miRNA-target Gene Pairs Research Articles

Integration analysis of miRNA-mRNA uncovers the molecular immune mechanism of macrophage response to Aeromonas veronii infection

Macrophages are innate immunity cells which play pivotal roles in infectious immunity. Aeromonas veronii is a zoonotic agent capable of causing sepsis and poses a serious threat to public health. However, few studies have focused on miRNA-mRNA integration analysis to address the immune mechanisms of macrophage response to A. veronii infection. Herein, we characterized the immunophysiological, biochemical, and transcriptome changes of macrophage under A. veronii infection. We found that macrophages infected with A. veronii released large amounts of cytokines and triggered NLRP3-dependent pyroptosis. Subsequently, 603 differentially expressed miRNAs (DEMIs) and 3693 differentially expressed mRNAs (DEMs) were identified by RNA-seq analysis under A. veronii infection. Moreover, integrated analysis of miRNA-mRNA yielded 66 miRNA-target gene pairs composed of 41 DEMIs and 27 DEMs. We next identified the Toll-like receptor, NOD-like receptor, TNF and NF-κB pathways as necessary for macrophage to respond to A. veronii infection. miR-847 and miR-627 were involved in macrophage response to A. veronii infection by negatively regulating Pannexin-1 and thioredoxin interacting protein (TXNIP). Our findings elucidate the molecular mechanism of macrophage response to A. veronii infection at the miRNA level, providing many candidate miRNAs and mRNAs therapeutic targets for the prevention and treatment of A. veornii infectious diseases.

Restricting Colorectal Cancer Cell Metabolism with Metformin: An Integrated Transcriptomics Study.

Metformin is a first-line therapy for type 2 diabetes as it disrupts cellular metabolism. Despite the association between metformin and lower cancer incidence, the anti-tumour activity of the drug in colorectal cancer (CRC) is incompletely understood. This study identifies underlying molecular mechanisms by which metformin slows colorectal cancer cell proliferation by investigating metformin-associated microRNA (miRNA) and target gene pairs implicated in signalling pathways. The present study analysed changes in miRNAs and the coding transcriptome in CRC cells treated with a sublethal dose of metformin, followed by the contextual validation of potential miRNA-target gene pairs. Analyses of small RNA and transcriptome sequencing data revealed 104 miRNAs and 1221 mRNAs to be differentially expressed in CRC cells treated with metformin for 72 h. Interaction networks between differentially expressed miRNAs and putative target mRNAs were identified. Differentially expressed genes were mainly implicated in metabolism and signalling processes, such as the PI3K-Akt and MAPK/ERK pathways. Further validation of potential miRNA-target mRNA pairs revealed that metformin induced miR-2110 and miR-132-3p to target PIK3R3 and, consequently, regulate CRC cell proliferation, cell cycle progression and the PI3K-Akt signalling pathway. Metformin also induced miR-222-3p and miR-589-3p, which directly target STMN1 to inhibit CRC cell proliferation and cell cycle progression. This study identified novel changes in the coding transcriptome and small non-coding RNAs associated with metformin treatment of CRC cells. Integration of these datasets highlighted underlying mechanisms by which metformin impedes cell proliferation in CRC. Importantly, it identified the post-transcriptional regulation of specific genes that impact both metabolism and cell proliferation.

Systematic identification and characterization of microRNAs with target genes involved in high night temperature stress at the filling stage of rice.

High night temperature stress is one of the main environmental factors affecting rice yield and quality. More and more evidence shows that microRNA (miRNA) plays an important role in various abiotic stresses. However, the molecular network of miRNA regulation on rice tolerance to high night temperatures remains unclear. Here, small RNA, transcriptome and degradome sequencing were integrated to identify differentially expressed miRNAs, genes, and key miRNA-target gene pairs in rice heat-sensitive and heat-tolerant lines at the filling stage suffering from high night temperature stress. It was discovered that there were notable differences in the relative expression of 102 miRNAs between the two rice lines under stress. Meanwhile, 5263 and 5405 mRNAs were differentially expressed in the heat-sensitive line and heat-tolerant line, and functional enrichment analysis revealed that these genes were involved in heat-related processes and pathways. The miRNAs-mRNAs target relationship was further verified by degradome sequencing. Eventually, 49 miRNAs-222 mRNAs target pairs with reverse expression patterns showed significant relative expression changes between the heat-tolerant and the heat-sensitive line, being suggested to be responsible for the heat tolerance difference of these two rice lines. Functional analysis of these 222 mRNA transcripts showed that high night temperature-responsive miRNAs targeted these mRNAs involved in many heat-related biological processes, such as transcription regulation, chloroplast regulation, mitochondrion regulation, protein folding, hormone regulation and redox process. This study identified possible miRNA-mRNA regulation relationships in response to high night temperature stress in rice and potentially contributed to heat resistance breeding of rice in the future.

