CircRNA Expression Pattern and ceRNA and miRNA-mRNA Networks Involved in Anther Development in the CMS Line of Brassica campestris.
ABSTRACT: Male-sterile plants provide an important breeding tool for the heterosis of hybrid crops, such as Brassicaceae. In the last decade, circular RNAs (circRNAs), as a novel class of covalently closed and single-stranded endogenous non-coding RNAs (ncRNAs), have received much attention because of their functions as "microRNA (miRNA) sponges" and "competing endogenous RNAs" (ceRNAs). However, the information about circRNAs in the regulation of male-sterility and anther development is limited. In this study, we established the Polima cytoplasm male sterility (CMS) line "Bcpol97-05A", and the fertile line, "Bcajh97-01B", in Brassica campestris L. ssp. chinensis Makino, syn. B. rapa ssp. chinensis, and performed RNA expression profiling comparisons between the flower buds of the sterile line and fertile line by whole-transcriptome sequencing. A total of 31 differentially expressed (DE) circRNAs, 47 DE miRNAs, and 4779 DE mRNAs were identified. By using Cytoscape, the miRNA-mediated regulatory network and ceRNA network were constructed, and the circRNA A02:23507399|23531438 was hypothesized to be an important circRNA regulating anther development at the post-transcriptional level. The gene ontology (GO) analysis demonstrated that miRNAs and circRNAs could regulate the orderly secretion and deposition of cellulose, sporopollenin, pectin, and tryphine; the timely degradation of lipids; and the programmed cell death (PCD) of tapetum cells, which play key roles in anther development. Our study revealed a new circRNA-miRNA-mRNA network, which is involved in the anther development of B. campestris, which enriched the understanding of CMS in flowering plants, and laid a foundation for further study on the functions of circRNAs and miRNAs during anther development.
Project description:BACKGROUND:Wucai (Brassica campestris L. ssp. chinensis var. rosularis Tsen) is a variant of nonheading Chinese cabbage (Brassica campestris L.), which is one of the major vegetables in China. Cytoplasmic male sterility (CMS) has been used for Wucai breeding in recent years. However, the underlying molecular mechanism of Wucai CMS remains unclear. In this study, the phenotypic and cytological features of Wucai CMS were observed by anatomical analysis, and a comparative transcriptome analysis was carried out to identify genes related to male sterility using Illumina RNA sequencing technology (RNA-Seq). RESULTS:Microscopic observation demonstrated that tapetum development was abnormal in the CMS line, which failed to produce fertile pollen. Bioinformatics analysis detected 4430 differentially expressed genes (DEGs) between the fertile and sterile flower buds. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed to better understand the functions of these DEGs. Among the DEGs, 35 genes (53 DEGS) were implicated in anther and pollen development, and 11 genes were involved in pollen cell wall formation and modification; most of these showed downregulated expression in sterile buds. In addition, several genes related to tapetum development (A6, AMS, MS1, MYB39, and TSM1) and a few genes annotated to flowering (CO, AP3, VIN3, FLC, FT, and AGL) were detected and confirmed by qRT-PCR as being expressed at the meiosis, tetrad, and uninucleate microspore stages, thus implying possible roles in specifying or determining the fate and development of the tapetum, male gametophyte and stamen. Moreover, the top four largest transcription factor families (MYB, bHLH, NAC and WRKY) were analyzed, and most showed reduced expression in sterile buds. These differentially expressed transcription factors might result in abortion of pollen development in Wucai. CONCLUSION:The present comparative transcriptome analysis suggested that many key genes and transcription factors involved in anther development show reduced gene expression patterns in the CMS line, which might contribute to male sterility in Wucai. This study provides valuable information for a better understanding of CMS molecular mechanisms and functional genome studies in Wucai.
