Small RNA Sequencing Data Research Articles

A microRNA Transcriptome-wide Association Study of Prostate Cancer Risk.

Large genome-wide association studies have identified hundreds of single-nucleotide polymorphisms associated with increased risk of prostate cancer (PrCa), and many of these risk loci is presumed to confer regulatory effects on gene expression. While eQTL studies of long RNAs has yielded many potential risk genes, the relationship between PrCa risk genetics and microRNA expression dysregulation is understudied. We performed an microRNA transcriptome-wide association study of PrCa risk using small RNA sequencing and genome-wide genotyping data from N = 441 normal prostate epithelium tissue samples along with N = 411 prostate adenocarcinoma tumor samples from the Cancer Genome Atlas (TCGA). Genetically regulated expression prediction models were trained for all expressed microRNAs using the FUSION TWAS software. TWAS for PrCa risk was performed with both sets of models using single-SNP summary statistics from the recent PRACTICAL consortium PrCa case-control OncoArray GWAS meta-analysis. A total of 613 and 571 distinct expressed microRNAs were identified in the normal and tumor tissue datasets, respectively (overlap: 480). Among these, 79 (13%) normal tissue microRNAs demonstrated significant cis-heritability (median cis-h2 = 0.15, range: 0.03–0.79) for model training. Similar results were obtained from TCGA tumor samples, with 48 (9%) microRNA expression models successfully trained (median cis-h2 = 0.14, range: 0.06–0.60). Using normal tissue models, we identified two significant TWAS microRNA associations with PrCa risk: over-expression of mir-941 family microRNAs (PTWAS = 2.9E-04) and reduced expression of miR-3617-5p (PTWAS = 1.0E-03). The TCGA tumor TWAS also identified a significant association with miR-941 overexpression (PTWAS = 9.7E-04). Subsequent finemapping of the TWAS results using a multi-tissue database indicated limited evidence of causal status for each microRNA with PrCa risk (posterior inclusion probabilities <0.05). Future work will examine downstream regulatory effects of microRNA dysregulation as well as microRNA-mediated risk mechanisms via competing endogenous RNA relationships.

Competing endogenous RNA-networks reveal key regulatory microRNAs involved in the response of Atlantic salmon to a novel orthomyxovirus

One of the most intriguing discoveries of the genomic era is that only a small fraction of the genome is dedicated to protein coding. The remaining fraction of the genome contains, amongst other elements, a number of non-coding transcripts that regulate the transcription of protein coding genes. Here we used transcriptome sequencing data to explore these gene regulatory networks using RNA derived from gill tissue of Atlantic salmon (Salmo salar) infected with Pilchard orthomyxovirus (POMV), but showing no clinical signs of disease. We examined fish sampled early during the challenge trial (8–12 days after infection) to uncover potential biomarkers of early infection and innate immunity, and fish sampled late during the challenge trial (19 dpi) to elucidate potential markers of resistance to POMV. We analysed total RNA-sequencing data to find differentially expressed messenger RNAs (mRNA) and identify new long-noncoding RNAs (lncRNAs). We also evaluated small RNA sequencing data to find differentially transcribed microRNAs (miRNAs) and explore their role in gene regulatory networks. Whole-genome expression data (both coding and non-coding transcripts) were used to explore the crosstalk between RNA molecules by constructing competing endogenous RNA networks (ceRNA). The teleost specific miR-462/miR-731 cluster was strongly induced in POMV infected fish and deemed a potential biomarker of early infection. Gene networks also identified a selenoprotein (selja), downregulated in fish sampled late during the challenge, which may be associated to viral clearance and the return to homeostasis after infection. This study provides the basis for further investigations using molecular tools to overexpress or inhibit miRNAs to confirm the functional impact of the interactions presented here on gene expression and their potential application at commercial level.

