Short Time-series Expression Miner Analysis Research Articles

Improving iPSC Differentiation Using a Nanodot Platform.

Differentiation of induced pluripotent stem cells (iPSCs) is an extremely complex process that has proven difficult to study. In this research, we utilized nanotopography to elucidate details regarding iPSC differentiation by developing a nanodot platform consisting of nanodot arrays of increasing diameter. Subjecting iPSCs cultured on the nanodot platform to a cardiomyocyte (CM) differentiation protocol revealed several significant gene expression profiles that were associated with poor differentiation. The observed expression trends were used to select existing small-molecule drugs capable of modulating differentiation efficiency. BRD K98 was repurposed to inhibit CM differentiation, while iPSCs treated with NSC-663284, carmofur, and KPT-330 all exhibited significant increases in not only CM marker expression but also spontaneous beating, suggesting improved CM differentiation. In addition, quantitative polymerase chain reaction was performed to determine the gene regulation responsible for modulating differentiation efficiency. Multiple genes involved in extracellular matrix remodeling were correlated with a CM differentiation efficiency, while genes involved in the cell cycle exhibited contrasting expression trends that warrant further studies. The results suggest that expression profiles determined via short time-series expression miner analysis of nanodot-cultured iPSC differentiation can not only reveal drugs capable of enhancing differentiation efficiency but also highlight crucial sets of genes related to processes such as extracellular matrix remodeling and the cell cycle that can be targeted for further investigation. Our findings confirm that the nanodot platform can be used to reveal complex mechanisms behind iPSC differentiation and could be an indispensable tool for optimizing iPSC technology for clinical applications.

Time-series analysis of hematopoietic stem cells.

This study aimed to investigate the molecular mechanisms underlying the aging of hematopoietic stem cells (HSCs). Gene expression profile GSE32719 was downloaded from the Gene Expression Omnibus database, including 14 young, 5 middle, and 8 old HSCs. Differential expression analysis, short time-series expression miner analysis, and weighted co-expression network analysis were conducted to screen for hub genes whose expression changed over time during HSC aging. Subsequently, functional enrichment and multiple regulatory network analyses of the hub genes were performed. A total of 124 intersecting time-dependent differentially expressed and module genes were obtained, which were considered hub genes whose expression changed over time during HSC aging. Hub genes were significantly enriched in pathways such as the Hippo and AMP-activated protein kinase (AMPK) signaling pathways. Moreover, AP-1 Transcription Factor Subunit (FOS) and sirtuin 1 (SIRT1) had higher degrees in the protein-protein interaction network, were regulated by more transcription factors (TFs), such as Sp1 transcription factor (SP1) and BRCA1 DNA repair-associated (BRCA1), in the TF-mRNA-miRNA network, were associated with more diseases in the disease-gene network, and could be targeted by more drugs in the drug-gene network. Furthermore, SIRT1 was targeted by miR-9-5p in the TF-mRNA-miRNA network. Hub genes such as FOS and SIRT1 and key pathways such as the Hippo and AMPK signaling pathways may play crucial roles in HSC aging. Moreover, FOS and SIRT1 were regulated by SP1 and BRCA1, respectively, during HSC aging. Furthermore, miR-9-5p may modulate HSC aging by targeting SIRT1. Thus, FOS and SIRT1 may be potential therapeutic targets for age-related hematopoietic dysfunction.