Integration of mRNA and miRNA analysis reveals the molecular mechanisms of sugar beet (Beta vulgaris L.) response to salt stress

The continuous increase of saline-alkali areas worldwide has led to the emergence of saline-alkali conditions, which are the primary abiotic stress or hindering the growth of plants. Beet is among the main sources of sugar, and its yield and sugar content are notably affected by saline-alkali stress. Despite sugar beet being known as a salt-tolerant crop, there are few studies on the mechanisms underlying its salt tolerance, and previous studies have mainly delineated the crop’s response to stress induced by NaCl. Recently, advancements in miRNA-mRNA network analysis have led to an increased understanding of how plants, including sugar beet, respond to stress. In this study, seedlings of beet variety "N98122" were grown in the laboratory using hydroponics culture and were exposed to salt stress at 40 days of growth. According to the phenotypic adaptation of the seedlings' leaves from a state of turgidity to wilting and then back to turgidity before and after exposure, 18 different time points were selected to collect samples for analysis. Subsequently, based on the data of real-time quantitative PCR (qRT-PCR) of salt-responsive genes, the samples collected at the 0, 2.5, 7.5, and 16 h time points were subjected to further analysis with experimental materials. Next, mRNA-seq data led to the identification of 8455 differentially expressed mRNAs (DEMs) under exposure to salt stress. In addition, miRNA-seq based investigation retrieved 3558 miRNAs under exposure to salt stress, encompassing 887 known miRNAs belonging to 783 families and 2,671 novel miRNAs. With the integrated analysis of miRNA-mRNA network, 57 miRNA-target gene pairs were obtained, consisting of 55 DEMIs and 57 DEMs. Afterwards, we determined the pivotal involvement of aldh2b7, thic, and δ-oat genes in the response of sugar beet to the effect of salt stress. Subsequently, we identified the miRNAs novel-m035-5p and novel-m0365-5p regulating the aldh gene and miRNA novel-m0979-3p regulating the thic gene. The findings of miRNA and mRNA expression were validated by qRT-PCR.

Characterization of the microRNA408-LACCASE5 module as a regulatory axis for photosynthetic efficiency in Medicago ruthenica: implications for forage yield enhancement

Medicago ruthenica is closely related to Medicago sativa, a commonly cultivated forage. Characterized by its high tolerance to environmental stress, M. ruthenica is a valuable genetic resource. However, low yield limits its large-scale utilization. Leaf morphology, an important agronomic trait, is closely related to forage yield and photosynthetic efficiency. In the presented study, “Correlation of Leaf Morphology and Photosynthetic Performance with Forage Yield in Medicago ruthenica: The Underlying Molecular Mechanisms,” comprehensive data analysis revealed a significant positive association between leaf width and leaf area with forage yield in Medicago ruthenica (p < 0.05). The specific cultivar “Mengnong No.1 (MN No.1) had a large leaf area, and its physiological parameters related to photosynthetic characteristics were superior. Anatomical examination revealed that the leaves of MN No.1 had strong palisade tissue and compact cell structure. Subsequent investigations, utilizing small RNA and transcriptome sequencing, discerned critical miRNA-target gene networks that underpin the high photosynthetic efficiency in M. ruthenica. A total of 63 differentially expressed miRNAs (DEMs) were identified, inclusive of several well-characterized miRNAs such as miR408, miR171, and miR398. These miRNAs were predicted to target 55 genes (mRNAs), of which 6 miRNA-target gene pairs, particularly those involving miR408and miR171, exhibited inverse expression patterns. Among the six postulated miRNA-target gene pairs, the targeted cleavage of LACCASE5 (LAC5) by miR408 was conclusively validated through degradome sequencing, with the cleavage site pinpointed between the 9th and 10th nucleotides from the 5′end of miR408 via the 5′-RLM-RACE assay. Therefore, it is posited that the miR408-MrLAC5 module constitutes a central mechanism in fostering high photosynthetic efficiency in M. ruthenica. Moreover, these findings also provide valuable information for further study of the regulatory genes and miRNA functions of forage yield in legume forage.

Ovarian cancer classification and prognosis assessment model based on prognostic target genes in key microRNA-target gene networks.