A comparative analysis of single cell small RNA sequencing data reveals heterogeneous isomiR expression and regulation

MicroRNAs (miRNAs) are non-coding small RNAs which play a critical role in the regulation of gene expression in cells. It is known that miRNAs are often expressed as multiple isoforms, called isomiRs, which may have alternative regulatory functions. Despite the recent development of several single cell small RNA sequencing protocols, these methods have not been leveraged to investigate isomiR expression and regulation to better understand their role on a single cell level. Here we integrate sequencing data from three independent studies and find substantial differences in isomiR composition that suggest that cell autonomous mechanisms may drive isomiR processing. We also find evidence of altered regulatory functions of different classes of isomiRs, when compared to their respective wild-type miRNA, which supports a biological role for many of the isomiRs that are expressed.

New Tricks with Old Dogs: Computational Identification and Experimental Validation of New miRNA-mRNA Regulation in hiPSC-CMs.

Cardiovascular disease is still the leading cause of morbidity and mortality worldwide. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have become a valuable widespread in vitro model to study cardiac disease. Herein, we employ the hiPSC-CM model to identify novel miRNA–mRNA interaction partners during cardiac differentiation and β-adrenergic stress. Whole transcriptome and small RNA sequencing data were combined to identify novel miRNA–mRNA interactions. Briefly, mRNA and miRNA expression profiles were integrated with miRNA target predictions to identify significant statistical dependencies between a miRNA and its candidate target set. We show by experimental validation that our approach discriminates true from false miRNA target predictions. Thereby, we identified several differentially expressed miRNAs and focused on the two top candidates: miR-99a-5p in the context of cardiac differentiation and miR-212-3p in the context of β-adrenergic stress. We validated some target mRNA candidates by 3′UTR luciferase assays as well as in transfection experiments in the hiPSC-CM model system. Our data show that iPSC-derived cardiomyocytes and computational modeling can be used to uncover new valid miRNA–mRNA interactions beyond current knowledge.

Genome-Wide Identification and Functional Characterization of Noncoding RNAs (ncRNAs) Differentially Expressed During Insect Development.

MicroRNAs (miRNAs) are important regulatory noncoding RNAs (ncRNAs) at the posttranscriptional level of gene expression. Linear long noncoding RNAs (lncRNAs) and circular RNAs (circRNAs) can function as competing endogenous RNAs (ceRNAs) of miRNAs and regulate the expression of protein-coding genes. This chapter presents a procedure for the bioinformatic analysis of these three ncRNAs that are differentially expressed during insect development. In the first step, lncRNAs and circRNAs are identified based on RNA-sequencing data. In the second step, miRNAs are identified based on small RNA-sequencing data and combined with the two ncRNAs from the previous step for functional characterization.

Phylogenetic analysis and target gene prediction of miR477 gene family in grape

To understand the molecular characteristics of the miR477 gene family of grape (Vvi-miR477) and to predict its target genes, the Vvi-miR477 genes were identified from previous small RNA sequencing data, then phylogenetic analysis and prediction of target gene were conducted. The Vvi-miR477 family consists of two precursor sequences and three mature sequences. The miR477 family members were mostly 19-22nt in length. The sequence is relatively conservative. Vvi-MIR477a and Vvi-MIR477b are located on chromosomes 1 and 2, respectively. These precursor sequences can form the typical stable stem-loop structure. Their minimum folding free energy is −39.10 kcal/mol and −50.90 kcal/mol, respectively. The MIR477 family can be divided into three groups. The prediction of target genes showed that Vvi-miR477 targets 26S proteasome, DEAD-box, GRAS family protein, Protein Phosphatase 2C, etc. The GO function of target genes was mainly enriched to six categories. The catabolic process, carboxylic ester hydrolase activity is shown to be high. This study provided a theoretical basis for further exploration of the molecular mechanism of miR477 in grape berry ripening.

