Identification Of microRNAs Research Articles

Identification and Analysis of Plant miRNAs: Evolution of In-silico Resources and Future Challenges

Abstract: Endogenous small RNAs (miRNA) are the key regulators of numerous eukaryotic lineages playing an important role in a broad range of plant development. Computational analysis of miRNAs facilitates the understanding of miRNA-based regulations in plants. The discovery of small non-coding RNAs has led to a greater understanding of gene regulation, and the development of bioinformatic tools has enabled the identification of microRNAs (miRNAs) and their targets. The need for comprehensive miRNA analysis is being accomplished by the development of advanced computational tools/algorithms and databases. Each resource has its own specificity and limitations for the analysis. This review provides a comprehensive overview of various algorithms used by computational tools, software, and databases for plant miRNA analysis. However, over a period of about two decades, a lot of knowledge has been added to our understanding of the biogenesis and functioning of miRNAs in other plants. Several parameters were already integrated and others need to be incorporated in order to give more accurate and efficient results. The reassessment of computational recourses (based on old algorithms) is required on the basis of new miRNA research and development. Generally, computational methods, including ab-initio and homology search-based methods, are used for miRNA identification and target prediction. This review presents the new challenges faced by the existing computational methods and the need to develop new tools and advanced algorithms and highlight the limitations of existing computational tools and methods, and emphasizing the need for a comprehensive platform for miRNA gene exploration.

The microRNAome of Strongylus vulgaris larvae and their excretory/secretory products with identification of parasite-derived microRNAs in horse arterial tissue

The equine bloodworm, Strongylus vulgaris, is a highly pathogenic parasite causing potentially fatal vascular and intestinal damage. Parasites express and release microRNAs (miRNAs) for internal regulation and to modulate host immunity. The complete set of miRNAs expressed by S. vulgaris (the S. vulgaris miRNAome) remains unannotated and the aim of this study was to annotate the miRNAome of L4 and L5 stages of S. vulgaris, and to examine differences in miRNA abundance between larval stages and sexes. Furthermore, we aimed to determine if miRNAs were detectable in excretory/secretory products (ESPs) from larvae and in arterial tissue from their predilection site, the cranial mesenteric artery (CMA). Larvae were collected from naturally infected foals, and categorized by sex and stage. A subset of larvae was snap-frozen, while those remaining were incubated and the (ESPs) collected. Arterial tissue samples were collected from the CMA. Small RNA sequencing, followed by a custom bioinformatic pipeline, was used for annotation. We identified 142 S. vulgaris miRNAs in larvae and 136 in ESPs. Significant differences in miRNA abundance were observed between larvae and ESPs, and between L5 females (L5Fs) and L5 males (L5Ms), L4s and L5Fs, and L4s and L5Ms. No differences were found between L4s and L5s overall. In ESPs, several miRNAs were differentially abundant across all groups. Validation through quantitative real-time PCR (qPCR) detected selected miRNAs and their differential abundance in larvae and ESPs. One parasite-derived miRNA was detected in some of the horse arterial tissue samples but at very low levels. This study provided the first annotation of the S. vulgaris miRNAome. Most of the annotated larval miRNAs were also detectable in ESPs, and differences in miRNA abundance between sexes were found for larvae, and between sexes and stages for ESPs. Parasite-derived miRNAs were, however, not consistently detectable in the surrounding host arterial tissue.

