Antifungal mechanism of volatile compounds emitted by Actinomycetota Paenarthrobacter ureafaciens from a disease-suppressive soil on Saccharomyces cerevisiae.

Increasing evidence suggests that in disease-suppressive soils, microbial volatile compounds (mVCs) released from bacteria may inhibit the growth of plant-pathogenic fungi. However, the antifungal activities and molecular responses of fungi to different mVCs remain largely undescribed. In this study, we first evaluated the responses of pathogenic fungi to treatment with mVCs from Paenarthrobacter ureafaciens. Then, we utilized the well-characterized fungal model organism Saccharomyces cerevisiae to study the potential mechanistic effects of the mVCs. Our data showed that exposure to P. ureafaciens mVCs leads to reduced growth of several pathogenic fungi, and in yeast cells, mVC exposure prompts the accumulation of reactive oxygen species. Further experiments with S. cerevisiae deletion mutants indicated that Slt2/Mpk1 and Hog1 MAPKs play major roles in the yeast response to P. ureafaciens mVCs. Transcriptomic analysis revealed that exposure to mVCs was associated with 1,030 differentially expressed genes (DEGs) in yeast. According to gene ontology and Kyoto Encyclopedia of Genes and Genomes analyses, many of these DEGs are involved in mitochondrial dysfunction, cell integrity, mitophagy, cellular metabolism, and iron uptake. Genes encoding antimicrobial proteins were also significantly altered in the yeast after exposure to mVCs. These findings suggest that oxidative damage and mitochondrial dysfunction are major contributors to the fungal toxicity of mVCs. Furthermore, our data showed that cell wall, antioxidant, and antimicrobial defenses are induced in yeast exposed to mVCs. Thus, our findings expand upon previous research by delineating the transcriptional responses of the fungal model. IMPORTANCE Since the use of bacteria-emitted volatile compounds in phytopathogen control is of considerable interest, it is important to understand the molecular mechanisms by which fungi may adapt to microbial volatile compounds (mVCs). Paenarthrobacter ureafaciens is an isolated bacterium from disease-suppressive soil that belongs to the Actinomycetota phylum. P. ureafaciens mVCs showed a potent antifungal effect on phytopathogens, which may contribute to disease suppression in soil. However, our knowledge about the antifungal mechanism of mVCs is limited. This study has proven that mVCs are toxic to fungi due to oxidative stress and mitochondrial dysfunction. To deal with mVC toxicity, antioxidants and physical defenses are required. Furthermore, iron uptake and CAP proteins are required for antimicrobial defense, which is necessary for fungi to deal with the thread from mVCs. This study provides essential foundational knowledge regarding the molecular responses of fungi to inhibitory mVCs.

Volatile cinnamaldehyde induces systemic salt tolerance in the roots of rice (Oryza sativa).

Cinnamaldehyde (CA) is a volatile plant secondary metabolite that exhibits strong anti-pathogenic activities. Nonetheless, less is known about the effect of CA on plant tolerance to abiotic stresses. In this study, we delineated the effects of CA fumigation on rice roots (Oryza Sativa L cv. TNG67) under salinity stress (200 mM NaCl). Our result showed that CA vapor significantly alleviated salinity-induced ROS accumulation and cell death. This CA-induced alleviation appears to be mediated primarily by the upregulation of proline metabolism genes, the rapid proline accumulation, and the decrease of Na+ /K+ ratio as early as 3 h after NaCl treatment. Of note, the activities of peroxidase (POD; EC 1.11.1.7) isozymes a and b were decreased by CA fumigation, and the activities of catalase (CAT; EC 1.11.1.6) and superoxide dismutase (SOD; EC 1.15.1.1) were not significantly affected. Our findings suggest that CA vapor might be useful for priming rice roots to withstand salinity stress, which is more prevalent due to the ongoing global climate change. To the best of our knowledge, this is the first study to show modulation of macro- and micro-elements as well as antioxidative factors after CA fumigation of salinity-stressed rice roots.

The involvement of AtMKK1 and AtMKK3 in plant-deleterious microbial volatile compounds-induced defense responses.