Integrated single-molecule real-time sequencing and RNA sequencing reveal the molecular mechanisms of salt tolerance in a novel synthesized polyploid genetic bridge between maize and its wild relatives

BackgroundTripsacum dactyloides (2n = 4x = 72) and Zea perennis (2n = 4x = 40) are tertiary gene pools of Zea mays L. and exhibit many abiotic adaptations absent in modern maize, especially salt tolerance. A previously reported allopolyploid (hereafter referred to as MTP, 2n = 74) synthesized using Zea mays, Tripsacum dactyloides, and Zea perennis has even stronger salt tolerance than Z. perennis and T. dactyloides. This allopolyploid will be a powerful genetic bridge for the genetic improvement of maize. However, the molecular mechanisms underlying its salt tolerance, as well as the key genes involved in regulating its salt tolerance, remain unclear.ResultsSingle-molecule real-time sequencing and RNA sequencing were used to identify the genes involved in salt tolerance and reveal the underlying molecular mechanisms. Based on the SMRT-seq results, we obtained 227,375 reference unigenes with an average length of 2300 bp; most of the unigenes were annotated to Z. mays sequences (76.5%) in the NR database. Moreover, a total of 484 and 1053 differentially expressed genes (DEGs) were identified in the leaves and roots, respectively. Functional enrichment analysis of DEGs revealed that multiple pathways responded to salt stress, including “Flavonoid biosynthesis,” “Oxidoreductase activity,” and “Plant hormone signal transduction” in the leaves and roots, and “Iron ion binding,” “Acetyl-CoA carboxylase activity,” and “Serine-type carboxypeptidase activity” in the roots. Transcription factors, such as those in the WRKY, B3-ARF, and bHLH families, and cytokinin negatively regulators negatively regulated the salt stress response. According to the results of the short time series-expression miner analysis, proteins involved in “Spliceosome” and “MAPK signal pathway” dynamically responded to salt stress as salinity changed. Protein–protein interaction analysis revealed that heat shock proteins play a role in the large interaction network regulating salt tolerance.ConclusionsOur results reveal the molecular mechanism underlying the regulation of MTP in the response to salt stress and abundant salt-tolerance-related unigenes. These findings will aid the retrieval of lost alleles in modern maize and provide a new approach for using T. dactyloides and Z. perennis to improve maize.

Transcriptome analysis provides insights into lignin synthesis and MAPK signaling pathway that strengthen the resistance of bitter gourd (Momordica charantia) to Fusarium wilt

Fusarium wilt is a typical soil-borne disease caused by Fusarium oxysporum f. sp. momordicae (FOM) in bitter gourd. In this study, by comparing sequencing data at multiple time points and considering the difference between resistant (R) and susceptible (S) varieties, differentially expressed genes were screened out. Short time-series expression miner analysis revealed the upregulated expression trend of genes, which were enriched in phenylpropanoid biosynthesis, plant–pathogen interaction, and mitogen-activated protein kinase signaling pathway. Further, observation of the microstructure revealed that the R variety may form tyloses earlier than the S variety to prevent mycelium diffusion from the xylem vessel. After Fusarium wilt infection, the enzymatic activities of superoxide dismutase, peroxidase, phenylalanine ammonia lyase, and catalaseas well as levels of superoxide anion and malondialdehyde were increased in the R variety higher than those in the S variety. This study provides a reference to elucidate the disease resistance mechanism of bitter gourd.

Identification of Key Genes Associated with Risk and Prognosis of Neuroblastoma.

Neuroblastoma is a childhood malignancy with high morbidity and mortality. We identified key biomarkers associated with neuroblastoma risk and prognosis. The gene modules most associated with neuroblastoma risk were derived by WGCNA. Modular genes were intersected with differentially expressed genes between patients with high-risk (HR) and non-high-risk (NHR) to obtain risk genes, and enrichment analysis was performed. After incorporating risk genes into Cox regression analysis, LASSO algorithm, and K-M survival analysis, key genes were identified and introduced into four external datasets for validation. We performed short time-series expression miner analysis and single-sample genome enrichment analysis. Finally, we evaluated the difference in DNA methylation levels to identify meaningful methylation marks. We identified 5 key genes (ANO6, CPNE2, DST, PLXNC1, SCN3A) for neuroblastoma risk and prognosis, which correlated closely with known neuroblastoma biomarkers. All key genes showed a progressive downregulation trend with increasing risk levels of neuroblastoma. The immune infiltration of 14 immune cells was significantly different between HR-NB and NHR-NB, and most immune cells were negatively correlated with key genes. Furthermore, the expression of ANO6, CPNE2, DST, and PLXNC1 was modified by DNA methylation. This study identified 5 key genes for neuroblastoma risk and prognosis that were potential biomarkers.