Maternal Serum miRNAs as Biomarkers for Abnormal Placentation

To identify maternal serum miRNAs that may be used as antenatal diagnostic markers of abnormal placentation. A multi-site cohort study of maternal serum collected between 14-41 weeks gestational age (GA) from patients with normal and abnormal placentation. Candidate miRNAs were chosen for validation based on analysis of small RNA sequencing data. Validation was performed using Taqman reverse transcription quantitative PCR (RT-qPCR) amplification of each candidate miRNA. Experiments were performed in quadruplicate and medians analyzed. Student’s T test and one-way ANOVA were used to compare means, Pearson’s chi-square test to analyze categorical variables, and linear regression modeling to calculate adjusted significance. Twenty-one candidate miRNAs were selected for RT-qPCR amplification on maternal serum samples from pregnancies with pathology-confirmed diagnosis of placenta accreta spectrum (n = 45), placenta previa (n = 31), and normal controls (n = 50). Multiparity (p < 0.001), race (p < 0.001), BMI (p < 0.001), placental laterality (p = 0.003), GA at blood draw (p < 0.001) were significantly different across the three groups. Analysis of the RT-qPCR Cq values identified miRNA 548av-3p as the strongest candidate to differentiate between accreta and previa (mean Cq 29.11 vs 28.655, p = 0.087). Postulating that bivariate biomarkers of paired miRNA would yield more robust results by providing an internal control, a significant difference was found in the Cq ratio of 548av-3p / 628-3p across the three final diagnoses (p = 0.004), and specifically between accreta vs previa (p = 0.024). After controlling for parity, race, BMI, placenta laterality, and GA at blood draw, the Cq ratio of mi-548av-3p / 628-3p remained a significant predictor for the final diagnosis (partial eta2 = 0.093, p = 0.001). Specific maternal serum miRNAs are differentially expressed among patients with abnormal placentation. These data suggest miRNA 548av-3p may be a potential non-invasive biomarker for antenatal risk assessment of abnormal placentation.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Investigation of the expression of P-element-induced wimpy testis-interacting RNAs in human acute myeloid leukemia

P-element induced wimpy testis (PIWI)-interacting RNAs (piRNAs) are a newly known class of small non-coding RNAs with 24–31 nucleotides in length. There are more than 20,000 piRNA genes in the human genome and some of them have a role in cancer. In this study, we investigated the expression of piRNAs in acute myeloid leukemia (AML). We used publicly available small RNA sequencing data derived from the peripheral blood of patients with AML and control samples to be re-analyzed for investigation of piRNA expression pattern and then was validated via the real-time polymerase chain reaction (PCR). Differential expression analysis showed that four upregulated piRNAs and six downregulated piRNAs in AML samples, among which, the piR-32,877 and piR-33,195 were the top two upregulated piRNAs. Real-time PCR was performed to validate the increased expression of these two piRNAs in AML compared to controls. Pathway analysis was also performed. The results provide evidence that piR-32,877 and piR-33,195 may serve as a new diagnostic and prognostic biomarker in AML.

Faecal miRNA profiles associated with age, sex, BMI, and lifestyle habits in healthy individuals

For their stability and detectability faecal microRNAs represent promising molecules with potential clinical interest as non-invasive diagnostic and prognostic biomarkers. However, there is no evidence on how stool miRNA profiles change according to an individual’s age, sex, and body mass index (BMI) or how lifestyle habits influence the expression levels of these molecules. We explored the relationship between the stool miRNA levels and common traits (sex, age, BMI, and menopausal status) or lifestyle habits (physical activity, smoking status, coffee, and alcohol consumption) as derived by a self-reported questionnaire, using small RNA-sequencing data of samples from 335 healthy subjects. We detected 151 differentially expressed miRNAs associated with one variable and 52 associated with at least two. Differences in miR-638 levels were associated with age, sex, BMI, and smoking status. The highest number of differentially expressed miRNAs was associated with BMI (n = 92) and smoking status (n = 84), with several miRNAs shared between them. Functional enrichment analyses revealed the involvement of the miRNA target genes in pathways coherent with the analysed variables. Our findings suggest that miRNA profiles in stool may reflect common traits and lifestyle habits and should be considered in relation to disease and association studies based on faecal miRNA expression.

Integrated Small RNA Sequencing, Transcriptome and GWAS Data Reveal microRNA Regulation in Response to Milk Protein Traits in Chinese Holstein Cattle.

