miRNA Transcriptome Research Articles

Dynamic patterns of microRNA expression during acute myeloid leukemia state-transition.

MicroRNAs (miRNAs) have been shown to hold prognostic value in acute myeloid leukemia (AML); however, the temporal dynamics of miRNA expression in AML are poorly understood. Using serial samples from a mouse model of AML to generate time-series miRNA sequencing data, we are the first to show that the miRNA transcriptome undergoes state-transition during AML initiation and progression. We modeled AML state-transition as a particle undergoing Brownian motion in a quasi-potential and validated the AML state-space and state-transition model to accurately predict time to AML in an independent cohort of mice. The critical points of the model provided a framework to align samples from mice that developed AML at different rates. Our mathematical approach allowed discovery of dynamic processes involved during AML development and, if translated to humans, has the potential to predict an individual’s disease trajectory.

Coding and regulatory transcriptome comparisons between fertile and infertile spermatozoa identify RNA signatures of male infertility

The aim of the present study was to identify RNA-based signatures of male infertility by sperm transcriptome analysis. In this study, deep sequencing analyses of coding (mRNA) and regulatory (miRNA) transcriptomes were performed by pooling 15 oligo/oligoasthenozoospermic infertile sperm and 9 normozoospermic fertile sperm samples. Furthermore, interesting candidates were selected for validation by real-time PCR. The comparison of miRNAs between cases and controls identified 94 differentially expressed miRNAs, of which at least 38 have known functions in spermatogenesis. In transcriptome (mRNA) data, a total of 60,505 transcripts were obtained. The comparison of coding RNAs between cases and controls revealed 11,688 differentially expressed genes. miRNA-mRNA paired analysis revealed that 94 differentially expressed miRNAs could potentially target 13,573 genes, of which 6419 transcripts were actually differentially expressed in our data. Out of these, 3303 transcripts showed inverse correlation with their corresponding regulatory miRNAs. Moreover, we found that most of the genes of miRNA-mRNA pairs were involved in male germ cell differentiation, apoptosis, meiosis, spermiogenesis and male infertility. In conclusion, we found that a number of sperm transcripts (miRNAs and mRNAs) have a very high potential of serving as infertility/sperm quality markers.

Identification of microRNA Transcriptome Involved in Bovine Intramuscular Fat Deposition.

BackgroundIntramuscular fat deposition in beef is a major determinant of carcass quality and value in the USA. The objective of this study was to examine changes in microRNA (miRNA) transcriptome that are involved with intramuscular fat deposition with time-on-concentrates (TOC). Yearling steers were individually fed a high concentrate diet and changes in intramuscular fat deposition were monitored by real-time ultrasound at 28 to 33 d intervals. Longissimus muscle biopsies collected on d 0, 92 and 124 TOC to examine changes in miRNA transcriptome that are involved in intramuscular fat deposition.ResultsSteer body weight increased (P < 0.0001) at each weigh day during TOC. Fat thickness increased (P < 0.005) from d 28 to 124. Ribeye area was larger (P < 0.001) on d 124 than d 61, which was larger than d 0 and 28. Ultrasound intramuscular fat content was greater (P < 0.001) on d 92 and 124 compared to d 0, 28 or 61. Sequencing of the muscle biopsy samples identified one miRNA, bta-miR-122, that was up-regulated (P < 0.005) at d 92 and 124 compared to d 0. At d 92 TOC, mRNA expression levels of fatty acid binding protein 4 (FABP4) and elongase 6 (ELOVL6) were up-regulated (P < 0.01) compared to d 0; whereas at d 124, lipogenic genes involved in de novo fatty acid synthesis, fatty acid transport, elongation and desaturation were highly up-regulated compared to d0.ConclusionsSmall RNA sequencing identified bta-miR-122 as a potential miRNA of interest that may be involved in intramuscular fat deposition with increasing TOC. Increased intramuscular fat content, as measured by real-time ultrasound, combined with differential gene expression suggests that preadipocyte differentiation may be stimulated first, which is followed by a global up-regulation of lipogenic genes involved in de novo fatty acid synthesis that provide fatty acids for subsequent hypertrophy.