Identification of Specific microRNAs in Adipose Tissue Affected by Lipedema

Lipedema is a chronic disorder affecting women with a 10% incidence worldwide. It is often confused with obesity. This study was undertaken to study microRNAs in lipedema tissue assessed by direct hybridization using the robust n-counter flex DX CE-IVD platform. The mean age of the subjects participating in the study was 40.29 (±12.17). The mean body weight and BMI were 67.37 (±10.02) and 25.75 (±4.10), respectively. The lipedema stages included were I and II. The differential expressed human (hsa)-miRNAs were determined according to a log2 fold-change (LFC) of 0.5 and p value < 0.05. To these, increased expression of hsa-let-7g-5p was evident, as well as reduced levels of hsa-miR-371a-5p, -4454+7975, -365a+b-3p, -205-5p, -196a-5p, -4488, -2116-5p, -141-3p, -208a-3p, -302b-3p, 374a-5p, and -1297. Then, several bioinformatics tools were used to analyze microarray data focusing on validated target genes in silico. KEGG and Gene Ontology (GO) pathway enrichment analysis was conducted. Furthermore, the protein–protein interaction and co-expression network were analyzed using STRING and Cytoscape, respectively. The most upregulated miRNA mainly affected genes related to cell cycle, oocyte meiosis, and inflammatory bowel disease. The downregulated microRNAs were related to endocrine resistance, insulin resistance, hypersensitivity to AGE-RAGEs, and focal adhesion. Finally, we validated by RT-PCR the upregulated hsa-let-7g-5p and two down-regulated ones, hsa-miR-205-5p and hsa-miR-302b-3p, confirming microarray results. In addition, three mRNA target miRNAs were monitored, SMAD2, the target of the hsa-let-7g-5p, and ESR1 and VEGFA, the target of hsa-miR-205-5p and hsa-miR-302b-3p, respectively. Our results open a new direction for comprehending biochemical mechanisms related with the pathogenesis of lipedema, shedding light on this intricate pathophysiological condition that could bring to light possible biomarkers in the future.

Identification of potential microRNAs involved in pathogenesis of venous thromboembolism (VTE): A meta analysis

ObjectiveMicroRNAs (MiRNAs) are master regulators of gene expression and have been suggested as potential diagnostic and therapeutic biomarkers in variety of complex diseases. Venous thromboembolism is a major cause of morbidity and mortality worldwide. Several miRNA expression studies have been conducted to identify miRNAs linked to VTE prognosis. These studies reveal that various miRNAs are significantly up-regulated and down-regulated in VTE patients in comparison to healthy controls. Present meta-analysis deliberate findings of such studies to identify most potential miRNA targets associated with VTE. MethodologyComprehensive literature review on Pubmed was conducted. Present analysis assessed 20 research article out of a total of 383 articles screened, based on inclusion and exclusion criteria. The up-regulated and down-regulated miRNAs obtained from selected research articles were subjected to meta-analysis. The differentially expressed miRNAs so obtained and were used for further bioinformatic analysis, including gene ontology and pathway analysis of their target genes, to study their potential involvement in VTE pathogenesis. ResultsTwenty articles selected for meta-analysis included a total of 808 patients. These included 26 up-regulated miRNAs and 11 down-regulated miRNAs. The meta-analysis based on Odds ratio suggested that one up-regulated miRNA (hsa-miR-1233-3p) and two down-regulated miRNAs (hsa-miR-103a-3p and hsa-miR-200c) returned significantly higher Odds compared to other miRNAs. Further bioinformatics analysis of their target genes revealed that these miRNAs target a large number of genes involved in cell adhesion, cell migration, endothelial activation and inflammation genes. ConclusionsThree miRNAs, viz.; hsa-miR-1233-3p, hsa-miR-103a-3p and hsa-miR-200c may play a crucial role in VTE pathogenesis and has potential to serve as reliable diagnostic or therapeutic biomarkers for VTE.

Screening and identification of microRNAs mediating cartilage endplate degeneration in human intervertebral disks.