The present study was designed to screen key microRNA (miRNA)-target gene networks for ovarian cancer (OC) and to classify and construct a risk assessment system for OC based on the target genes. OC sample data of The Cancer Genome Atlas dataset and GSE26193, GSE30161, GSE63885 and GSE9891 datasets were retrospectively collected. Pearson correlation analysis and targeted analysis of miRNA and target gene were performed to screen key miRNA-target gene networks. Target genes associated with the prognosis of OC were screened from key miRNA-target gene networks for consensus clustering and least absolute shrinkage and selection operator-based regression machine learning analysis of OC samples. Twenty target genes of 2651 key miRNA-target gene pairs had significant prognostic correlation in each OC cohort, and OC was divided into three clusters. There were differences in prognostic outcome, biological pathways, immune cell abundance and susceptibility to immune checkpoint blockade (ICB) therapy and anti-tumor drugs among the three molecular clusters. S2 exhibited the least advantage in prognosis and immunotherapy response rate in the three molecular clusters, and the pathways regulating immunity, hypoxia, metabolism and promoting malignant progression of cancer, as well as infiltrating immune and stromal cell population abundance, were the highest in this cluster. An eight-target gene prognostic model was created, and the risk index obtained by using this model not only significantly distinguished the immune characteristics of the sample, but also predicted the response of the sample to ICB treatment, and helped to screen 36 potential anti-OC drugs. The present study provides a classification strategy for OC based on prognostic target genes in key miRNA-target gene networks, and creates a risk assessment system for predicting prognosis and response to ICB therapy in OC patients, providing molecular basis for prognosis and precise treatment of OC.

Integrated transcriptome and microRNA sequencing analyses reveal gene responses in poplar leaves infected by the novel pathogen bean common mosaic virus (BCMV).

Recently, a novel poplar mosaic disease caused by bean common mosaic virus (BCMV) was investigated in Populus alba var. pyramidalis in China. Symptom characteristics, physiological performance of the host, histopathology, genome sequences and vectors, and gene regulation at the transcriptional and posttranscriptional levels were analyzed and RT-qPCR (quantitative reverse transcription PCR) validation of expression was performed in our experiments. In this work, the mechanisms by which the BCMV pathogen impacts physiological performance and the molecular mechanisms of the poplar response to viral infection were reported. The results showed that BCMV infection decreased the chlorophyll content, inhibited the net photosynthesis rate (Pn) and stomatal conductance (Gs), and significantly changed chlorophyll fluorescence parameters in diseased leaves. Transcriptome analysis revealed that the expression of the majority of DEGs (differentially expressed genes) involved in the flavonoid biosynthesis pathway was promoted, but the expression of all or almost all DEGs associated with photosynthesis-antenna proteins and the photosynthesis pathway was inhibited in poplar leaves, suggesting that BCMV infection increased the accumulation of flavonoids but decreased photosynthesis in hosts. Gene set enrichment analysis (GSEA) illustrated that viral infection promoted the expression of genes involved in the defense response or plant-pathogen interaction. MicroRNA-seq analysis illustrated that 10 miRNA families were upregulated while 6 families were downregulated in diseased poplar leaves; moreover, miR156, the largest family with the most miRNA members and target genes, was only differentially upregulated in long-period disease (LD) poplar leaves. Integrated transcriptome and miRNA-seq analyses revealed 29 and 145 candidate miRNA-target gene pairs; however, only 17 and 76 pairs, accounting for 2.2% and 3.2% of all DEGs, were authentically negatively regulated in short-period disease (SD) and LD leaves, respectively. Interestingly, 4 miR156/SPL (squamosa promoter-binding-like protein) miRNA-target gene pairs were identified in LD leaves: the miR156 molecules were upregulated, but SPL genes were downregulated. In conclusion, BCMV infection significantly changed transcriptional and posttranscriptional gene expression in poplar leaves, inhibited photosynthesis, increased the accumulation of flavonoids, induced systematic mosaic symptoms, and decreased physiological performance in diseased poplar leaves. This study elucidated the fine-tuned regulation of poplar gene expression by BCMV; moreover, the results also suggested that miR156/SPL modules played important roles in the virus response and development of viral systematic symptoms in plant virus disease.

MiRNA expression profiles of peripheral white blood cells from beef heifers with varying reproductive potential.