Biosynthetic mechanisms of isoflavone accumulation affected by different growth patterns in Astragalus mongholicus products

BackgroundAt present, Astragalus mongholicus products on the market represent two growth patterns: imitative wild A. mongholicus (WAM) and cultivated A. mongholicus (CAM). The 6-year-old WAM (A6) and 2-year-old CAM (B2) products are often sold as commodities. This study aimed to explore the effects of the abovementioned growth patterns on the biosynthetic mechanisms of isoflavone accumulation in A. mongholicus products.ResultsIn this paper, the content of calycosin-7-O-β-D-glucoside in 6-year-old WAM (A6) was significantly higher than that in 2-year-old CAM (B2) based on high-performance liquid chromatography. Tissue anatomy indicated that A6 has developed phloem fibers, thickened secondary walls, and a more well-developed vascular system than B2. Thirteen differentially accumulated metabolites were found in A6 and B2 by UHPLC-ESI-Q-TOF-MS/MS, of which isoflavones were highly and significantly enriched in A6. By combining transcriptomics and metabolomics analysis, we found that the metabolomics profile was the same as the transcriptomics profile in both A6 and B2. In total, 11 novel isoflavone-related genes were isolated using BLAST and functional annotation through RNA-Seq and Iso-Seq. The results of integrated analysis, Short Time-series Expression Miner analysis, and Pearson correlation analysis showed that the regulation of four key enzymes, cinnamate 4-hydroxylase, 6-deoxychalcone synthase, chalcone reductase, and chalcone isomerase, led to the high accumulation of isoflavones in A6. In addition, AmUFGT (c778119) and AmUCGT (c303354) were predicted to be 7-O-glycosyltransferases by phylogenetic analysis; these genes catalyze formononetin and calycosin, respectively.ConclusionsThe findings of this work will clarify the differences in the biosynthetic mechanism of isoflavone accumulation between A6 and B2, which will guide the cultivation of A. mongholicus.

Dynamic transcriptome analysis of ovarian follicles in artificial maturing Japanese eel (Anguilla japonica)

Inducing maturation of the ovaries to enable the production of good-quality eggs is critical for the successful artificial breeding of Anguilla japonica. During the spawning season, however, the ovaries of A. japonica have been found to develop into asynchronous clutches, impeding the success of artificial breeding on a commercial scale. The dynamic molecular regulation of follicular development in the same individual was assessed by transcriptome analysis of the five stages of follicles, the pre-vitellogenic, early vitellogenic, midvitellogenic, late vitellogenic, and migratory nucleus stages in artificial maturing A. japonica. Comparisons across these developmental stages identified a total of 19,298 differentially expressed transcripts (DETs). Short time-series expression miner analysis across these DETs revealed four significant expression profiles. Gene Ontology function and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses found that some of the significantly enriched biological processes and metabolic pathways included those related to steroid hormone biosynthesis (cyp11a1, cyp17a1, cyp17a2, hsd17b1, and hsd17b12), cargo receptor activity (vtgr and vldlr), meiosis and ovulation (pgrs and mPRγ), hydration (cts and aqp1), and egg coat formation (zp). These genes and pathways were associated with serum 17β-estradiol concentrations and morphological changes. The levels of hsd17b12 and mPRγ mRNAs were much higher during the migratory nucleus stage, suggesting their respective involvement in the biosynthesis and functional pathway of the maturation-inducing steroid 17α,20β-dihydroxy-4-pregnen-3-one. The gene subtypes aqp1b and ctsd may regulate water influx into oocytes and yolk protein proteolysis, respectively. To our knowledge, the present study is the first to describe combined transcriptome profiling of asynchronously developing follicles in the same individual. The findings suggest that steroid hormone synthesis and nutrient absorption in follicular somatic cells play important roles during follicular development and maturation, despite the same external physiological surroundings.