KEY MESSAGE: Plant-deleterious microbial volatiles activate the transactivation of hypoxia, MAMPs and wound responsive genes in Arabidopsis thaliana. AtMKK1 and AtMKK3 are involved in the plant-deleterious microbial volatiles-induced defense responses. Microbial volatile compounds (mVCs) are a collection of volatile metabolites from microorganisms with biological effects on all living organisms. mVCs function as gaseous modulators of plant growth and plant health. In this study, the defense events induced by plant-deleterious mVCs were investigated. Enterobacter aerogenes VCs lead to growth inhibition and immune responses in Arabidopsis thaliana. E. aerogenes VCs negatively regulate auxin response and transport gene expression in the root tip, as evidenced by decreased expression of DR5::GFP, PIN3::PIN3-GFP and PIN4::PIN4-GFP. Data from transcriptional analysis suggests that E. aerogenes VCs trigger hypoxia response, innate immune responses and metabolic processes. In addition, the transcript levels of the genes involved in the synthetic pathways of antimicrobial metabolites camalexin and coumarin are increased after the E. aerogenes VCs exposure. Moreover, we demonstrate that MKK1 serves as a regulator of camalexin biosynthesis gene expression in response to E. aerogenes VCs, while MKK3 is the regulator of coumarin biosynthesis gene expression. Additionally, MKK1 and MKK3 mediate the E. aerogenes VCs-induced callose deposition. Collectively, these studies provide molecular insights into immune responses by plant-deleterious mVCs.

Mysteries of gene regulation: Promoters are not the sole triggers of gene expression.

Cis-regulatory elements of promoters are essential for gene regulation by transcription factors (TFs). However, the regulatory roles of nonpromoter regions, TFs, and epigenetic marks remain poorly understood in plants. In this study, we characterized the cis-regulatory regions of 53 TFs and 19 histone marks in 328 chromatin immunoprecipitation (ChIP-seq) datasets from Arabidopsis. The genome-wide maps indicated that both promoters and regions around the transcription termination sites of protein-coding genes recruit the most TFs. The maps also revealed a diverse of histone combinations. The analysis suggested that exons play critical roles in the regulation of non-coding genes. Additionally, comparative analysis between heat-stress-responsive and nonresponsive genes indicated that the genes with distinct functions also exhibited substantial differences in cis-regulatory regions, histone regulation, and topologically associating domain (TAD) boundary organization. By integrating multiple high-throughput sequencing datasets, this study generated regulatory models for protein-coding genes, non-coding genes, and TAD boundaries to explain the complexity of transcriptional regulation.

LncRNA LINC00460 facilitates the proliferation and metastasis of renal cell carcinoma via PI3K/AKT signaling pathway.

Renal cell carcinoma (RCC) is one of the most prevalent cancers diseases in the worldwide. Long noncoding RNAs (LncRNAs) have been indicated as a mediator acted in tumorigenesis of RCC. LINC00460 has been reported to participate in many kinds of malignancies and promotes cancer progressions. However, the mechanism of LINC00460 on RCC is yet to be investigated. This study aimed to explore the potential function and regulation mechanism of LINC00460 in RCC. We analysed the LINC00460 expression and the prognosis in RCC patients using Gene Expression Profiling Interactive Analysis (GEPIA) and The Cancer Genome Atlas (TCGA) databases. LINC00460 level in normal renal cell line and RCC cell lines were examined by qRT-PCR. We study the effects of LINC00460 on proliferation, migration, invasion, apoptosis in RCC cells lines using a series of in vivo and in vitro experiments. RNA sequencing (RNA-seq) analysis was applied to searching potential LINC00460 related signal pathway in RCC. We identified the significant up-regulated expression of LINC00460 both in RCC tissues and cell. RCC patients with elevated LINC00460 expression have shorter survival. Up-expression of LINC00460 promoted cell proliferation, invasion and migration, meanwhile down-regulation of LINC00460 exerted inhibitory effect on these activities. We crucially identified that LNC00460 promotes development of RCC by influencing the PI3K/AKT pathway. Knockdown of LNC00460 decreased the phosphorylation of AKT and mTOR. The key finding of our study showed that LINC00460 functions as an oncogene in RCC pathogenesis by mediating the PI3K/AKT.

Role of Circular RNA in Kidney-Related Diseases.

The kidney is vital in maintaining fluid, electrolyte, and acid-base balance. Kidney-related diseases, which are an increasing public health issue, can happen to people of any age and at any time. Circular RNAs (circRNAs) are endogenous RNA that are produced by selective RNA splicing and are involved in progression of various diseases. Studies have shown that various kidney diseases, including renal cell carcinoma, acute kidney injury, and chronic kidney disease, are linked to circRNAs. This review outlines the characteristics and biological functions of circRNAs and discusses specific studies that provide insights into the function and potential of circRNAs for application in the diagnosis and treatment of kidney-related diseases.