This study aimed to discover micro-ribonucleic acids (microRNAs) involved in the degeneration of cartilage endplates through next-generation sequencing and lay the foundation for further research. The cartilage endplate was obtained from patients who underwent interbody fusion surgery at the Department of Spine Surgery, Shanghai East Hospital Affiliated to Tongji University, from 1 January 2020 to 1 January 2023. Total RNA was extracted from the cartilage endplate tissue. Discover differential genes through NGS. To annotate gene functions, all target genes were aligned against the Kyoto Encyclopedia of Genes (KEGG) and Gene Ontology (GO) databases. The GO enrichment and KEGG enrichment analyses of target genes were performed using phyper, a function of R. The p-value was corrected using the Bonferroni method, and a corrected p-value of ≤0.05 was taken as the threshold. GO terms or KEGG terms fulfilling this condition were defined as significantly enriched terms. The screened miRNAs and their target protein were verified in vitro using quantitative polymerase chain reaction (qPCR) and Western blotting (WB). RNA was extracted from normal and degenerated cartilage endplate tissues for NGS. Eight downregulated differentially expressed genes (DEGs) and 22 upregulated DEGs were found. The KEGG pathway analysis of these target genes revealed that differential microRNAs and target genes were enriched in different signaling pathways, and the regulated signaling pathways were mainly mitochondrial autophagy and autophagy. The qPCR results demonstrated a significant upregulation of miR-25-3p and miR-345-5p in degenerative cartilage endplate tissues (p ≤ 0.001). Western blot analysis revealed that BRD4 exhibited a marked increase in protein expression levels in degenerative cartilage endplate tissues (p ≤ 0.0001), while BECN1 showed a significant decrease in protein expression levels within these samples (p ≤ 0.0001). We found that DEG hsa-miR-25-3p and hsa-miR-345-5p can be used as diagnostic and therapeutic targets for IDD. The significant target proteins of miR-25-3p and miR-345-5p were BRD4 and BECN1, respectively.

Profiling of MicroRNAs for the Identification of Unique and Common MicroRNAs in Preeclamptic Patients of South India Using Next-Generation Sequencing.

Preeclampsia (PE) is a serious pregnancy complication with an unclear cause. Recent studies suggest that microRNAs (miRNAs), particularly miR-1, may play a role in controlling the genes associated with this condition. This study aimed to compare the expression of miRNAs in the blood and placental tissues of women with PE to those with normal pregnancies. We conducted small RNA sequencing on blood and placental samples from three groups: (a) early-onset preeclampsia (EOPE), (b) late-onset preeclampsia (LOPE), and (c) normal pregnancies. Bioinformatics tools were used to compare the miRNA profiles across these groups. A total of 744 miRNAs were detected in placental samples, while 913 miRNAs were found in blood samples. We further analyzed the target genes using protein-protein interaction (PPI) maps to understand how these miRNAs may influence gene functions. Our analysis revealed significant differences in miRNA expression between the EOPE, LOPE, and control groups. Eight miRNAs were consistently detected in both blood and placental samples across all groups, while other miRNAs were either specific to PE or certain tissue types. The 492 target genes identified formed dense interaction networks, with several key genes occupying central roles. These findings suggest that altered miRNA expression and the resulting disruption of gene networks may contribute to the development of PE. The distinct differences between EOPE and LOPE indicate that these two subtypes may be driven by different underlying mechanisms. This paves the way for future research to explore new treatments targeting these miRNAs and their associated genes.

Identification and function of microRNAs in hemipteran pests: A review.

Hemiptera is one of the most significant orders of insect pests, including whiteflies, true bugs, aphids, planthoppers, psyllids, and so forth, which have led to substantial economic losses in agricultural industries and have significantly affected food yields through their ability to suck the phloem sap of crops and transmit numerous bacterial and viral pathogens. Therefore, explorations of pest-specific, eco-friendly and easy-to-adopt technologies for hemipteran pest control are urgently needed. To the best of our knowledge, microRNAs (miRNAs), which are endogenous non-coding small RNAs approximately 22 nucleotides in length, are involved in regulating gene expression via the direct recognition and binding of the 3'-untranslated region (3'-UTR) of target messenger RNAs (mRNAs) or by acting as a center of a competitive endogenous RNA (ceRNA) network at the post-transcriptional level. This review systematically outlines the characterization and functional investigation of the miRNA biogenesis pathway in hemipteran pests, such as whiteflies, true bugs, aphids and planthoppers. In addition, we explored the results of small RNA sequencing and functional observations of miRNAs in these pests, and the results suggest that the numerous miRNAs obtained and annotated via high-throughput sequencing technology and bioinformatic analyses contribute to molting development, fitness, wing polyphenism, symbiont interactions and insecticide resistance in hemipteran pests. Finally, we summarize current advances and propose a framework for future research to extend the current data and address potential limitations in the investigation and application of hemipteran miRNAs.