Spatiotemporal Regulation and Functional Analysis of Circular RNAs in Skeletal Muscle and Subcutaneous Fat during Pig Growth

Simple SummaryThis study provides a clear and accurate dynamic profile of circRNAs in skeletal muscle and subcutaneous fat of Ningxiang pig. The results include high-quality genomic data, and help to elucidate the roles of circRNAs in regulation of protein and lipid synthesis and metabolism providing new ideas for further understanding of the molecular mechanisms governing pork quality traits. Recently, thousands of circular RNAs have been reported in different pig breeds. However, researches on the temporal and spatial expression patterns of circRNA over the period of animal growth are limited. Here, we systematically analyzed circRNAs in skeletal muscle and subcutaneous fat in four growth time points (30 days, 90 days, 150 days and 210 days after birth) of a Chinese native pig breed, Ningxiang pigs. A total of 1171 differentially expressed (DE) circRNAs between muscle and fat were identified, including 562 upregulated and 609 downregulated circRNAs. KEGG pathway enrichment analysis of these DE circRNAs revealed that host genes were mainly involved in glycogen metabolism signaling pathways, muscle development signaling pathways such as ErbB pathway and adipocytokine signaling pathways and AMPK signaling pathways and fatty acid biosynthesis. The circRNAs have striking spatiotemporal specificity in the form of dynamic expression at 90 d. Short Time-Series Expression Miner analysis showed multiple model spectra that are significantly enriched with time changes in muscle and fat. Our findings provide new ideas and perspectives about the role of circular RNAs and their targeting relations with mRNA and miRNA in skeletal muscle and fat tissue during pig growth.

Deciphering the miRNA transcriptome of breast muscle from the embryonic to post-hatching periods in chickens

BackgroundmiRNAs play critical roles in growth and development. Various studies of chicken muscle development have focused on identifying miRNAs that are important for embryo or adult muscle development. However, little is known about the role of miRNAs in the whole muscle development process from embryonic to post-hatching periods. Here, we present a comprehensive investigation of miRNA transcriptomes at 12-day embryo (E12), E17, and day 1 (D1), D14, D56 and D98 post-hatching stages.ResultsWe identified 337 differentially expressed miRNAs (DE-miRNAs) during muscle development. A Short Time-Series Expression Miner analysis identified two significantly different expression profiles. Profile 4 with downregulated pattern contained 106 DE-miRNAs, while profile 21 with upregulated pattern contained 44 DE-miRNAs. The DE-miRNAs with the upregulated pattern mainly played regulatory roles in cellular turnover, such as pyrimidine metabolism, DNA replication, and cell cycle, whereas DE-miRNAs with the downregulated pattern directly or indirectly contributed to protein turnover metabolism such as glycolysis/gluconeogenesis, pyruvate metabolism and biosynthesis of amino acids.ConclusionsThe main functional miRNAs during chicken muscle development differ between embryonic and post-hatching stages. miRNAs with an upregulated pattern were mainly involved in cellular turnover, while miRNAs with a downregulated pattern mainly played a regulatory role in protein turnover metabolism. These findings enrich information about the regulatory mechanisms involved in muscle development at the miRNA expression level, and provide several candidates for future studies concerning miRNA-target function in regulation of chicken muscle development.