Reduced expression of annexin A1 promotes gemcitabine and 5-fluorouracil drug resistance of human pancreatic cancer.

Intrinsic chemoresistance is the main reason for the failure of human pancreatic ductal adenocarcinoma (PDAC) therapy. To identify the candidate protein, we compared the protein expression profiling of PDAC cells and its distinct surviving cells following primary treatment with gemcitabine (GEM) and 5-fluorouracil (5-FU) by two-dimensional electrophoresis combined with liquid chromatography-mass spectrometry or mass spectrometry. A total of 20 differentially expressed proteins were identified, and annexin A1 (ANXA1) was analyzed for further validation. The functional validation showed that the downregulation of ANXA1 contributes to GEM and 5-FU resistance in PDAC cells through protein kinase C/c-Jun N-terminal kinase/P-glycoprotein signaling pathway. Our findings provide a platform for the further elucidation of the underlying mechanisms of PDAC intrinsic chemoresistance and demonstrated that ANXA1 may be a valid marker for anticancer drug development.

Functional roles of circular RNAs during epithelial-to-mesenchymal transition.

Cancer has become a major health issue worldwide, contributing to a high mortality rate. Tumor metastasis is attributed to the death of most patients. Epithelial-to-mesenchymal transition (EMT) plays a vital role in inducing metastasis. During EMT, epithelial cells lose their characteristics, such as cell-to-cell adhesion and cell polarity, and cells gain motility, migratory potential, and invasive properties to become mesenchymal stem cells. Circular RNAs (circRNAs) are closely associated with tumor metastasis and patient prognosis, as revealed by increasing lines of evidence. CircRNA is a type of single-stranded RNA that forms a covalently closed continuous loop. CircRNAs are insensitive to ribonucleases and are widespread in body fluids. This work is the first review on EMT-related circRNAs. In this review, we briefly discuss the characteristics and functions of circRNAs. The correlation of circRNAs with EMT has been reported, and we discuss the ways circRNAs can regulate EMT progression through EMT transcription factors, EMT-related signaling pathways, and other mechanisms. This work summarizes current studies on EMT-related circRNAs in various cancers and provides a theoretical basis for the use of EMT-related circRNAs in targeted management and therapy.

Emerging Roles of p53 Related lncRNAs in Cancer Progression: A Systematic Review.

p53 is the major mediator of the tumor suppressor response. It participates in apoptosis and senescence and can respond to DNA damage. As a crucial sequence-specific transcription factor, p53 regulates the expression of many genes, such as small noncoding RNAs (ncRNAs), microRNAs, and long ncRNAs (lncRNAs). Given the emergence of novel and high-throughput sequencing technologies, many lncRNAs have been discovered. LncRNAs may function as vital gene regulators in a variety of biological processes through extensive mechanisms. Recently, lncRNAs have been demonstrated to be associated with the p53 regulatory pathway. In this review, we discuss the current and fast growing knowledge about the influence of lncRNAs to the p53 signaling pathway, the different mechanisms by which they affect gene expression in cancer. Our findings show that p53-associated lncRNAs may be used as biomarkers for cancer diagnosis or targets for disease therapy.

The roles of Wnt/ß-catenin signaling pathway related lncRNAs in cancer.

Long noncoding RNAs (lncRNAs), with length of more than 200 nucleotides, are not translated into proteins but involved in multiple diverse diseases, especially tumorigenesis. The dysregulation of lncRNAs greatly contributes to the progression of various tumors through specific signaling pathways, including Wnt/ß-catenin signaling pathway, which is associated with malignant features of tumors. The interactions between lncRNAs, which have specific expression characteristics in diverse cancer tissues, and Wnt/ß-catenin signaling pathway, exhibit potential as novel biomarkers and therapeutic targets. In this review, we aim to present research findings on the roles of Wnt pathway-related lncRNAs and their effects on Wnt/ß-catenin signaling to regulate tumorigenesis in different cancer types. Results may be used as basis to develop or improve strategies for treatment of different carcinomas.

CUL1 promotes breast cancer metastasis through regulating EZH2-induced the autocrine expression of the cytokines CXCL8 and IL11.