Identification and characterization of functional microRNAs and their significant targets in maize plants

Identification and characterization of functional microRNAs and their significant targets in maize plants

Identification and characterization of microRNAs in virus-resistant and susceptible barley cultivars

Identification and characterization of microRNAs in virus-resistant and susceptible barley cultivars

Identification of MicroRNAs in children with increased asthma bronchodilator usage in genetics of asthma in Costa Rica Study.

Uncontrolled or severe asthma results in symptomatic usage of short-acting ß2-agonist (SABA) usage. MicroRNAs (miRNAs) are posttranslational regulators that can influence asthma biology. This study aims to identify miRNAs that are associated with increased SABA usage. Small RNA sequenced from blood serum of 1,132 children with asthma aged 6 to 14 years in the Genetics of Asthma in Costa Rica Study was used for this analysis. Logistic regression identified miRNAs in patients who required increased SABA usage. These miRNA were validated for association with SABA-induced bronchodilator responsiveness (BDR). Gene target pathway analysis was performed on validated miRNAs. Twenty-one miRNAs were significantly associated with increased SABA usage with OR ranging from 0.87 to 1.23. Two miRNAs, miR-378a-3p and miR-144-3p, had odds ratio 1.14 (1-1.29, p = .05) and 1.11 (1.01-1.22, p = .035), respectively, for increased SABA usage and were also significantly associated with bronchodilator response. Furthermore, a linear regression analysis involving these miRNA and bronchodilator response revealed that increased miR-378a-3p correlated with decreased BDR and increased expression of miR-144-3p correlated with improving pulmonary function with bronchodilators. In gene target Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, the erythroblastosis viral oncogene signaling pathway had among one of the highest fold enrichment and p-value. Increased expression of miR-378a-3p and miR-144-3p was seen in this patient population who required increased SABA usage. There were different bronchodilatory effects seen in these two miRNAs, suggesting different potential mechanisms underlying increased SABA usage.

Genome-Wide Identification of microRNAs Associated with Starch Biosynthesis and Endosperm Development in Foxtail Millet.

Foxtail millet is one of the oldest crops, and its endosperm contains up to 70% of starch. Grain filling is an important starch accumulation process associated with foxtail millet yield and quality. However, the molecular mechanisms of grain filling in foxtail millet are relatively unclear. Here, we investigate the genes and regulated miRNAs associated with starch synthesis and metabolism in foxtail millet using high-throughput small RNA, mRNA and degradome sequencing. The regulation of starch synthesis and quality is carried out mainly at the 15 DAA to 35 DAA stage during grain filling. The DEGs between waxy and non-waxy foxtail millet were significant, especially for GBSS. Additionally, ptc-miR169i_R+2_1ss21GA, fve-miR396e_L-1R+1, mtr-miR162 and PC-5p-221_23413 regulate the expression of genes associated with the starch synthesis pathway in foxtail millet. This study provides new insights into the molecular mechanisms of starch synthesis and quality formation in foxtail millet.

Identification of microRNA and their potential role in regulation diapause termination in seven spot ladybird beetle, Coccinella septempunctata

Diapause is an alternative development stage in seven spot ladybird beetle, Coccinella septempunctata. However, the regulatory mechanism governing the initiation, maintenance, and termination of diapause in the seven-spot ladybird have not been extensively studied. MicroRNAs (miRNAs), a type of non-coding RNA, might be involved in diapause regulation and related physiological processes. The objective of this study is to investigate the potential involvement of miRNAs in diapause termination in C. septempunctata. High-throughput sequencing was used to identify miRNAs associated with diapause termination in C. septempunctata. A total of 769 miRNAs were identified, potentially implicated in diapause termination, including 673 evolutionarily conserved miRNA and 96 putatively novel-miRNAs. Among these, two evolutionarily conserved miRNAs, aae-miR-305-5P and tca-miR-277-5P, exhibited differential abundance during diapause termination compared to diapause. aae-miR-305-5P was overexpressed in diapause termination ladybird beetle and may be responsible for silencing the expression of candidate genes in peroxisome pathway associated with diapause termination. Conversely, tca-miR-277-5P was under-expressed in diapause termination and may promote the expression of genes related to the longevity regulating pathway, thereby increase the lifetime, a characteristic feature of diapause termination. In addition, a putatively novel-miRNA (unconservative_c62764) was overexpressed in diapause termination ladybird beetle, potentially contributing to the decreased expression of genes related to Wnt signaling pathway during diapause termination. These findings highlighting the significant roles of microRNAs in pathway such as longevity regulation, perisome function, and Wnt signaling, which may regulate diapause termination in C. septempunctata. This study might help us to unveil the miRNA involvement in gene expression regulation of diapause termination in insects.