Milk protein is one of the most important economic traits in the dairy industry. Yet, the regulatory network of miRNAs for the synthesis of milk protein in mammary is poorly understood. Samples from 12 Chinese Holstein cows with three high ( ≥ 3.5%) and three low ( ≤ 3.0%) phenotypic values for milk protein percentage in lactation and non-lactation were examined through deep small RNA sequencing. We characterized 388 known and 212 novel miRNAs in the mammary gland. Differentially expressed analysis detected 28 miRNAs in lactation and 52 miRNAs in the non-lactating period with a highly significant correlation with milk protein concentration. Target prediction and correlation analysis identified some key miRNAs and their targets potentially involved in the synthesis of milk protein. We analyzed for enrichments of GWAS signals in miRNAs and their correlated targets. Our results demonstrated that genomic regions harboring DE miRNA genes in lactation were significantly enriched with GWAS signals for milk protein percentage traits and that enrichments within DE miRNA targets were significantly higher than in random gene sets for the majority of milk production traits. This integrated study on the transcriptome and posttranscriptional regulatory profiles between significantly differential phenotypes of milk protein concentration provides new insights into the mechanism of milk protein synthesis, which should reveal the regulatory mechanisms of milk secretion.

Integrated analysis of dysregulated microRNA and mRNA expression in intestinal epithelial cells following ethanol intoxication and burn injury

Gut barrier dysfunction is often implicated in pathology following alcohol intoxication and burn injury. MicroRNAs (miRNAs) are negative regulators of gene expression that play a central role in gut homeostasis, although their role after alcohol and burn injury is poorly understood. We performed an integrated analysis of miRNA and RNA sequencing data to identify a network of interactions within small intestinal epithelial cells (IECs) which could promote gut barrier disruption. Mice were gavaged with ~ 2.9 g/kg ethanol and four hours later given a ~ 12.5% TBSA full thickness scald injury. One day later, IECs were harvested and total RNA extracted for RNA-seq and miRNA-seq. RNA sequencing showed 712 differentially expressed genes (DEGs) (padj < 0.05) in IECs following alcohol and burn injury. Furthermore, miRNA sequencing revealed 17 differentially expressed miRNAs (DEMs) (padj < 0.1). Utilizing the miRNet, miRDB and TargetScan databases, we identified both validated and predicted miRNA gene targets. Integration of small RNA sequencing data with mRNA sequencing results identified correlated changes in miRNA and target expression. Upregulated miRNAs were associated with decreased proliferation (miR-98-3p and miR-381-3p) and cellular adhesion (miR-29a-3p, miR-429-3p and miR3535), while downregulated miRNAs were connected to upregulation of apoptosis (Let-7d-5p and miR-130b-5p) and metabolism (miR-674-3p and miR-185-5p). Overall, these findings suggest that alcohol and burn injury significantly alters the mRNA and miRNA expression profile of IECs and reveals numerous miRNA–mRNA interactions that regulate critical pathways for gut barrier function after alcohol and burn injury.

Hsa-mir-548 family expression in human reproductive tissues

BackgroundHsa-miR-548ba expressed in ovarian granulosa cells targets PTEN and LIFR, which are essential for ovarian follicle activation and growth. The expression pattern of hsa-miR-548ba correlates with its host gene follicle-stimulating hormone receptor (FSHR), and FSH has a positive influence on hsa-miR-548ba expression. However, hsa-miR-548ba is a member of a large hsa-mir-548 family with potentially overlapping targets. The current study aims to investigate the co-expression of hsa-mir-548 family members in FSHR-positive reproductive tissues and to explore the potential co-regulation of pathways.ResultsFor the above-described analysis, small RNA sequencing data from public data repositories were used. Sequencing results revealed that hsa-miR-548ba was expressed at the highest level in the ovarian granulosa cells and uterine myometrial samples together with another twelve and one hsa-miR-548 family members, respectively. Pathway enrichment analysis of microRNA targets in the ovarian samples revealed the hsa-miR-548ba and hsa-miR-548b-5p co-regulation of RAB geranylgeranylation in mural granulosa cells. Moreover, other hsa-mir-548 family members co-regulate pathways essential for ovarian functions (PIP3 activates AKT signalling and signalling by ERBB4). In addition to hsa-miR-548ba, hsa-miR-548o-3p is expressed in the myometrium, which separately targets the peroxisome proliferator-activated receptor alpha (PPARA) pathway.ConclusionThis study reveals that hsa-mir-548 family members are expressed in variable combinations in the reproductive tract, where they potentially fulfil different regulatory roles. The results provide a reference for further studies of the hsa-mir-548 family role in the reproductive tract.