CUL1 is an essential component of SCF (SKP1-CUL1-F-box protein) E3 ubiquitin ligase complex. Our previous study has showed that CUL1 is positively associated with poor overall and disease-specific survival of breast cancer patients. Here, we further explored its roles in breast cancer metastasis. Our data showed that CUL1 significantly promoted breast cancer cell migration, invasion, tube formation in vitro, as well as angiogenesis and metastasis in vivo. In mechanism, the human gene expression profiling was used to determine global transcriptional changes in MDA-MB-231 cells, and we identified autocrine expression of the cytokines CXCL8 and IL11 as the target genes of CUL1 in breast cancer cell migration, invasion, metastasis, and angiogenesis. CUL1 regulated EZH2 expression to promote the production of cytokines, and finally significantly aggravating the breast cancer cell metastasis and angiogenesis through the PI3K-AKT-mTOR signaling pathway. Combined with the previous report about CUL1, we proposed that CUL1 may serve as a promising therapeutic target for breast cancer metastasis.

KIF4A facilitates cell proliferation via induction of p21-mediated cell cycle progression and promotes metastasis in colorectal cancer.

Kinesin family member 4A (KIF4A) was found to be implicated in the regulation of chromosome condensation and segregation during mitotic cell division, which is essential for eukaryotic cell proliferation. However, little is known about the role of KIF4A in colorectal carcinoma (CRC). This study explored the biological function of KIF4A in CRC progression and investigated the potential molecular mechanisms involved. Here, we found that KIF4A was remarkably upregulated in primary CRC tissues and cell lines compared with paired non-cancerous tissues and normal colorectal epithelium. Elevated expression of KIF4A in CRC tissues was significantly correlated with clinicopathological characteristics in patients as well as with shorter overall and disease-free cumulative survival. Multivariate Cox regression analysis revealed that KIF4A was an independent prognostic factor for poor survival in human CRC patients. Functional assays, including a CCK-8 cell proliferation assay, colony formation analysis, cancer xenografts in nude mice, cell cycle and apoptosis analysis, indicated that KIF4A obviously enhanced cell proliferation by promoting cell cycle progression in vitro and in vivo. Furthermore, gene set enrichment analysis, Luciferase reporter assays, and ChIP assays revealed that KIF4A facilitates cell proliferation via regulating the p21 promoter, whereas KIF4A had no effect on cell apoptosis. In addition, Transwell analysis indicated that KIF4A promotes migration and invasion in CRC. Taken together, these findings not only demonstrate that KIF4A contributes to CRC proliferation via modulation of p21-mediated cell cycle progression but also suggest the potential value of KIF4A as a clinical prognostic marker and target for molecular treatments.

Delineation of condition specific Cis- and Trans-acting elements in plant promoters under various Endo- and exogenous stimuli.

BACKGROUND: Transcription factors (TFs) play essential roles during plant development and response to environmental stresses. However, the relationships among transcription factors, cis-acting elements and target gene expression under endo- and exogenous stimuli have not been systematically characterized. RESULTS: Here, we developed a series of bioinformatics analysis methods to infer transcriptional regulation by using numerous gene expression data from abiotic stresses and hormones treatments. After filtering the expression profiles of TF-encoding genes, 291 condition specific transcription factors (CsTFs) were obtained. Differentially expressed genes were then classified into various co-expressed gene groups based on each CsTFs. In the case studies of heat stress and ABA treatment, several known and novel cis-acting elements were identified following our bioinformatics approach. Significantly, a palindromic sequence of heat-responsive elements is recognized, and also obtained from a 3D protein structure of heat-shock protein-DNA complex. Notably, overrepresented 3- and 4-mer motifs in an enriched 8-mer motif could be a core cis-element for a CsTF. In addition, the results suggest DNA binding preferences of the same CsTFs are different according to various conditions. CONCLUSIONS: The overall results illustrate this study may be useful in identifying condition specific cis- and trans- regulatory elements and facilitate our understanding of the relationships among TFs, cis-acting elements and target gene expression.

Paddy-upland rotation for sustainable agriculture with regards to diverse soil microbial community.