Weighted gene co-expression network indicates that the DYNLL2 is an important regulator of chicken breast muscle development and is regulated by miR-148a-3p

BackgroundThe characteristics of muscle fibers determine the growth and meat quality of poultry. In this study, we performed a weighted gene co-expression network analysis (WGCNA) on the muscle fiber characteristics and transcriptome profile of the breast muscle tissue of Gushi chicken at 6, 14, 22, and 30 weeks.ResultsA total of 27 coexpressed biological functional modules were identified, of which the midnight blue module had the strongest correlation with muscle fiber and diameter. In addition, 7 hub genes were found from the midnight blue module, including LC8 dynein light chain 2 (DYNLL2). Combined with miRNA transcriptome data, miR-148a-3p was found to be a potential target miRNA of DYNLL2. Experiments on chicken primary myoblasts (CPMs) demonstrated that miR-148a-3p promotes the expression of myosin heavy chain (MYHC) protein by targeting DYNLL2, proving that it can promote differentiation of myoblasts.ConclusionsThis study proved that the hub gene DYNLL2 and its target miR-148-3p are important regulators in chicken myogenesis. These results provide novel insights for understanding the molecular regulation mechanisms related to the development of chicken breast muscle.

Multi-Omics Reveals Different Strategies in the Immune and Metabolic Systems of High-Yielding Strains of Laying Hens

Lohmann Brown (LB) and Lohmann Selected Leghorn (LSL) are two commercially important laying hen strains due to their high egg production and excellent commercial suitability. The present study integrated multiple data sets along the genotype-phenotype map to better understand how the genetic background of the two strains influences their molecular pathways. In total, 71 individuals were analyzed (LB, n = 36; LSL, n = 35). Data sets include gut miRNA and mRNA transcriptome data, microbiota composition, immune cells, inositol phosphate metabolites, minerals, and hormones from different organs of the two hen strains. All complex data sets were pre-processed, normalized, and compatible with the mixOmics platform. The most discriminant features between two laying strains included 20 miRNAs, 20 mRNAs, 16 immune cells, 10 microbes, 11 phenotypic traits, and 16 metabolites. The expression of specific miRNAs and the abundance of immune cell types were related to the enrichment of immune pathways in the LSL strain. In contrast, more microbial taxa specific to the LB strain were identified, and the abundance of certain microbes strongly correlated with host gut transcripts enriched in immunological and metabolic pathways. Our findings indicate that both strains employ distinct inherent strategies to acquire and maintain their immune and metabolic systems under high-performance conditions. In addition, the study provides a new perspective on a view of the functional biodiversity that emerges during strain selection and contributes to the understanding of the role of host–gut interaction, including immune phenotype, microbiota, gut transcriptome, and metabolome.

Expression characteristics and interaction networks of microRNAs in spleen tissues of grass carp (Ctenopharyngodon idella).

The spleen is an important immune organ in fish. MicroRNAs (miRNAs) have been shown to play an important role in the regulation of immune function. However, miRNA expression profiles and their interaction networks associated with the postnatal late development of spleen tissue are still poorly understood in fish. The grass carp (Ctenopharyngodon idella) is an important economic aquaculture species in China. Here, two small RNA libraries were constructed from the spleen tissue of healthy grass carp at one-year-old and three-year-old. A total of 324 known conserved miRNAs and 9 novel miRNAs were identified by using bioinformatic analysis. Family analysis showed that 23 families such as let-7, mir-1, mir-10, mir-124, mir-8, mir-7, mir-9, and mir-153 were highly conserved between vertebrates and invertebrates. In addition, 14 families such as mir-459, mir-430, mir-462, mir-7147, mir-2187, and mir-722 were present only in fish. Expression analysis showed that the expression patterns of miRNAs in the spleen of one-year-old and three-year-old grass carp were highly consistent, and the percentage of miRNAs with TPM > 100 was above 39%. Twenty significant differentially expressed (SDE) miRNAs were identified. Gene ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis showed that these SDE miRNAs were primarily involved in erythrocyte differentiation, lymphoid organ development, immune response, lipid metabolic process, the B cell receptor signaling pathway, the T cell receptor signaling pathway, and the PPAR signaling pathway. In addition, the following miRNA-mRNA interaction networks were constructed: immune and hematopoietic, cell proliferation and differentiation, and lipid metabolism. This study determined the miRNA transcriptome as well as miRNA-mRNA interaction networks in normal spleen tissue during the late development stages of grass carp. The results expand the number of known miRNAs in grass carp and are a valuable resource for better understanding the molecular biology of the spleen development in grass carp.