DBtRend: A Web-Server of tRNA Expression Profiles from Small RNA Sequencing Data in Humans

Transfer RNA (tRNA), a key component of the translation machinery, plays critical roles in stress conditions and various diseases. While knowledge regarding the importance of tRNA function is increasing, its biological roles are still not well understood. There is currently no comprehensive database or web server providing the expression landscape of tRNAs across a variety of human tissues and diseases. Here, we constructed a user-friendly and interactive database, DBtRend, which provides a profile of mature tRNA expression across various biological conditions by reanalyzing the small RNA or microRNA sequencing data from the Cancer Genome Atlas (TCGA) and NCBI’s Gene Expression Omnibus (GEO) in humans. Users can explore not only the expression values of mature individual tRNAs in the human genome, but also those of isodecoders and isoacceptors based on our specific pipelines. DBtRend provides the expressed patterns of tRNAs, the differentially expressed tRNAs in different biological conditions, and the information of samples or patients, tissue types, and molecular subtype of cancers. The database is expected to help researchers interested in functional discoveries of tRNAs.

Extracellular vesicles from adipose tissue-derived stem cells alleviate osteoporosis through osteoprotegerin and miR-21-5p.

Osteoporosis is one of the most common skeletal disorders caused by the imbalance between bone formation and resorption, resulting in quantitative loss of bone tissue. Since stem cell‐derived extracellular vesicles (EVs) are growing attention as novel cell‐free therapeutics that have advantages over parental stem cells, the therapeutic effects of EVs from adipose tissue‐derived stem cells (ASC‐EVs) on osteoporosis pathogenesis were investigated. ASC‐EVs were isolated by a multi‐filtration system based on the tangential flow filtration (TFF) system and characterized using transmission electron microscopy, dynamic light scattering, zeta potential, flow cytometry, cytokine arrays, and enzyme‐linked immunosorbent assay. EVs are rich in growth factors and cytokines related to bone metabolism and mesenchymal stem cell (MSC) migration. In particular, osteoprotegerin (OPG), a natural inhibitor of receptor activator of nuclear factor‐κB ligand (RANKL), was highly enriched in ASC‐EVs. We found that the intravenous administration of ASC‐EVs attenuated bone loss in osteoporosis mice. Also, ASC‐EVs significantly inhibited osteoclast differentiation of macrophages and promoted the migration of bone marrow‐derived MSCs (BM‐MSCs). However, OPG‐depleted ASC‐EVs did not show anti‐osteoclastogenesis effects, demonstrating that OPG is critical for the therapeutic effects of ASC‐EVs. Additionally, small RNA sequencing data were analysed to identify miRNA candidates related to anti‐osteoporosis effects. miR‐21‐5p in ASC‐EVs inhibited osteoclast differentiation through Acvr2a down‐regulation. Also, let‐7b‐5p in ASC‐EVs significantly reduced the expression of genes related to osteoclastogenesis. Finally, ASC‐EVs reached the bone tissue after they were injected intravenously, and they remained longer. OPG, miR‐21‐5p, and let‐7b‐5p in ASC‐EVs inhibit osteoclast differentiation and reduce gene expression related to bone resorption, suggesting that ASC‐EVs are highly promising as cell‐free therapeutic agents for osteoporosis treatment.