Diverse soil microbial community is determinant for sustainable agriculture. Rich microbial diversity has presumably improved soil health for economic crops to grow. In this work, the benefits of paddy-upland rotation on soil microbial diversity and specific microbes are thus intensively explored. The microbiome from multiple factor experiment (three fertilizations coupled with two rotation systems) were investigated by novel enrichment and co-occurrence analysis in a field well maintained for 25 years. Using next-generation sequencing technique, we firstly present explicit evidence that different rotation systems rather than fertilizations mightily governed the soil microbiome. Paddy-upland rotation (R1) obviously increase more microbial diversity than upland rotation (R2) whether organic (OF), chemical (CF) or integrated fertilizers (IF) were concomitantly applied. Besides, the specific bacterial composition dominated in OF soil is more similar to that of R1 than to CF, suggesting that paddy-upland rotation might be the best option for sustainable agriculture if chemical fertilizer is still required. Interestingly, the pot bioassay verified clearly the novel analysis prediction, illustrating that greater microbial diversity and specific microbial composition correlated significantly with disease resistance. This finding highlights the eminence of paddy-upland rotation in promoting microbial diversity and specific microbial compositions, preserving soil health for sustainable agriculture.

XRCC1 serves as a potential prognostic indicator for clear cell renal cell carcinoma and inhibits its invasion and metastasis through suppressing MMP-2 and MMP-9.

X-ray repair cross-complementing group 1 (XRCC1) is a major DNA repair gene that is responsible for fixing DNA base damage and single-strand breaks by interacting with DNA components at the damage site. This study explored the clinical significance of XRCC1 in human clear cell renal cell carcinoma (ccRCC) and further examined the mechanism of the role of XRCC1 in ccRCC. The clinical relevance of XRCC1 in ccRCC was evaluated using tissue microarrays and immunohistochemical staining of two independent human ccRCC cohorts. Our data demonstrated that XRCC1 expression was dramatically decreased in ccRCC tissues compared with that in normal renal tissues and paired adjacent non-tumor tissues. Low XRCC1 expression was significantly correlated with lymph node metastasis and with worse overall and disease-specific survival in patients, as determined by log-rank tests. However, Cox regression analysis revealed that XRCC1 expression was not an independent prognostic factor in ccRCC patients. Furthermore, XRCC1 suppressed ccRCC migration and invasion by inhibiting MMP-2 and MMP-9 expression through the regulation of TIMP-2 and TIMP-1. These findings indicated that decreased XRCC1 expression was associated with lymph node metastasis but was not an independent prognostic factor in ccRCC patients. XRCC1 may serve as a potential therapeutic target for inhibiting ccRCC metastasis but cannot be used as an independent prognostic factor.

Dicer suppresses MMP-2-mediated invasion and VEGFA-induced angiogenesis and serves as a promising prognostic biomarker in human clear cell renal cell carcinoma.

Dicer, a key component of the microRNA processing machinery, has been reported to exert discrepant prognostic values and biological roles in different types of cancers. Here, we investigated the function and prognostic value of Dicer in clear cell renal cell carcinoma (ccRCC). Using the retrospective ccRCC patients' cohorts with tissue microarray (TMA), we demonstrated that Dicer expression was significantly down-regulated in ccRCC compared with renal non-tumor tissues, and negatively associated with pN status (P = 0.005), pM status (P = 0.009) and TNM stage (P =0.013). Multivariate Cox proportional hazards regression analyses showed that positive Dicer expression was an independent favorable factor for prognosis of ccRCC patients (hazard ratio (HR) = 0.709, P = 0.025 for 5-year overall survival; HR = 0.655, P = 0.008 for disease specific survival). Moreover, we found that Dicer decreased the abilities of cell migration, invasion and angiogenesis through suppressing MMP-2 and VEGFA expression. Tumor metastasis model in vivo showed much more metastatic nodules of lung in the Dicer knockdown group than the control group via increased MMP-2 expression. Our findings imply that Dicer inhibits ccRCC metastasis and may serve as promising prognostic biomarkers for ccRCC patients.

PinX1: structure, regulation and its functions in cancer.

PIN2/TRF1-interacting telomerase inhibitor 1 (PinX1) is a novel cloned gene located at human chromosome 8p23, playing a vital role in maintaining telomeres length and chromosome stability. It has been demonstrated to be involved in tumor genesis and progression in most malignancies. However, some researches showed opposing molecular status of PinX1 gene and its expression patterns in several other types of tumors. The pathogenic mechanism of PinX1 expression in human malignancy is not yet clear. Moreover, emerging evidence suggest that PinX1 (especially its TID domain) might be a potential new target cancer treatment. Therefore, PinX1 may be a new potential diagnostic biomarker and therapeutic target for human cancers, and may play different roles in different human cancers. The functions and the mechanisms of PinX1 in various human cancers remain unclear, suggesting the necessity of further extensive works of its role in tumor genesis and progression.