MicroRNA expression profiling reveals potential roles for microRNA in the liver during pigeon (Columba livia) development

The liver is the central organ for metabolism and influence the growth and development of the animals. To date, little is known about the microRNA (miRNA) in pigeon livers, particularly in different developmental stages. A comprehensive investigation into miRNA transcriptomes in livers across 3 pigeon developmental stages (1, 14, 28 d old) and an adult stage (2 y old) was performed by small RNA sequencing. We identified 312 known miRNA, 433 conserved miRNA, and 192 novel miRNA in pigeon livers. A set of differentially expressed (DE) miRNA in livers were screened out during pigeon development. This set of miRNA might be involved in hepatospecific phenotype and liver development. A Short Time-series Expression Miner analysis indicated significant expression variations in DE miRNA during liver development of pigeons. These DE miRNA with different expression patterns might play essential roles in response to growth factor, cell morphogenesis, and gland development, etc. Protein–protein interaction network and Molecular Complex Detection analysis identified several vital target genes (e.g., TNRC6B, FRS2, PTCH1, etc.) of DE miRNA, which is closely linked in liver development and enriched in PI3K cascade and regulation of growth. Our results expanded the repertoire of pigeon miRNA and may be of help in better understanding the mechanism of squab's rapid development from the perspective of liver development.

MRNA and miRNA Transcriptome Profiling of Granulosa and Theca Layers From Geese Ovarian Follicles Reveals the Crucial Pathways and Interaction Networks for Regulation of Follicle Selection.

Follicle development is characterized by the recruitment, growth, selection, and dominance of follicles, and follicle selection determines the lifetime reproductive performance. However, in birds, the molecular mechanisms underlying follicle selection still remain elusive. This study analyzed genome-wide changes in the mRNA and miRNA expression profiles in both the granulosa and theca layers of geese ovarian follicles before selection (4–6- and 8–10-mm follicles) and after selection (F5). The sequencing results showed that a higher number of both differentially expressed (DE) mRNAs and DE miRNAs were identified between 8–10-mm and F5 follicles compared with those between the 4–6- and 8–10-mm follicles, especially in the granulosa layer. Moreover, a Short Time-series Expression Miner analysis identified a large number of DE mRNAs and DE miRNAs that are associated with follicle selection. The functional enrichment analysis showed that DE genes in the granulosa layer during follicle selection were mainly enriched in five pathways related to junctional adhesion and two pathways associated with lipid metabolism. Additionally, an interaction network was constructed to visualize interactions among protein-coding genes, which identified 53 junctional adhesion- and 15 lipid regulation-related protein-coding genes. Then, a co-expression network between mRNAs and miRNAs in relation to junctional adhesion was also visualized and mainly included acy-miR-2954, acy-miR-218, acy-miR-2970, acy-miR-100, acy-miR-1329, acy-miR-199, acy-miR-425, acy-miR-181, and acy-miR-147. Furthermore, miRNA–mRNA interaction pairs related to lipid regulation were constructed including acy-miR-107, acy-miR-138, acy-miR-130, acy-miR-128, and acy-miR-101 during follicular selection. In summary, these data highlight the key roles of junctional adhesion and lipid metabolism during follicular selection and contribute to a better understanding of the mechanisms underlying follicle selection in birds.

Testicular transcriptome alterations in zebrafish (Danio rerio) exposure to 17β-estradiol

The hormone 17β-estradiol (E2) can be found in rivers, effluents, and even drinking water. Researches have demonstrated that E2 affects various metabolic pathways through gene activation and may cause reproductive toxicity in fish. Therefore, the aim of this study was to evaluate E2-induced toxicity via testicular transcriptome of zebrafish (Danio rerio) exposed to different concentrations (10 ng L−1, and 100 ng L−1) of E2. A total of >600 significant differentially expressed genes (DEGs) were enriched among the three treatments. Short time-series expression miner analysis revealed five KEGG pathways including drug metabolism, other enzymes, calcium signaling pathway, ECM-receptor interaction, gap junction, and cell adhesion molecules. Twenty genes were selected to verify the accuracy of RNA-Seq. Other reported genes related to sex differentiation, development, energy metabolism, and other processes were found. One set of genes significantly increased/decreased/fluctuated over time, especially 12 h after E2 exposure. Genes associated with ovaries (zp3c), and development (bmp15, gdf9, and sycp2l) were significantly upregulated with increasing E2 concentration. E2 and testosterone was significantly decreased by 10 (except for T) and 100 ng L−1 E2 exposure at 12 h. The current study demonstrated that sex differentiation, development, energy metabolism, immunity, and ribosome biogenesis in male zebrafish were all significantly affected by 17β-estradiol exposure through transcriptional alterations.