Identification of microRNAs expressed in an animal model of periodontal disease and their impact on pathological processes

MicroRNAs represent a class of small RNAs that act to silence genes post-transcriptionally by inhibiting the translation of target messenger RNAs, and this study aimed to understand how miRNAs influence the set-up of periodontal disease. Periodontitis was induced by inserting a ligature into the left first mandibular molar in a rat model, which was kept for the entire 56 days-time of experiment. After 56 days post-periodontitis induction, the histopathological analysis showed an apical extension of the junctional epithelium, with areas of hyperplasia, exocytosis, and a mixed inflammatory infiltrate with a predominance of neutrophils, lymphocytes, and eventual plasma cells in the deeper layers. The cement surface showed areas of irregularity, covered by cementoblasts and irregular surfaces, confirming the set-up of periodontitis. In the sequencing analysis, 26,404 genes were identified, with 132 reaching statistical significance. Among genes with a statistical difference, 18 were found to encode for microRNAs. The identified microRNAs are primarily involved in bone remodeling by acting on fibroblast growth factors, and collagen production. These outcomes demonstrate a signaling role in bone resorption, which is consistent with the histopathological observations that show the installation of inflammation with epithelial migration and the beginning of the repair process, with cementum resorption. The disclosure of how miRNAs may influence the maintaining of periodontal disease will help the development of new dental materials for the prophylaxis and treatment of alveolar bone resorption.

Identification of plasma microRNAs in association with urinary arsenic exposure

Identification of plasma microRNAs in association with urinary arsenic exposure

Identification of Stage-Specific microRNAs that Govern the Early Stages of Sequential Oral Oncogenesis by Strategically Bridging Human Genetics with Epigenetics and Utilizing an Animal Model.

Oral squamous cell carcinoma (OSCC) is a highly prevalent and aggressive malignancy, with mortality rates reaching 60%, mainly due to its excessive diagnostic delay. MiRNAs, a class of crucial epigenetic gene-expression regulators, have emerged as potential diagnostic biomarkers, with >200 molecules exhibiting expressional dysregulation in OSCC. We had previously established an in silico methodology for the identification of the most disease-specific molecules by bridging genetics and epigenetics. Here, we identified the stage-specific miRNAs that govern the asymptomatic early stages of oral tumorigenesis by exploiting seed-matching and the reverse interplay between miRNA levels and their target genes' expression. Incorporating gene-expression data from our group's experimental hamster model of sequential oral oncogenesis, we bioinformatically detected the miRNAs that simultaneously target/regulate >75% of the genes that are characteristically upregulated or downregulated in the consecutive stages of hyperplasia, dysplasia, and early invasion, while exhibiting the opposite expressional dysregulation in OSCC-derived tissue and/or saliva specimens. We found that all stages share the downregulation of miR-34a-5p, miR124-3p, and miR-125b-5p, while miR-1-3p is under-expressed in dysplasia and early invasion. The malignant early-invasion stage is distinguished by the downregulation of miR-147a and the overexpression of miR-155-5p, miR-423-3p, and miR-34a-5p. The identification of stage-specific miRNAs may facilitate their utilization as biomarkers for presymptomatic OSCC diagnosis.

