Barley Seeds miRNome Stability during Long-Term Storage and Aging

Seed aging is a complex biological process that has been attracting scientists’ attention for many years. High-throughput small RNA sequencing was applied to examine microRNAs contribution in barley seeds senescence. Unique samples of seeds that, despite having the same genetic makeup, differed in viability after over 45 years of storage in a dry state were investigated. In total, 61 known and 81 novel miRNA were identified in dry seeds. The highest level of expression was found in four conserved miRNA families, i.e., miR159, miR156, miR166, and miR168. However, the most astonishing result was the lack of significant differences in the level of almost all miRNAs in seed samples with significantly different viability. This result reveals that miRNAs in dry seeds are extremely stable. This is also the first identified RNA fraction that is not deteriorating along with the loss of seed viability. Moreover, the novel miRNA hvu-new41, with higher expression in seeds with the lowest viability as detected by RT-qPCR, has the potential to become an indicator of the decreasing viability of seeds during storage in a dry state.     

Identification of Novel miRNAs and Their Target Genes in the Response to Abscisic Acid in Arabidopsis

miRNAs are involved in various biological processes, including adaptive responses to abiotic stress. To understand the role of miRNAs in the response to ABA, ABA-responsive miRNAs were identified by small RNA sequencing in wild-type Arabidopsis, as well as in abi1td, mkkk17, and mkkk18 mutants. We identified 10 novel miRNAs in WT after ABA treatment, while in abi1td, mkkk17, and mkkk18 mutants, three, seven, and nine known miRNAs, respectively, were differentially expressed after ABA treatment. One novel miRNA (miRn-8) was differentially expressed in the mkkk17 mutant. Potential target genes of the miRNA panel were identified using psRNATarget. Sequencing results were validated by quantitative RT-PCR of several known and novel miRNAs in all genotypes. Of the predicted targets of novel miRNAs, seven target genes of six novel miRNAs were further validated by 5′ RLM-RACE. Gene ontology analyses showed the potential target genes of ABA-responsive known and novel miRNAs to be involved in diverse cellular processes in plants, including development and stomatal movement. These outcomes suggest that a number of the identified miRNAs have crucial roles in plant responses to environmental stress, as well as in plant development, and might have common regulatory roles in the core ABA signaling pathway.     

Identification of miRNAs and Target Genes at Key Stages of Sexual Differentiation in Androdioecious Osmanthus fragrans

Androdioecy is the crucial transition state in the evolutionary direction of hermaphroditism to dioecy, however, the molecular mechanisms underlying the formation of this sex system remain unclear. While popular in China for its ornamental and cultural value, Osmanthus fragrans has an extremely rare androdioecy breeding system, meaning that there are both male and hermaphroditic plants in a population. To unravel the mechanisms underlying the formation of androdioecy, we performed small RNA sequencing studies on male and hermaphroditic O. fragrans. A total of 334 miRNAs were identified, of which 59 were differentially expressed. Functional categorization revealed that the target genes of differentially expressed miRNAs were mainly involved in the biological processes of reproductive development and the hormone signal transduction pathway. We speculated that the miRNA160, miRNA167, miRNA393 and miRNA396 families may influence the sex differentiation in O. fragrans. Overall, our study is the first exploration of miRNAs in the growth and development process of O. fragrans, and is also the first study of androdioecious plants from the miRNA sequencing perspective. The analysis of miRNAs and target genes that may be involved in the sex differentiation process lay a foundation for the ultimate discovery of the androdioecious molecular mechanism in O. fragrans.     