Comprehensive Analysis of MicroRNA⁻Messenger RNA from White Yak Testis Reveals the Differentially Expressed Molecules Involved in Development and Reproduction.

Testis development is a vital and tightly regulated process in mammals. Understanding the biological mechanisms underlying testis development will benefit the animal reproduction industry. Expression changes in microRNA and messenger RNA in response to dynamic regulation effects have been associated with this process. However, very little is known about the roles of these molecules in yak development. Using whole-genome small RNA and messenger RNA sequencing, we performed a comprehensive analysis of the microRNA–messenger RNA interaction network expression in the testicles of Tianzhu white yaks during three developmental stages. Using Short Time-series Expression Miner analysis we identified 589 differentially expressed microRNAs (DERs) and 3383 differentially expressed messenger RNAs (DEGs) in the three age groups. A total of 93 unique DEGs are primarily involved in reproduction and testis development. Subsequently, four integration networks were constructed according to the DEGs and DERs in three biological processes. Nineteen DEGs were potentially regulated by 60 DERs, of which miR-574 and target gene AURKA played a crucial role in yak testis development and reproduction. The results of this study provide a basis for further exploration of the microRNA–messenger RNA interactions in testis development and reproduction and aid in uncovering the molecular mechanisms of spermatogenesis in male mammals.

Comprehensive long non-coding RNA expression profiling reveals their potential roles in systemic lupus erythematosus

Long non-coding RNAs can regulate gene transcription, modulate protein function, and act as competing endogenous RNA. Yet, their roles in systemic lupus erythematosus remain to be elucidated. We determined the expression profiles of lncRNAs in T cells of SLE patients and healthy controls using microarrays. Up to 1935 lncRNAs and 1977 mRNAs were differentially expressed. QRT-PCR showed downregulated uc001ykl.1 and ENST00000448942 in SLE patients. Expression of uc001ykl.1 correlated with erythrocyte sedimentation rate (ESR) and C-reactive protein, whereas ENST00000448942 level correlated with ESR and anti-Sm antibodies. Short time-series expression miner analysis revealed some lncRNAs whose expressions might correlate with disease activity of SLE patients. Coding-non-coding gene coexpression analyses showed differential lncRNAs might operate via modulating expressions of their correlated, relevant mRNAs in SLE. Differential lncRNAs might also function through their ceRNAs. Our study established that the aberrant expression profiles of lncRNAs may play a role in SLE and thus warrant further investigation.

Deciphering the microRNA transcriptome of skeletal muscle during porcine development.

MicroRNAs (miRNAs) play critical roles in many important biological processes, such as growth and development in mammals. Various studies of porcine muscle development have mainly focused on identifying miRNAs that are important for fetal and adult muscle development; however, little is known about the role of miRNAs in middle-aged muscle development. Here, we present a comprehensive investigation of miRNA transcriptomes across five porcine muscle development stages, including one prenatal and four postnatal stages. We identified 404 known porcine miRNAs, 118 novel miRNAs, and 101 miRNAs that are conserved in other mammals. A set of universally abundant miRNAs was found across the distinct muscle development stages. This set of miRNAs may play important housekeeping roles that are involved in myogenesis. A short time-series expression miner analysis indicated significant variations in miRNA expression across distinct muscle development stages. We also found enhanced differentiation- and morphogenesis-related miRNA levels in the embryonic stage; conversely, apoptosis-related miRNA levels increased relatively later in muscle development. These results provide integral insight into miRNA function throughout pig muscle development stages. Our findings will promote further development of the pig as a model organism for human age-related muscle disease research.