Identification of reference microRNAs in skeletal muscle of a canine model of Duchenne muscular dystrophy

Background Duchenne muscular dystrophy (DMD) is a fatal muscle wasting disease caused by mutations in the dystrophin gene. DE50-MD dogs are a canine model of DMD used as final translational models for evaluation of promising treatments. MicroRNA (miR) expressions in the muscle of DE50-MD dogs represent potential biomarkers, but stable reference miRs must first be identified. The aim of this paper was to establish a panel of reference miRs for WT and DE50-MD dogs over a range of ages and muscle groups. Methods RNA was extracted from WT and DE50-MD dog (N=6 per genotype) vastus lateralis muscle samples collected longitudinally at 3, 6, 9, 12, 15 and 18 months of age, and from muscles collected post-mortem (N=3 per genotype; cranial tibial, semimembranosus, lateral triceps and diaphragm). 87 RNAs were quantified in a subset of 6-month-old WT and DE50-MD muscles (N=4 per genotype) using the QIAcuity miFinder panel. GeNorm, BestKeeper and Normfinder were used to identify a candidate panel of the 8 most stable small RNAs, which were then quantified in all RNA samples, alongside the commonly used reference RNA snRNA U6. Results The most stable miRs of this subset were used to normalise quantities of dystromiRs miR-1, miR-133a and miR-206, and fibromiR miR-214. MicroRNAs miR-191, let-7b, miR-125a and miR-15a were the most stable miRs tested, while snRNA U6 performed poorly. DystromiR expression, normalised to the geometric mean of the panel of reference miRs, was lower for miR-1 and miR-133a in DE50-MD compared to WT muscles, while miR-206 levels did not significantly differ between genotypes. FibromiR miR-214 was 2- to 4-fold higher in DE50-MD versus WT muscles. Conclusions A normalisation factor derived from miR-191, let-7b, miR-125a and miR-15a is suitable for normalising miR expression data from WT and DE50-MD muscle over a range of ages and muscle types.

Identification of exosomal microRNAs and related hub genes associated with imatinib resistance in chronic myeloid leukemia.

Chemotherapy resistance is a major obstacle in cancer therapy, and identifying novel druggable targets to reverse this phenomenon is essential. The exosome-mediated transmittance of drug resistance has been shown in various cancer models including ovarian and prostate cancer models. In this study, we aimed to investigate the role of exosomal miRNA transfer inchronic myeloid leukemia drug resistance. For this purpose, firstly exosomes were isolated from imatinib sensitive (K562S) and resistant (K562R) chronic myeloid leukemia (CML)cells and named as Sexo and Rexo, respectively. Then, miRNA microarray was used to compare miRNA profiles of K562S, K562R, Sexo, Rexo, and Rexo-treated K562S cells. According to our results, miR-125b-5p and miR-99a-5p exhibited increased expression in resistant cells, their exosomes, and Rexo-treated sensitive cells compared to their sensitive counterparts. On the other hand, miR-210-3p and miR-193b-3p were determined to be the two miRNAs which exhibited decreased expression profile in resistant cells and their exosomes compared to their sensitive counterparts. Gene targets, signaling pathways, and enrichment analysis were performed for these miRNAs by TargetScan, KEGG, and DAVID. Potential interactions between gene candidates at the protein level were analyzed via STRING and Cytoscape software. Our findings revealed CCR5, GRK2, EDN1, ARRB1, P2RY2, LAMC2, PAK3, PAK4, and GIT2 as novel gene targets that may play roles in exosomal imatinib resistance transfer as well as mTOR, STAT3, MCL1, LAMC1, and KRAS which are already linked to imatinib resistance. MDR1 mRNA exhibited higher expression in Rexo compared to Sexo as well as in K562S cells treated with Rexo compared to K562S cells which may suggest exosomal transfer of MDR1 mRNA.

Exonuclease III assisted exponential amplification reaction (EXPAR) for specific miRNA-155 analysis during post-anesthetic nursing

The persistent obstacle in precise and sensitive identification of microRNAs (miRNAs) pertains to the advancement of expeditious and effective isothermal amplification methodologies suitable for point-of-care environments and monitoring the cancer prognosis in patients receiving post-anesthetic nursing. The exponential amplification reaction (EXPAR) has attracted considerable interest due to its simplicity and ability to rapidly amplify signals. The practical application of the EXPAR is, nevertheless, severely hampered by the inability to differentiate closely related homologous sequences and to modify the designed templates to suit other targets. A loop-stem template for the EXPAR system was developed in this study to facilitate specific target recognition with the aid of exonuclease III (Exo III). This innovation effectively eliminated non-specific hybridization that could occur between the template and interfering sequences, thereby ensuring minimal background amplification of EXPAR. By modulating Exo III-based target recycling, EXPAR based chain amplification and G4/hemin based color reaction, this method facilitated the precise and sensitive examination of miRNA-155, yielding acceptable yields and a minimal detection limit of 0.43 fM. The approach expedites simple and expeditious molecular diagnostic applications involving short nucleic acids and offers an innovative method for enhancing the selectivity of EXPAR-based techniques, providing a robust tool for monitoring the expression level from patients receiving post-anesthetic nursing and guiding the treatment strategy.