Reproductive performance is the most critical factor affecting production efficiency in the cow-calf industry. Heifers with low reproductive efficiency may fail to become pregnant during the breeding season or maintain a pregnancy. The cause of reproductive failure often remains unknown, and the non-pregnant heifers are not identified until several weeks after the breeding season. Therefore, improving heifer fertility utilizing genomic information has become increasingly important. One approach is using microRNAs (miRNA) in the maternal blood that play an important role in regulating the target genes underlying pregnancy success and thereby in selecting reproductively efficient heifers. Therefore, the current study hypothesized that miRNA expression profiles from peripheral white blood cells (PWBC) at weaning could predict the future reproductive outcome of beef heifers. To this end, we measured the miRNA profiles using small RNA-sequencing in Angus-Simmental crossbred heifers sampled at weaning and retrospectively classified as fertile (FH, n = 7) or subfertile (SFH, n = 7). In addition to differentially expressed miRNAs (DEMIs), their target genes were predicted from TargetScan. The PWBC gene expression from the same heifers were retrieved and co-expression networks were constructed between DEMIs and their target genes. We identified 16 differentially expressed miRNAs between the groups (p-value ≤0.05 and absolute (log2 fold change ≥0.05)). Interestingly, based on a strong negative correlation identified from miRNA-gene network analysis with PCIT (partial correlation and information theory), we identified miRNA-target genes in the SFH group. Additionally, TargetScan predictions and differential expression analysis identified bta-miR-1839 with ESR1 , bta-miR-92b with KLF4 and KAT2B, bta-miR-2419-5p with LILRA4, bta-miR-1260b with UBE2E1, SKAP2 and CLEC4D, and bta-let-7a-5p with GATM, MXD1 as miRNA-gene targets. The miRNA-target gene pairs in the FH group are over-represented for MAPK, ErbB, HIF-1, FoxO, p53, mTOR, T-cell receptor, insulin and GnRH signaling pathways, while those in the SFH group include cell cycle, p53 signaling pathway and apoptosis. Some miRNAs, miRNA-target genes and regulated pathways identified in this study have a potential role in fertility; other targets are identified as novel and need to be validated in a bigger cohort that could help to predict the future reproductive outcomes of beef heifers.

Comparative Analysis of miRNA-mRNA Regulation in the Testes of Gobiocypris rarus following 17α-Methyltestosterone Exposure.

17α-Methyltestosterone (17MT), a synthetic organic compound commonly found in sewage waters, can affect reproduction in aquatic animals, such as tilapia and yellow catfish. In the present study, male Gobiocypris rarus were exposed to 25, 50, and 100 ng/L of 17α-methyltestosterone (17MT) for 7 days. We first analyzed miRNA- and RNA-seq results to determine miRNA-target gene pairs and then developed miRNA-mRNA interactive networks after 17MT administration. Total weights, total lengths, and body lengths were not significantly different between the test groups and control groups. The paraffin slice method was applied to testes of G. rarus in the MT exposure and control groups. We found that there were more mature sperm (S) and fewer secondary spermatocytes (SSs) and spermatogonia (SGs) in the testes of control groups. As 17MT concentration increased, fewer and fewer mature sperm (S) were observed in the testes of male G. rarus. The results showed that FSH, 11-KT, and E2 were significantly higher in individuals exposed to 25 ng/L 17MT compared with the control groups. VTG, FSH, LH, 11-KT, and E2 were significantly lower in the 50 ng/L 17MT exposure groups compared to the control groups. VTG, FSH, LH, 11-KT, E2, and T were significantly lower in the groups exposed to 100 ng/L 17MT. High-throughput sequencing revealed 73,449 unigenes, 1205 known mature miRNAs, and 939 novel miRNAs in the gonads of G. rarus. With miRNA-seq, 49 (MT25-M vs. Con-M), 66 (MT50-M vs. Con-M), and 49 (MT100-M vs. Con-M) DEMs were identified in the treatment groups. Five mature miRNAs (miR-122-x, miR-574-x, miR-430-y, lin-4-x, and miR-7-y), as well as seven differentially expressed genes (soat2, inhbb, ihhb, gatm, faxdc2, ebp, and cyp1a1), which may be associated with testicular development, metabolism, apoptosis, and disease response, were assayed using qRT-PCR. Furthermore, miR-122-x (related to lipid metabolism), miR-430-y (embryonic development), lin-4-x (apoptosis), and miR-7-y (disease) were differentially expressed in the testes of 17MT-exposed G. rarus. This study highlights the role of miRNA-mRNA pairs in the regulation of testicular development and immune response to disease and will facilitate future studies on the miRNA-RNA-associated regulation of teleost reproduction.

Identification of miRNA-mRNA-TFs regulatory network and crucial pathways involved in asthma through advanced systems biology approaches.

Asthma is a life-threatening and chronic inflammatory lung disease that is posing a true global health challenge. The genetic basis of the disease is fairly well examined. However, the molecular crosstalk between microRNAs (miRNAs), target genes, and transcription factors (TFs) networks and their contribution to disease pathogenesis and progression is not well explored. Therefore, this study was aimed at dissecting the molecular network between mRNAs, miRNAs, and TFs using robust computational biology approaches. The transcriptomic data of bronchial epithelial cells of severe asthma patients and healthy controls was studied by different systems biology approaches like differentially expressed gene detection, functional enrichment, miRNA-target gene pairing, and mRNA-miRNA-TF molecular networking. We detected the differential expression of 1703 (673 up-and 1030 down-regulated) genes and 71 (41 up-and 30 down-regulated) miRNAs in the bronchial epithelial cells of asthma patients. The DEGs were found to be enriched in key pathways like IL-17 signaling (KEGG: 04657), Th1 and Th2 cell differentiation (KEGG: 04658), and the Th17 cell differentiation (KEGG: 04659) (p-values = 0.001). The results from miRNAs-target gene pairs-transcription factors (TFs) have detected the key roles of 3 miRs (miR-181a-2-3p; miR-203a-3p; miR-335-5p), 6 TFs (TFAM, FOXO1, GFI1, IRF2, SOX9, and HLF) and 32 miRNA target genes in eliciting autoimmune reactions in bronchial epithelial cells of the respiratory tract. Through systemic implementation of comprehensive system biology tools, this study has identified key miRNAs, TFs, and miRNA target gene pairs as potential tissue-based asthma biomarkers.