Project description:microRNAs (miRNAs) are a class of newly identified, noncoding, small RNA molecules that negatively regulate gene expression. Many miRNAs are reportedly involved in plant growth, development and stress response processes. However, their roles in the sexual reproduction mechanisms in flowering plants remain unknown. Pollen development is an important process in the life cycle of a flowering plant, and it is closely related to the yield and quality of crop seeds. This study aimed to identify miRNAs involved in pollen development. A microarray assay was conducted using the known complementary sequences of plant miRNAs as probes on inflorescences of a sterile male line (Bcajh97-01A) and a fertile male line (Bcajh97-01B) of the Brassica campestris ssp. chinensis cv. 'Aijiaohuang' genic male sterility sister line system (Bcajh97-01A/B). The results showed that 44 miRNAs were differently expressed in the two lines. Of these, 15 had over 1.5-fold changes in their transcript levels, with 9 upregulated and 6 downregulated miRNAs in inflorescences of 'Bcajh97-01A' sterile line plants. We then focused on 3 of these 15 miRNAs (miR158, miR168 and miR172). Through computational methods, 13 family members were predicted for these 3 miRNAs and 22 genes were predicted to be their candidate target genes. By using 5' modified RACE, 2 target genes of miR168 and 5 target genes of miR172 were identified. Then, qRT-PCR was applied to verify the existence and expression patterns of the 3 miRNAs in the flower buds at five developmental stages. The results were generally consistent with those of the microarray. Thus, this study may give a valuable clue for further exploring the miRNA group that may function during pollen development.
Project description:BACKGROUND: microRNAs (miRNAs) are endogenous, noncoding, small RNAs that have essential regulatory functions in plant growth, development, and stress response processes. However, limited information is available about their functions in sexual reproduction of flowering plants. Pollen development is an important process in the life cycle of a flowering plant and is a major factor that affects the yield and quality of crop seeds. RESULTS: This study aims to identify miRNAs involved in pollen development. Two independent small RNA libraries were constructed from the flower buds of the male sterile line (Bcajh97-01A) and male fertile line (Bcajh97-01B) of Brassica campestris ssp. chinensis. The libraries were subjected to high-throughput sequencing by using the Illumina Solexa system. Eight novel miRNAs on the other arm of known pre-miRNAs, 54 new conserved miRNAs, and 8 novel miRNA members were identified. Twenty-five pairs of novel miRNA/miRNA* were found. Among all the identified miRNAs, 18 differentially expressed miRNAs with over two-fold change between flower buds of male sterile line (Bcajh97-01A) and male fertile line (Bcajh97-01B) were identified. qRT-PCR analysis revealed that most of the differentially expressed miRNAs were preferentially expressed in flower buds of the male fertile line (Bcajh97-01B). Degradome analysis showed that a total of 15 genes were predicted to be the targets of seven miRNAs. CONCLUSIONS: Our findings provide an overview of potential miRNAs involved in pollen development and interactions between miRNAs and their corresponding targets, which may provide important clues on the function of miRNAs in pollen development.
Project description:BACKGROUND:Increasing evidence has shown that circular RNAs (circRNAs) are involved in neurodegenerative disorders, but their roles in neurological toxoplasmosis are yet to know. This study examined miRNA and circRNA expressions in mouse brain following oral infection with T. gondii Pru strain. RESULTS:Total RNA extracted from acutely infected (11?days post infection (DPI)), chronically infected (35 DPI) and uninfected mouse brain samples were subjected to genome-wide small RNA sequencing. In the acutely infected mice, 9 circRNAs and 20 miRNAs were upregulated, whereas 67 circRNAs and 28 miRNAs were downregulated. In the chronically infected mice, 2 circRNAs and 42 miRNAs were upregulated, whereas 1 circRNA and 29 miRNAs were downregulated. Gene ontology analysis predicted that the host genes that produced the dysregulated circRNAs in the acutely infected brain were primarily involved in response to stimulus and ion binding activities. Furthermore, predictive interaction networks of circRNA-miRNA and miRNA-mRNA were constructed based on genome-wide transcriptome sequencing and computational analyses, which might suggest the putative functions of miRNAs and circRNAs as a large class of post-transcriptional regulators. CONCLUSIONS:These findings will shed light on circRNA-miRNA interactions during the pathogenesis of toxoplasmosis, and they will lay solid foundation for studying the potential regulation roles of miRNAs and circRNAs in T. gondii induced pathogenesis.
