DE miRNAs were predominantly implicated in the phosphorylation, cell migration, and protein phosphorylation processes. Moreover, the DE miRNAs were mainly found to be involved in the pathways of axon guidance, endocytosis, and focal adhesion, as well as in the Wnt signaling pathway, which is related to colitis. The expression patterns of the candidate miRNAs (rno-miR-27a-5p and rno-miR-187-3p), as assessed by RT-qPCR, were in accordance with the expression patterns obtained by miRNA-sequencing. The miRNA/mRNA/pathway network revealed that rno-miR-27a-5p and rno-miR-187-3p might be involved in NEC via the Wnt signaling pathway. We found an altered miRNA expression pattern in rats with NEC. We hypothesize that rno-miR-27a-5p and rno-miR-187-3p might mediate the NEC pathophysiological processes via the Wnt signaling pathway.

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