Dissecting microRNA−Target Gene Pairs Involved in Rubber Biosynthesis in Eucommia ulmoides

MicroRNAs (miRNAs) play essential roles in regulating various development processes in plants. However, their role in regulating rubber biosynthesis in Eucommia ulmoides is largely unknown. Rubber is mainly distributed in the tissue covering the seed (GZ) rather than the periphery (GB) of the pericarp of E. ulmoides during accumulation in May. To investigate the roles of miRNAs in rubber biosynthesis, we conducted high-throughput small RNA sequencing using GZ and GB collected on 11 May (rapid accumulation) and 11 June (reduced accumulation). In total, 12 and 25 miRNAs were either up- or downregulated in GZ in May (GZ511) compared to GB (GB511) in May, while 27 and 38 miRNAs were either up- or downregulated in GZ in May compared to GZ in June (GZ611), respectively. Functional analyses of differentially expressed (DE−) genes targeted by DE-miRNAs revealed that miRNAs may regulate genes involved in rubber biosynthesis. For instance, when Eu-miR45 expression declined, the expression of its predicted target, small rubber particle protein 1 (EuSRPP1), increased in GZ511 vs. GB511 and GZ511 vs. GZ611, possibly resulting in higher rubber accumulation in GZ511. Additionally, we identified potential lncRNA−miRNA−mRNA networks in rubber biosynthesis. Overall, these results indicate that miRNAs play pivotal roles in regulating rubber biosynthesis via miRNA-target gene pairs and lncRNA−miRNA−mRNA networks in E. ulmoides. Our findings will enhance research on the mechanisms of rubber biosynthesis in plants.

Integration of mRNA and microRNA analysis reveals the molecular mechanisms underlying drought stress tolerance in maize (Zea mays L.).

Drought is among the most serious environmental issue globally, and seriously affects the development, growth, and yield of crops. Maize (Zea mays L.), an important crop and industrial raw material, is planted on a large scale worldwide and drought can lead to large-scale reductions in maize corn production; however, few studies have focused on the maize root system mechanisms underlying drought resistance. In this study, miRNA–mRNA analysis was performed to deeply analyze the molecular mechanisms involved in drought response in the maize root system under drought stress. Furthermore, preliminary investigation of the biological function of miR408a in the maize root system was also conducted. The morphological, physiological, and transcriptomic changes in the maize variety “M8186” at the seedling stage under 12% PEG 6000 drought treatment (0, 7, and 24 h) were analyzed. With prolonged drought stress, seedlings gradually withered, the root system grew significantly, and abscisic acid, brassinolide, lignin, glutathione, and trehalose content in the root system gradually increased. Furthermore, peroxidase activity increased, while gibberellic acid and jasmonic acid gradually decreased. Moreover, 32 differentially expressed miRNAs (DEMIRs), namely, 25 known miRNAs and 7 new miRNAs, and 3,765 differentially expressed mRNAs (DEMRs), were identified in maize root under drought stress by miRNA-seq and mRNA-seq analysis, respectively. Through combined miRNA–mRNA analysis, 16 miRNA–target gene pairs, comprising 9 DEMIRs and 15 DEMRs, were obtained. In addition, four metabolic pathways, namely, “plant hormone signal transduction”, “phenylpropane biosynthesis”, “glutathione metabolism”, and “starch and sucrose metabolism”, were predicted to have important roles in the response of the maize root system to drought. MiRNA and mRNA expression results were verified by real-time quantitative PCR. Finally, miR408a was selected for functional analysis and demonstrated to be a negative regulator of drought response, mainly through regulation of reactive oxygen species accumulation in the maize root system. This study helps to elaborate the regulatory response mechanisms of the maize root system under drought stress and predicts the biological functions of candidate miRNAs and mRNAs, providing strategies for subsequent mining for, and biological breeding to select for, drought-responsive genes in the maize root system.