Project description:The 7B-1 tomato line (Solanum lycopersicum cv. Rutgers) is a photoperiod-sensitive male-sterile mutant, with potential application in hybrid seed production. Small RNAs (sRNAs) in tomato have been mainly characterized in fruit development and ripening, but none have been studied with respect to flower development and regulation of male-sterility. Using sRNA sequencing, we identified miRNAs that are potentially involved in anther development and regulation of male-sterility in 7B-1 mutant.Two sRNA libraries from 7B-1 and wild type (WT) anthers were sequenced and thirty two families of known miRNAs and 23 new miRNAs were identified in both libraries. MiR390, miR166, miR159 were up-regulated and miR530, miR167, miR164, miR396, miR168, miR393, miR8006 and two new miRNAs, miR#W and miR#M were down-regulated in 7B-1 anthers. Ta-siRNAs were not differentially expressed and likely not associated with 7B-1 male-sterility. miRNA targets with potential roles in anther development were validated using 5'-RACE. QPCR analysis showed differential expression of miRNA/target pairs of interest in anthers and stem of 7B-1, suggesting that they may regulate different biological processes in these tissues. Expression level of most miRNA/target pairs showed negative correlation, except for few. In situ hybridization showed predominant expression of miR159, GAMYBL1, PMEI and cystatin in tapetum, tetrads and microspores.Overall, we identified miRNAs with potential roles in anther development and regulation of male-sterility in 7B-1. A number of new miRNAs were also identified from tomato for the first time. Our data could be used as a benchmark for future studies of the molecular mechanisms of male-sterility in other crops.
Project description:The regulatory roles of circular RNAs (circRNAs) in cancer are attracting increasing attention. The aim of the present study was to explore the roles of circRNAs in pancreatic ductal adenocarcinoma (PDAC) using microarray data. The circRNA and microRNA (miRNA) microarray data were downloaded from Gene Expression Omnibus. A total of 256 differentially expressed circRNAs were obtained by analyzing the circRNA microarray data from 26 pairs of PDAC and adjacent normal tissues. Differentially expressed miRNAs were analyzed using a dataset of 6 PDAC tissues and 5 non-neoplastic pancreas samples (GSE43796); 20 differentially expressed miRNAs were detected. circRNA/miRNA interactions were predicted between differentially expressed circRNAs and miRNAs using miRanda and RNAhybrid algorithms and 51 circRNA/miRNA interactions were obtained. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis using gene symbols of differentially expressed circRNAs demonstrated that 41 circRNAs were enriched in 17 pathways. Subnetworks that were associated with apoptosis or proliferation were extracted from the 17 pathways and a new network was constructed using Cytoscape software, which identified that mitogen-activated protein kinase, PI3K/AKT and WNT/β-catenin signaling pathways may be associated with PDAC development. In conclusion, 256 differentially expressed circRNAs and 20 differentially expressed miRNAs were identified in PDAC tissues compared with normal tissues; the circRNA/miRNA interactions and the networks of KEGG pathways provided a global view of the function of these differentially expressed circRNAs and miRNAs.
Project description:Circular RNAs (circRNAs) involve in the epigenetic regulation and its major mechanism is the sequestration of the target micro RNAs (miRNAs). We hypothesized that circRNAs might be related with the pathophysiology of chronic epilepsy and evaluated the altered circRNA expressions and their possible regulatory effects on their target miRNAs and mRNAs in a mouse epilepsy model. The circRNA expression profile in the hippocampus of the pilocarpine mice was analyzed and compared with control. The correlation between the expression of miRNA binding sites (miRNA response elements, MRE) in the dysregulated circRNAs and the expression of their target miRNAs was evaluated. As miRNAs also inhibit their target mRNAs, circRNA-miRNA-mRNA regulatory network, comprised of dysregulated RNAs that targets one another were searched. For the identified networks, bioinformatics analyses were performed. As the result, Forty-three circRNAs were dysregulated in the hippocampus (up-regulated, 26; down-regulated, 17). The change in the expression of MRE in those circRNAs negatively correlated with the change in the relevant target miRNA expression (r = -0.461, P<0.001), supporting that circRNAs inhibit their target miRNA. 333 dysregulated circRNA-miRNA-mRNA networks were identified. Gene ontology and pathway analyses demonstrated that the up-regulated mRNAs in those networks were closely related to the major processes in epilepsy. Among them, STRING analysis identified 37 key mRNAs with abundant (?4) interactions with other dysregulated target mRNAs. The dysregulation of the circRNAs which had multiple interactions with key mRNAs were validated by PCR. We concluded that dysregulated circRNAs might have a pathophysiologic role in chronic epilepsy by regulating multiple disease relevant mRNAs via circRNA-miRNA-mRNA interactions.