FP07.03 Diagnostic Potential of Novel Mesothelioma-Specific MicroRNAs

One of the main challenges in diagnosing malignant pleural mesothelioma (MPM) is the differential diagnosis between MPM and other cancers, especially lung adenocarcinoma, that have metastasized to the pleura. Tissue-specific biomarkers could overcome this issue, and in particular microRNAs have been suggested to be very useful in this context, with several studies suggesting that many tissue-specific microRNAs remain to be annotated. Recently, an in-depth analysis of the TCGA small-RNA sequencing data of 87 MPM tumours identified previously undetected microRNAs that could distinguish MPM from non-small cell lung cancer (Martinez VD et al, AJRCM 2019: 61(2)).

A New Specific and Sensitive RT-qPCR Method Based on Splinted 5' Ligation for the Quantitative Detection of RNA Species Shorter than microRNAs.

Recently, we discovered a new family of unusually short RNAs mapping to 5.8S ribosomal RNA (rRNA) and which we named dodecaRNAs (doRNAs), according to the number of core nucleotides (12 nt) their members contain. To confirm these small RNA-sequencing (RNA-Seq) data, validate the existence of the two overly abundant doRNAs—the minimal core 12-nt doRNA sequence and its + 1-nt variant bearing a 5′ Cytosine, C-doRNA—and streamline their analysis, we developed a new specific and sensitive splinted 5′ ligation reverse transcription (RT)-quantitative polymerase chain reaction (qPCR) method. This method is based on a splint-assisted ligation of an adapter to the 5′ end of doRNAs, followed by RT-qPCR amplification and quantitation. Our optimized protocol, which may discriminate between doRNA, C-doRNA, mutated and precursor sequences, can accurately detect as low as 240 copies and is quantitatively linear over a range of 7 logs. This method provides a unique tool to expand and facilitate studies exploring the molecular and cellular biology of RNA species shorter than microRNAs.

Regulation of retrotransposition in Arabidopsis.

Plant genomes are largely comprised of retrotransposons which can replicate through 'copy and paste' mechanisms. Long terminal repeat (LTR) retrotransposons are the major class of retrotransposons in plant species, and importantly they broadly affect the expression of nearby genes. Although most LTR retrotransposons are non-functional, active retrotranspositions have been reported in plant species or mutants under normal growth condition and environmental stresses. With the well-defined reference genome and numerous mutant alleles, Arabidopsis studies have significantly expanded our understanding of retrotransposon regulation. Active LTR retrotransposon loci produce virus-like particles to perform reverse transcription, and their complementary DNA can be inserted into new genomic loci. Due to the detrimental consequences of retrotransposition, plants like animals, have developed transcriptional and post-transcriptional silencing mechanisms. Recently several different genome-wide techniques have been developed to understand LTR retrotransposition in Arabidopsis and different plant species. Transposome, methylome, transcriptome, translatome and small RNA sequencing data have revealed how host silencing mechanisms can affect multiple steps of retrotransposition. These recent advances shed light on future mechanistic studies of retrotransposition as well as retrotransposon diversity.

Ancient viral integrations in marsupials: a potential antiviral defence.

Marsupial viruses are understudied compared to their eutherian mammal counterparts, although they may pose severe threats to vulnerable marsupial populations. Genomic viral integrations, termed ‘endogenous viral elements’ (EVEs), could protect the host from infection. It is widely known past viral infections and EVEs play an active role in antiviral defence in invertebrates and plants. This study aimed to characterise actively transcribed EVEs in Australian marsupial species, because they may play an integral role in cellular defence against viruses. This study screened publicly available RNA sequencing data sets (n = 35) and characterised 200 viral transcripts from thirteen Australian marsupial species. Of the 200 transcripts, 188 originated from either Bornaviridae, Filoviridae, or Parvoviridae EVEs. The other twelve transcripts were from putative active infections from members of the Herpesviridae and Anelloviridae, and Hepadnaviridae. EVE transcripts (n = 188) were mapped to marsupial genomes (where available, n = 5/13) to identify the genomic insertion sites. Of the 188 transcripts, 117 mapped to 39 EVEs within the koala, bare-nosed wombat, tammar wallaby, brushtail possum, and Tasmanian devil genomes. The remaining eight animals had no available genome (transcripts n = 71). Every marsupial has Bornaviridae, Filoviridae, and Parvoviridae EVEs, a trend widely observed in eutherian mammals. Whilst eutherian bornavirus EVEs are predominantly nucleoprotein-derived, marsupial bornavirus EVEs demonstrate a surprising replicase gene bias. We predicted these widely distributed EVEs were conserved within marsupials from ancient germline integrations, as many were over 65 million years old. One bornavirus replicase EVE, present in six marsupial genomes, was estimated to be 160 million years old, predating the American–Australian marsupial split. We considered transcription of these EVEs through small non-coding RNA as an ancient viral defence. Consistent with this, in koala small RNA sequence data sets, we detected Bornaviridae replicase and Filoviridae nucleoprotein produced small RNA. These were enriched in testis tissue, suggesting they could protect marsupials from vertically transmitted viral integrations.