The expression of Cullin1 is increased in renal cell carcinoma and promotes cancer cell proliferation, migration, and invasion.

Cullin1 (Cul1) is a scaffold protein of the ubiquitin E3 ligase Skp1/Cullin1/Rbx1/F-box protein complex, which ubiquitinates a broad range of proteins involved in cell-cycle progression, signal transduction, and transcription. To investigate the role of Cul1 in the development of renal cell carcinoma (RCC), we evaluated the Cul1 expression by immunohistochemistry using a tissue microarray (TMA) containing 307 cases of RCC tissues and 34 normal renal tissues. The Cul1 expression was increased significantly in RCC and was correlated with renal carcinoma histology grade (P = 0.007), tumor size (P = 0.013), and pT status (P = 0.023). Also, we found that silencing of Cul1 leads to increased expression of p21 and p27 that could inhibit the cyclin D1 and cyclin E2 expressions and arrest cell cycle at the G1 phase. Furthermore, knockdown of Cul1 inhibits RCC cell migration and invasion abilities by up-regulating the expression of TIMP-1 which could inhibit the expression of MMP-9. Finally, using bioluminescence imaging, we found that Cul1 knockdown significantly reduced the tumor growth in vivo. Cul1 may constitute a potential therapeutic target in RCC.

PlantPAN 2.0: an update of plant promoter analysis navigator for reconstructing transcriptional regulatory networks in plants.

Transcription factors (TFs) are sequence-specific DNA-binding proteins acting as critical regulators of gene expression. The Plant Promoter Analysis Navigator (PlantPAN; http://PlantPAN2.itps.ncku.edu.tw) provides an informative resource for detecting transcription factor binding sites (TFBSs), corresponding TFs, and other important regulatory elements (CpG islands and tandem repeats) in a promoter or a set of plant promoters. Additionally, TFBSs, CpG islands, and tandem repeats in the conserve regions between similar gene promoters are also identified. The current PlantPAN release (version 2.0) contains 16 960 TFs and 1143 TF binding site matrices among 76 plant species. In addition to updating of the annotation information, adding experimentally verified TF matrices, and making improvements in the visualization of transcriptional regulatory networks, several new features and functions are incorporated. These features include: (i) comprehensive curation of TF information (response conditions, target genes, and sequence logos of binding motifs, etc.), (ii) co-expression profiles of TFs and their target genes under various conditions, (iii) protein-protein interactions among TFs and their co-factors, (iv) TF-target networks, and (v) downstream promoter elements. Furthermore, a dynamic transcriptional regulatory network under various conditions is provided in PlantPAN 2.0. The PlantPAN 2.0 is a systematic platform for plant promoter analysis and reconstructing transcriptional regulatory networks.

AtmiRNET: a web-based resource for reconstructing regulatory networks of Arabidopsis microRNAs.

Compared with animal microRNAs (miRNAs), our limited knowledge of how miRNAs involve in significant biological processes in plants is still unclear. AtmiRNET is a novel resource geared toward plant scientists for reconstructing regulatory networks of Arabidopsis miRNAs. By means of highlighted miRNA studies in target recognition, functional enrichment of target genes, promoter identification and detection of cis- and trans-elements, AtmiRNET allows users to explore mechanisms of transcriptional regulation and miRNA functions in Arabidopsis thaliana, which are rarely investigated so far. High-throughput next-generation sequencing datasets from transcriptional start sites (TSSs)-relevant experiments as well as five core promoter elements were collected to establish the support vector machine-based prediction model for Arabidopsis miRNA TSSs. Then, high-confidence transcription factors participate in transcriptional regulation of Arabidopsis miRNAs are provided based on statistical approach. Furthermore, both experimentally verified and putative miRNA-target interactions, whose validity was supported by the correlations between the expression levels of miRNAs and their targets, are elucidated for functional enrichment analysis. The inferred regulatory networks give users an intuitive insight into the pivotal roles of Arabidopsis miRNAs through the crosstalk between miRNA transcriptional regulation (upstream) and miRNA-mediate (downstream) gene circuits. The valuable information that is visually oriented in AtmiRNET recruits the scant understanding of plant miRNAs and will be useful (e.g. ABA-miR167c-auxin signaling pathway) for further research. Database URL: http://AtmiRNET.itps.ncku.edu.tw/