Project description:MicroRNAs (miRNAs) are a type of endogenous non-coding small RNAs that play critical roles in plant growth and developmental processes. Cytoplasmic male sterility (CMS) is typically a maternally inherited trait and widely used in plant heterosis utilization. However, the miRNA-mediated regulatory network of CMS occurrence during anther development remains largely unknown in radish. In this study, a comparative small RNAome sequencing was conducted in floral buds of CMS line 'WA' and its maintainer line 'WB' by high-throughput sequencing. A total of 162 known miRNAs belonging to 25 conserved and 24 non-conserved miRNA families were isolated and 27 potential novel miRNA families were identified for the first time in floral buds of radish. Of these miRNAs, 28 known and 14 potential novel miRNAs were differentially expressed during anther development. Several target genes for CMS occurrence-related miRNAs encode important transcription factors and functional proteins, which might be involved in multiple biological processes including auxin signaling pathways, signal transduction, miRNA target silencing, floral organ development, and organellar gene expression. Moreover, the expression patterns of several CMS occurrence-related miRNAs and their targets during three stages of anther development were validated by qRT-PCR. In addition, a potential miRNA-mediated regulatory network of CMS occurrence during anther development was firstly proposed in radish. These findings could contribute new insights into complex miRNA-mediated genetic regulatory network of CMS occurrence and advance our understanding of the roles of miRNAs during CMS occurrence and microspore formation in radish and other crops.
Project description:As circular RNAs (circRNAs) regulates the effect of micro RNAs (miRNAs), circRNA-miRNA-mRNA network might be implicated in various disease pathogenesis. Therefore, we evaluated the dysregulated circRNAs in the Tg2576 mouse Alzheimer's disease (AD) model, their possible regulatory effects on downstream target mRNAs, and their pathomechanistic role during the disease progression. The microarray-based circRNA expression analysis at seven- and twelve-months of ages (7?M and 12?M) returned 101 dysregulated circRNAs at 7?M (55 up-regulated and 46 down-regulated) and twelve dysregulated circRNAs at 12?M (five up-regulated and seven down-regulated). For each dysregulated circRNA, potential target miRNAs and their downstream target mRNAs were searched. Dysregulation of circRNAs was associated with increased frequency of relevant dysregulation of their downstream target mRNAs. Those differentially expressed circRNA-miRNA-mRNA regulatory network included 2,275 networks (876 for up-regulated circRNAs and 1,399 for down-regulated circRNAs) at 7?M and 38 networks (25 for up-regulated circRNAs and 13 for down-regulated circRNAs) at 12?M. Gene ontology (GO) and pathway analyses demonstrated that the dysregulated mRNAs in those networks represent the AD pathomechanism at each disease stage. We concluded that the dysregulated circRNAs might involve in the AD pathogenesis by modulating disease relevant mRNAs via circRNA-miRNA-mRNA regulatory networks.
Project description:Olive flounder (Paralichthys olivaceus) is an important economical flatfish in Japan, Korea, and China, but its production has been greatly threatened by disease outbreaks. In this research, we aimed to explore the immune responsive mechanism of P. olivaceus against Edwardsiella tarda infection by profiling the expression of circRNA, miRNA, and mRNA by RNA-seq and constructing a regulatory circular circRNA-miRNA-mRNA network. Illumina sequencing of samples from normal control (H0), 2 h (H2), 8 h (H8), and 12 h (H12) post-challenge was conducted. Differentially expressed (DE) circRNA (DE-circRNAs), miRNAs (DE-miRNAs), and mRNAs [differential expression genes (DEGs)] between challenge and control groups were identified, resulting in a total of 62 DE-circRNAs, 39 DE-miRNAs, and 3,011 DEGs. Based on the differentially expressed gene results, miRNA target interactions (circRNA-miRNA pairs and miRNA-mRNA pairs) were predicted by MiRanda software. Once these paired were combined, a preliminary circRNA-miRNA-mRNA network was generated with 198 circRNA-miRNA edges and 3,873 miRNA-mRNA edges, including 44 DE-circRNAs, 32 DE-miRNAs, and 1,774 DEGs. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was performed to evaluate the function of the DEGs in this network, and we focused and identified two important intestinal immune pathways (herpes simplex infection and intestinal immune network for IgA production) that showed statistical significance between the challenge and control groups. Furthermore, three critical DEGs (nectin2, MHC II ?-chain, and MHC II ?-chain) were identified, mapped into the preliminary circRNA-miRNA-mRNA network, and new circRNA-miRNA-mRNA regulatory networks were constructed. In conclusion, we, for the first time, identified circRNA-miRNA-mRNA network from P. olivaceus in the pathogenesis of E. tarda and provided valuable resources for further analyses of the molecular mechanisms and signaling networks.