MiRNA-mRNA associations with inosine monophosphate specific deposition in the muscle of Jingyuan chicken

ABSTRACT 1. Inosine monophosphate (IMP), is an essential component for meat flavour and microRNAs (miRNAs) play a vital role in its post-transcriptional regulation. However, the mechanism of how miRNA expression affects muscle-specific IMP deposition is unclear. 2. The following study performed transcriptome sequencing and bioinformatics analysis of breast and leg muscle, which have significantly different IMP content in Jingyuan chicken. The differential miRNA-mRNAs were screened out and correlation analysis with IMP content was performed. 3. A total of 39 differentially expressed miRNAs (DE miRNAs) and 666 differentially expressed mRNAs (DE mRNAs) were identified between breast muscles and leg muscles. Using miRNA-mRNA integrated analysis, 29 miRNA-target gene pairs were obtained, composed of 13 DE miRNAs and 28 DE mRNAs. Next, purine metabolism, glycolysis/gluconeogenesis, pyruvate metabolism and the biosynthesis of amino acid pathways as necessary for muscle IMP-specific deposition were identified. The differentially expressed gene PKM2, which was significantly enriched in all four pathways, is involved in IMP anabolism in the form of energy metabolism and enzyme activity regulation. The correlation analysis suggested that the gga-miR-107-3p-KLHDC2 negative interaction may be a key regulator in IMP deposition. 4. This study explores the functional mechanism of IMP-specific deposition in Jingyuan chicken muscles at the miRNA and mRNA levels and highlights multiple candidate miRNAs and mRNAs for molecular-assisted breeding.

Parallel analysis of RNA ends reveals global microRNA-mediated target RNA cleavage in maize.

MicroRNAs (miRNAs) are endogenous 20-24-nucleotide non-coding RNAs that play important regulatory roles in many biological processes in eukaryotes. miRNAs modulate the expression of target genes at the post-transcriptional level by transcript cleavage or translational inhibition. The identification of miRNA target genes has been extensively investigated in Arabidopsis and rice, but an in-depth global analysis of miRNA-mediated target regulation is still lacking in maize. Here, we report a transcriptome-wide identification of miRNA targets by analyzing parallel analysis of RNA ends (PARE) datasets derived from nine different tissues at five developmental stages of the maize (Zea mays L.) B73 cultivar. In total, 246 targets corresponding to 60 miRNAs from 25 families were identified, including transcription factors and other genes. In addition, PARE analysis revealed that miRNAs guide specific target transcript cleavage in a tissue-preferential manner. Primary transcripts of MIR159c and MIR169e were found to be cleaved by mature miR159 and miR169, respectively, indicating a negative-feedback regulatory mechanism in miRNA biogenesis. Moreover, several miRNA-target gene pairs involved in seed germination were identified and experimentally validated. Our PARE analyses generated a wide and detailed miRNA-target interaction atlas, which provides a valuable resource for investigating the roles of miRNAs and their targets in maize.

Integrated microRNA and transcriptome profiling reveals the regulatory network of embryo abortion in jujube.

Hybridization is an important approach to the production of new varieties with exceptional traits. Although the kernel rate of wild jujube (Ziziphus jujuba Mill. var. spinosa Hu.) is generally high, that of cultivated jujube (Z. jujuba Mill.) is low, greatly hampering the jujube breeding process. However, the mechanism by which this trait changed during jujube domestication remains unclear. Here, we explored the potential regulatory network that governs jujube embryo abortion using correlation analysis of population traits, artificial pollination, sugar content measurements and multi-omics analysis. The results showed that embryo abortion was an important reason for the low kernel rate of cultivated jujube, and kernel rate was negatively correlated with edible rate. Twenty-one days after pollination was a critical period for embryo abortion. At this time, the sugar content of cultivated 'Junzao' kernels decreased significantly compared with that of the pulp, but sugar content remained relatively stable in kernels of wild 'Suanzao'. A total of 1142 differentially expressed genes targeted by 93 microRNAs (miRNAs) were identified by transcriptome, miRNA and degradome sequencing, and may be involved in the regulation of embryo abortion during kernel development. Among them, DELLA protein, TCP14 and bHLH93 transcription factors have been shown to participate in the regulation of embryonic development. Our findings suggest that carbohydrate flow between different tissues of cultivated jujube exhibits a bias toward the pulp at 21days after pollination, thereby restricting the process of kernel development. This information enhances our understanding of the embryo abortion process and reveals miRNA-target gene pairs that may be useful for molecular-assisted breeding.