MicroRNA-Regulated Protein-Protein Interaction Networks and Their Functions in Breast Cancer

MicroRNAs, which are small endogenous RNA regulators, have been associated with various types of cancer. Breast cancer is a major health threat for women worldwide. Many miRNAs were reported to be associated with the progression and carcinogenesis of breast cancer. In this study, we aimed to discover novel breast cancer-related miRNAs and to elucidate their functions. First, we identified confident miRNA-target pairs by combining data from miRNA target prediction databases and expression profiles of miRNA and mRNA. Then, miRNA-regulated protein interaction networks (PINs) were constructed with confident pairs and known interaction data in the human protein reference database (HPRD). Finally, the functions of miRNA-regulated PINs were elucidated by functional enrichment analysis. From the results, we identified some previously reported breast cancer-related miRNAs and functions of the PINs, e.g., miR-125b, miR-125a, miR-21, and miR-497. Some novel miRNAs without known association to breast cancer were also found, and the putative functions of their PINs were also elucidated. These include miR-139 and miR-383. Furthermore, we validated our results by receiver operating characteristic (ROC) curve analysis using our miRNA expression profile data, gene expression-based outcome for breast cancer online (GOBO) survival analysis, and a literature search. Our results may provide new insights for research in breast cancer-associated miRNAs.     

Identification of miRNAs and Their Targets in Cotton Inoculated with Verticillium dahliae by High-Throughput Sequencing and Degradome Analysis

MicroRNAs (miRNAs) are a group of endogenous small non-coding RNAs that play important roles in plant growth, development, and stress response processes. Verticillium wilt is a vascular disease in plants mainly caused by Verticillium dahliae Kleb., the soil-borne fungal pathogen. However, the role of miRNAs in the regulation of Verticillium defense responses is mostly unknown. This study aimed to identify new miRNAs and their potential targets that are involved in the regulation of Verticillium defense responses. Four small RNA libraries and two degradome libraries from mock-infected and infected roots of cotton (both Gossypium hirsutum L. and Gossypium barbadense L.) were constructed for deep sequencing. A total of 140 known miRNAs and 58 novel miRNAs were identified. Among the identified miRNAs, many were differentially expressed between libraries. Degradome analysis showed that a total of 83 and 24 genes were the targets of 31 known and 14 novel miRNA families, respectively. Gene Ontology analysis indicated that many of the identified miRNA targets may function in controlling root development and the regulation of Verticillium defense responses in cotton. Our findings provide an overview of potential miRNAs involved in the regulation of Verticillium defense responses in cotton and the interactions between miRNAs and their corresponding targets. The profiling of these miRNAs lays the foundation for further understanding of the function of small RNAs in regulating plant response to fungal infection and Verticillium wilt in particular.     

Identification and Dynamic Regulation of microRNAs Involved in Salt Stress Responses in Functional Soybean Nodules by High-Throughput Sequencing

Both symbiosis between legumes and rhizobia and nitrogen fixation in functional nodules are dramatically affected by salt stress. Better understanding of the molecular mechanisms that regulate the salt tolerance of functional nodules is essential for genetic improvement of nitrogen fixation efficiency. microRNAs (miRNAs) have been implicated in stress responses in many plants and in symbiotic nitrogen fixation (SNF) in soybean. However, the dynamic regulation of miRNAs in functioning nodules during salt stress response remains unknown. We performed deep sequencing of miRNAs to understand the miRNA expression profile in normal or salt stressed-soybean mature nodules. We identified 110 known miRNAs belonging to 61 miRNA families and 128 novel miRNAs belonging to 64 miRNA families. Among them, 104 miRNAs were dramatically differentially expressed (>2-fold or detected only in one library) during salt stress. qRT-PCR analysis of eight miRNAs confirmed that these miRNAs were dynamically regulated in response to salt stress in functional soybean nodules. These data significantly increase the number of miRNAs known to be expressed in soybean nodules, and revealed for the first time a dynamic regulation of miRNAs during salt stress in functional nodules. The findings suggest great potential for miRNAs in functional soybean nodules during salt stress.     