Distinct small RNAs are expressed at different stages of Phytophthora capsici and play important roles in development and pathogenesis.

Small RNAs (sRNAs) are important non-coding RNA regulators that play key roles in the development and pathogenesis of plant pathogens, as well as in other biological processes. However, whether these abundant and varying sRNAs are involved in Phytophthora development or infection remains enigmatic. In this study, sRNA sequencing of 4 asexual stages of Phytophthora capsici (P. capsici), namely, as mycelia (HY), sporangia (SP), zoospores (ZO), cysts (CY), and pepper infected with P. capsici (IN), were performed, followed by sRNA analysis, microRNA (miRNA) identification, and miRNA target prediction. sRNAs were mainly distributed at 25-26nt in HY, SP, and ZO but distributed at 18-34nt in CY and IN. 92, 42, 176, 39, and 148 known miRNAs and 15, 19, 54, 13, and 1 novel miRNA were identified in HY, SP, ZO, CY, and IN, respectively. It was found that the expression profiles of known miRNAs vary greatly at different stages and could be divided into 4 categories. Novel miRNAs mostly belong to part I. Gene ontology (GO) analysis of known miRNA-targeting genes showed that they are involved in the catalytic activity pathway, binding function, and other biological processes. Kyoto Encyclopedia of Gene and Genome (KEGG) analysis of novel miRNA-targeting genes showed that they are involved in the lysine degradation pathway. The expression of candidate miRNAs was validated using quantitative reverse transcription-polymerase chain reaction (qRT-PCR), and miRNAs were downregulated in PcDCL1 or PcAGO1 mutants. To further explore the function of the detected miRNAs, the precursor of a novel miRNA, miR91, was knockout by CRISPR-Cas9, the mutants displayed decreased mycelial growth, sporangia production, and zoospore production. It was found that 503142 (Inositol polyphosphate 5-phosphatase and related proteins) can be predicted as a target of miR91, and the interaction between miR91 and 503142 was verified using the tobacco transient expression system. Overall, our results indicate that the diverse and differentially expressed sRNAs are involved in the development and pathogenesis of P. capsici.

Identification and expression profiling of microRNAs in leaf tissues of Foeniculum vulgare Mill. under salinity stress

ABSTRACT Foeniculum vulgare Mill. commonly known as fennel, is a globally recognized aromatic medicinal plant and culinary herb with widespread popularity due to its antimicrobial, antioxidant, carminative, and diuretic properties, among others. Although the phenotypic effects of salinity stress have been previously explored in fennel, the molecular mechanisms underlying responses to elevated salinity in this plant remain elusive. MicroRNAs (miRNAs) are tiny, endogenous, and extensively conserved non-coding RNAs (ncRNAs) typically ranging from 20 to 24 nucleotides (nt) in length that play a major role in a myriad of biological functions. In fact, a number of miRNAs have been extensively associated with responses to abiotic stress in plants. Consequently, employing computational methodologies and rigorous filtering criteria, 40 putative miRNAs belonging to 25 different families were characterized from fennel in this study. Subsequently, employing the psRNATarget tool, a total of 67 different candidate target transcripts for the characterized fennel miRNAs were predicted. Additionally, the expression patterns of six selected fennel miRNAs (i.e. fvu-miR156a, fvu-miR162a-3p, fvu-miR166a-3p, fvu-miR167a-5p, fvu-miR171a-3p, and fvu-miR408-3p) were analyzed under salinity stress conditions via qPCR. This article holds notable significance as it identifies not only 40 putative miRNAs in fennel, a non-model plant, but also pioneers the analysis of their expression under salinity stress conditions.