Integrated SRNA-Seq and RNA-Seq Analysis Reveals the Regulatory Roles of miRNAs in the Low-Temperature Responses of Canarium album

Chinese olive (Canarium album), a characteristic fruit tree in tropical and subtropical areas, suffers greatly from low-temperature stress (LTS). The regulatory roles of microRNA (miRNA) in plant LTS responses have been confirmed in many plant species but not in C. album. In this study, a cold-tolerant cultivar ‘Rui’an 3′ (RA) and a susceptible cultivar ‘Qinglan 1’ (QL) treated at 25 °C (control, CK) and −3 °C (cold temperature treatment, CT) were subjected to small RNA (sRNA) and transcriptome sequencing for the exploration of the cold responses of C. album. Comparative sRNA sequencing analysis identified much fewer LTS-responsive, differentially expressed miRNAs (DEMs) in RA (4 DEMs) than in QL (23 DEMs). Cal-miR482-22 was found to be specifically induced by LTS in RA. Cal-miR397-3 was upregulated, while cal-miR398_2-3 and cal-undef-190 were downregulated after LTS only in QL. However, when compared with QL, a higher basic expression of cal-miR397-3, and lower expression of cal-miR398_2-3 and cal-undef-190 were found in RA, suggesting that they may contribute to the cold tolerance of RA. Comparative transcriptome analysis showed that the number of LTS-responsive differentially expressed genes (DEGs) identified in QL was larger than that in RA, and some DEGs were also predicted as the target genes of the identified DEMs, forming multiple differentially expressed miRNA–target gene pairs, such as cal-miR397-3_laccase 2, 4, 17, cal-miR482-22_suppressor of npr1-1, etc. Quantitative real time PCR results showed that the expression changes of DEGs and DEMs in different samples were generally consistent with the sequencing results. Our study indicated that the basic expression levels of some miRNAs (especially the cal-miR397-3, cal-miR398_2-3, and cal-miR482-22), and their target genes contribute greatly to the cold-tolerance characteristics of C. album. Our study is helpful for understanding the roles of miRNAs in the cold resistance and responses of C. album.

Identification of Key Endometrial MicroRNAs and Their Target Genes Associated With Pathogenesis of Recurrent Implantation Failure by Integrated Bioinformatics Analysis.

Purpose: Recurrent implantation failure (RIF) is an enormous challenge for in vitro fertilization (IVF) clinicians. An understanding of the molecular mechanisms of RIF helps to predict prognosis and develop new therapeutic strategies. The study is designed to identify diagnostic biomarkers for RIF as well as the potential mechanisms underlying RIF by utilizing public databases together with experimental validation. Methods: Two microarray datasets of RIF patients and the healthy control endometrium were downloaded from the Gene Expression Omnibus (GEO) database. First, differentially expressed microRNAs (miRNAs) (DEMs) were identified and their target genes were predicted. Then, we identified differentially expressed genes (DEGs) and selected hub genes through protein-protein interaction (PPI) analyses. Functional enrichment analyses of DEGs and DEMs were conducted. Furthermore, the key DEMs which targeted these hub genes were selected to obtain the key miRNA–target gene network. The key genes in the miRNA-target gene network were validated by a single-cell RNA-sequencing dataset of endometrium from GEO. Finally, we selected two miRNA–target gene pairs for further experimental validation using dual-luciferase assay and quantitative polymerase chain reaction (qPCR). Results: We identified 49 DEMs between RIF patients and the fertile group and found 136,678 target genes. Then, 325 DEGs were totally used to construct the PPI network, and 33 hub genes were selected. Also, 25 DEMs targeted 16 key DEGs were obtained to establish a key miRNA–target gene network, and 16 key DEGs were validated by a single-cell RNA-sequencing dataset. Finally, the target relationship of hsa-miR-199a-5p-PDPN and hsa-miR-4306-PAX2 was verified by dual-luciferase assay, and there were significant differences in the expression of those genes between the RIF and fertile group by PCR (p < 0.05). Conclusion: We constructed miRNA–target gene regulatory networks associated with RIF which provide new insights regarding the underlying pathogenesis of RIF; hsa-miR-199a-5p-PDPN and hsa-miR-4306-PAX2 could be further explored as potential biomarkers for RIF, and their detection in the endometrium could be applied in clinics to estimate the probability of successful embryo transfer.

Integrated mRNA and miRNA Transcriptome Analysis Suggests a Regulatory Network for UV-B-Controlled Terpenoid Synthesis in Fragrant Woodfern (Dryopteris fragrans).

Fragrant woodfern (Dryopteris fragrans) is a medicinal plant rich in terpenoids. Ultraviolet-B (UV–B) light could increase concentration of terpenoids. The aim of this study was to analyze how UV–B regulates the terpenoid synthesis of the molecular regulatory mechanism in fragrant woodfern. In this study, compared with the control group, the content of the terpenes was significantly higher in fragrant woodfern leaves under UV–B treatment for 4 days (d). In order to identify how UV–B regulates the terpenoid metabolic mechanism in fragrant woodfern, we examined the mRNAs and small RNAs in fragrant woodfern leaves under UV–B treatment. mRNA and miRNA–seq identified 4533 DEGs and 17 DEMs in the control group compared with fragrant woodfern leaves under UV–B treatment for 4 d. mRNA–miRNA analysis identified miRNA target gene pairs consisting of 8 DEMs and 115 miRNAs. The target genes were subjected to GO and KEGG analyses. The results showed that the target genes were mainly enriched in diterpene biosynthesis, terpenoid backbone biosynthesis, plant hormone signal transduction, MEP pathway and MVA pathway, in which miR156 and miR160 regulate these pathways by targeting DfSPL and DfARF, respectively. The mRNA and miRNA datasets identified a subset of candidate genes. It provides the theoretical basis that UV–B regulates the terpenoid synthesis of the molecular regulatory mechanism in fragrant woodfern.