Expression Profiling of Exosomal miRNAs Derived from Human Esophageal Cancer Cells by Solexa High-Throughput Sequencing

Cellular genetic materials, such as microRNAs (miRNAs), mRNAs and proteins, are packaged inside exosomes, small membrane vesicles of endocytic origin that are released into the extracellular environment. These cellular genetic materials can be delivered into recipient cells, where they exert their respective biological effects. However, the miRNA profiles and biological functions of exosomes secreted by cancer cells remain unknown. The present study explored the miRNA expression profile and distribution characteristics of exosomes derived from human esophageal cancer cells through Solexa high-throughput sequencing. Results showed that 56,421 (2.94%) unique sequences in cells and 7727 (0.63%) in exosomes matched known miRNAs. A total of 342 and 48 known miRNAs were identified in cells and exosomes, respectively. Moreover, 64 and 32 novel miRNAs were predicted in cells and exosomes, respectively. Significant differences in miRNA expression profiles were found between human esophageal cancer cells and exosomes. These findings provided new insights into the characteristics of miRNAs in exosomes derived from human esophageal cancer cells and the specific roles of miRNAs in intercellular communication mediated by exosomes in esophageal cancer.     

Analysis of MicroRNA Expression Profiles in Weaned Pig Skeletal Muscle after Lipopolysaccharide Challenge

MicroRNAs (miRNAs) constitute a class of non-coding RNAs that play a crucial regulatory role in skeletal muscle development and disease. Several acute inflammation conditions including sepsis and cancer are characterized by a loss of skeletal muscle due primarily to excessive muscle catabolism. As a well-known inducer of acute inflammation, a lipopolysaccharide (LPS) challenge can cause serious skeletal muscle wasting. However, knowledge of the role of miRNAs in the course of inflammatory muscle catabolism is still very limited. In this study, RNA extracted from the skeletal muscle of pigs injected with LPS or saline was subjected to small RNA deep sequencing. We identified 304 conserved and 114 novel candidate miRNAs in the pig. Of these, four were significantly increased in the LPS-challenged samples and five were decreased. The expression of five miRNAs (ssc-miR-146a-5p, ssc-miR-221-5p, ssc-miR-148b-3p, ssc-miR-215 and ssc-miR-192) were selected for validation by quantitative polymerase chain reaction (qPCR), which found that ssc-miR-146a-5p and ssc-miR-221-5p were significantly upregulated in LPS-challenged pig skeletal muscle. Moreover, we treated mouse C2C12 myotubes with 1000 ng/mL LPS as an acute inflammation cell model. Expression of TNF-α, IL-6, muscle atrophy F-box (MAFbx) and muscle RING finger 1 (MuRF1) mRNA was strongly induced by LPS. Importantly, miR-146a-5p and miR-221-5p also showed markedly increased expression in LPS-treated C2C12 myotubes, suggesting the two miRNAs may be involved in muscle catabolism systems in response to acute inflammation caused by a LPS challenge. To our knowledge, this study is the first to examine miRNA expression profiles in weaned pig skeletal muscle challenged with LPS, and furthers our understanding of miRNA function in the regulation of inflammatory muscle catabolism.     

MiR-10a in Pancreatic Juice as a Biomarker for Invasive Intraductal Papillary Mucinous Neoplasm by miRNA Sequencing

New biomarkers are needed to further stratify the risk of malignancy in intraductal papillary mucinous neoplasm (IPMN). Although microRNAs (miRNAs) are expected to be stable biomarkers, they can vary owing to a lack of definite internal controls. To identify universal biomarkers for invasive IPMN, we performed miRNA sequencing using tumor-normal paired samples. A total of 19 resected tissues and 13 pancreatic juice samples from 32 IPMN patients were analyzed for miRNA expression by next-generation sequencing with a two-step normalization of miRNA sequence data. The miRNAs involved in IPMN associated with invasive carcinoma were identified from this tissue analysis and further verified with the pancreatic juice samples. From the tumor-normal paired tissue analysis of the expression levels of 2792 miRNAs, 20 upregulated and 17 downregulated miRNAs were identified. In IPMN associated with invasive carcinoma (INV), miR-10a-5p and miR-221-3p were upregulated and miR-148a-3p was downregulated when compared with noninvasive IPMN. When these findings were further validated with pancreatic juice samples, miR-10a-5p was found to be elevated in INV (p = 0.002). Therefore, three differentially expressed miRNAs were identified in tissues with INV, and the expression of miR-10a-5p was also elevated in pancreatic juice samples with INV. MiR-10a-5p is a promising additional biomarker for invasive IPMN.