Circulating skeletal muscle related microRNAs profile in Piedmontese cattle during different age

Piedmontese cattle is known for double-muscle phenotype. MicroRNAs (miRNAs) play important role as regulators in skeletal muscle physiological processes, and we hypothesize that plasma miRNAs expression profiles could be affected by skeletal muscle growth status related to age. Plasma samples of cattle were collected during four different ages from first week of life until the time of commercial end of the fattening period before slaughter. Small-RNA sequencing data analysis revealed the presence of 40% of muscle-related miRNAs among the top 25 highly expressed miRNAs and, 19 miRNAs showed differential expression too. Using qRT-PCR, we validated in a larger bovine population, miRNAs involved in skeletal muscle physiology pathways. Comparing new-born with the other age groups, miR-10b, miR-126-5p, miR-143 and miR-146b were significantly up-regulated, whereas miR-21-5p, miR-221, miR-223 and miR-30b-5p were significantly down-regulated. High expression levels of miR-23a in all the groups were found. Myostatin, a negative regulator of skeletal muscle hypertrophy, was predicted as the target gene for miR-23a and miR-126-5p and we demonstrated their direct binding. Correlation analysis revealed association between miRNAs expression profiles and animals’ weights along the age. Circulating miRNAs could be promising for future studies on their biomarker potentialities to beef cattle selection.

Integrated small RNA, mRNA, and degradome sequencing reveals the important role of miRNAs in the interactions between parasitic plant Cuscuta australis and its host Trifolium repens

Cuscuta australis R.Br. is a stem parasite that obtains water and nutrients from host plants, with which it also exchanges proteins and mRNAs with host plants. Recently, a study found that novel miRNAs accumulated during C. campestris Yunk. parasitism of Arabidopsis thaliana L. However, the fate of miRNAs transferred from parasite to host plants as well as their targets has remained unexamined. To understand the miRNA–mRNA interaction network of host Trifolium repens L. in response to C. australis parasitism, integrated mRNA, small RNA, and degradome sequencing data were analyzed. In total, 60,824 unigenes were identified by RNA-seq, of which 1,601 were differentially expressed genes (DEGs). A total of 71 known miRNAs belonging to 38 miRNA families and 65 novel miRNAs were identified from small RNA sequencing data and 8,012 target genes were predicted. Of these target genes, 129 miRNA–mRNA pairs were identified via degradome analyzes. There were also eight differentially expressed miRNAs (DEMs). DEG and DEM correlation analyzes revealed 23 pairs of differentially expressed miRNA–mRNA pairs responsive to C. australis parasitism. The most enriched pathways were plant hormone signal transduction, ribosome and plant–pathogen interaction pathways. The miRNA–mRNA pairs in host T. repens, including trr-miR393a–RPS6, trr-miR398b–TGA, trr-miR395a/g–bHLH112, trr-miR395g–GH3, and trr-miRn41–WRKY/MYB, likely play important roles in interactions between host T. repens and parasitic C. australis. These results provide valuable information on the mechanisms of parasite–host plant interactions.