Integrated Analysis of Physiological, mRNA Sequencing, and miRNA Sequencing Data Reveals a Specific Mechanism for the Response to Continuous Cropping Obstacles in Pogostemon cablin Roots.

Pogostemon cablin (patchouli) is a commercially important medicinal and industrial crop grown worldwide for its medicinal and aromatic properties. Patchoulol and pogostone, derived from the essential oil of patchouli, are considered valuable components in the cosmetic and pharmaceutical industries. Due to its high application value in the clinic and industry, the demand for patchouli is constantly growing. Unfortunately, patchouli cultivation has suffered due to severe continuous cropping obstacles, resulting in a significant decline in yield and quality. Moreover, the physiological and transcriptional changes in patchouli in response to continuous cropping obstacles remain unclear. This has greatly restricted the development of the patchouli industry. To explore the mechanism underlying the rapid response of patchouli roots to continuous cropping stress, integrated analysis of the transcriptome and miRNA profiles of patchouli roots under continuous and noncontinuous cropping conditions in different growth periods was conducted using RNA sequencing (RNA-seq) and miRNA-seq and complemented with physiological data. The physiological and biochemical results showed that continuous cropping significantly inhibited root growth, decreased root activity, and increased the activity of antioxidant enzymes (superoxide dismutase, peroxidase, and catalase) and the levels of osmoregulators (malondialdehyde, soluble protein, soluble sugar, and proline). Subsequently, we found 4,238, 3,494, and 7,290 upregulated and 4,176, 3,202, and 8,599 downregulated differentially expressed genes (DEGs) in the three growth periods of continuously cropped patchouli, many of which were associated with primary carbon and nitrogen metabolism, defense responses, secondary metabolite biosynthesis, and transcription factors. Based on miRNA-seq, 927 known miRNAs and 130 novel miRNAs were identified, among which 67 differentially expressed miRNAs (DEMIs) belonging to 24 miRNA families were induced or repressed by continuous cropping. By combining transcriptome and miRNA profiling, we obtained 47 miRNA-target gene pairs, consisting of 18 DEMIs and 43 DEGs, that likely play important roles in the continuous cropping response of patchouli. The information provided in this study will contribute to clarifying the intricate mechanism underlying the patchouli response to continuous cropping obstacles. In addition, the candidate miRNAs and genes can provide a new strategy for breeding continuous cropping-tolerant patchouli.

An integrative analysis of miRNA and mRNA expression in the brains of Alzheimer's disease transgenic mice after real-world PM2.5 exposure

Fine particulate matter (PM2.5) is associated with increased risks of Alzheimer's disease (AD), yet the toxicological mechanisms of PM2.5 promoting AD remain unclear. In this study, wild-type and APP/PS1 transgenic mice (AD mice) were exposed to either filtered air (FA) or PM2.5 for eight weeks with a real-world exposure system in Taiyuan, China (mean PM2.5 concentration in the cage was 61 µg/m3). We found that PM2.5 exposure could remarkably aggravate AD mice's ethological and brain ultrastructural damage, along with the elevation of the pro-inflammatory cytokines (IL-6 and TNF-α), Aβ-42 and AChE levels and the decline of ChAT levels in the brains. Based on high-throughput sequencing results, some differentially expressed (DE) mRNAs and DE miRNAs in the brains of AD mice after PM2.5 exposure were screened. Using RT-qPCR, seven DE miRNAs (mmu-miR-193b-5p, 122b-5p, 466h-3p, 10b-5p, 1895, 384–5p, and 6412) and six genes (Pcdhgb8, Unc13b, Robo3, Prph, Pter, and Tbata) were evidenced the and verified. Two miRNA-target gene pairs (miR-125b-Pcdhgb8 pair and miR-466h-3p-IL-17Rα/TGF-βR2/Aβ-42/AChE pairs) were demonstrated that they were more related to PM2.5-induced brain injury. Results of Gene Ontology (GO) pathways and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways predicted that synaptic and postsynaptic regulation, axon guidance, Wnt, MAPK, and mTOR pathways might be the possible regulatory mechanisms associated with pathological response. These revealed that PM2.5-elevated pro-inflammatory cytokine levels and PM2.5-altered neurotransmitter levels in AD mice could be the important causes of brain damage and proposed the promising miRNA and mRNA biomarkers and potential miRNA-mRNA interaction networks of PM2.5-promoted AD.