MiR-24-3p Conservatively Regulates Muscle Cell Proliferation and Apoptosis by Targeting Common Gene CAMK2B in Rat and Cattle.

Simple SummaryMicroRNAs (miRNAs) play important roles in the development of skeletal muscle. Here, a comparative analysis of four skeletal muscle transcriptomes of cattle, rat, goat, and pig showed that miR-24-3p may conservatively regulate muscle development. There is a large proportion of the target genes of miR-24-3p shared by cattle, rat, goat, and pig. GO (Gene ontology) and KEGG (A Kyoto Encyclopedia of Genes and Genomes) enrichment analysis showed that these genes are enriched in multiple cell functions and signal pathways that are closely related to muscle development. In rat and cattle, a double luciferase test showed that three shared target genes, WNT4 (Wnt Family Member 4), CAMK2B (Calcium/Calmodulin Dependent Protein Kinase II Beta), TCF7 (Transcription Factor 7) were targeted by mmu-miR-24-3p and bta-miR-24-3p. The three shared target genes (WNT4, CAMK2B, and TCF7) were all targets in the Wnt signaling pathway, which plays an important role in muscle proliferation in rat and cattle. The shared target gene (CAMK2B) rat was increased significantly after the inhibition of miR-24-3p in rat and cattle. This study will be a good foundation by which to improve our understanding of the functions of miRNAs in the regulation of muscle development.Skeletal muscle plays an important role in the growth and development of meat animals. MicroRNAs (miRNAs) can participate in the regulation of muscle development-related functions; however, there have been few reports on whether there are related miRNAs that conservatively regulate muscle development among different species. In this study, the miRNA transcriptome sequencing data of the muscle tissue of cattle, rat, goat, and pig showed that miR-24-3p may conservatively regulate muscle development in these species. Furthermore, mmu-miR-24-3p can positively regulate C2C12 cell proliferation and apoptosis by regulating key proliferation and apoptosis genes in muscle development, which was verified by CCK-8 and RT-qPCR. Bta-miR-24-3p can also positively regulate the proliferation and apoptosis of bovine muscle primary cells by regulating key proliferation and apoptosis genes in the process of muscle development, as verified by CCK-8 and RT-qPCR. The target genes of miR-24-3p in cattle, rat, goat, and pig, which include a large proportion of target genes shared among the four species, are enriched in multiple cell functions and signal pathways that are closely related to muscle development, as revealed by GO and KEGG enrichment analysis. A double luciferase test showed that the shared target genes WNT4, CAMK2B, and TCF7 were targeted by mmu-miR-24-3p in rat and bta-miR-24-3p in cattle. These three shared target genes WNT4, CAMK2B, and TCF7 are involved in the Wnt signaling pathway, which showed that miR-24-3p plays an important role in rat and cattle. The shared target gene (CAMK2B) in rat and cattle increased significantly after the inhibition of miR-24-3p by RT-qPCR. The findings of this study contribute to a better understanding of the role of miR-24-3p in the regulation of muscle development.

The Mechanism of Carbonate Alkalinity Exposure on Juvenile Exopalaemon carinicauda With the Transcriptome and MicroRNA Analysis

Saline-alkali water is distributed all over the world and affects the development of fisheries. The ridge tail white prawn Exopalaemon carinicauda is an economically important shrimp in China, which has excellent environmental tolerance. However, due to its complex genetic structure, there have been few studies on its alkalinity-adaptation mechanisms. In order to explore the molecular mechanisms of E. carinicauda in adapting to the alkaline water, mRNA and miRNA transcriptomes from the gills of E. carinicauda were determined. The results showed that after alkalinity stress, the structures of the gill and hepatopancreas were disorganized; however, E. carinicauda could still maintain vital signs. Transcriptome results showed that ATP binding protein and carbonic anhydrase played an important role in alkalinity-adaptation. At the same time, a large number of immune-related genes were up-regulated, which protect E. carinicauda from bacterial infection. MiRNAs also played an important role in alkalinity-adaptation. A total of 24 miRNAs were identified as differentially expressed after alkalinity stress, and up-regulated miRNAs might active the GnRH signaling pathway and accelerate the synthesis and secretion of aldosterone, which might maintain the balance of osmotic pressure in E. carinicauda to adapt to alkaline environment. These results provide a better understanding of the alkalinity-adaptation mechanism of economic aquatic animals and provide theoretical basis for breeding in the future.

Comparative Transcriptomic Profiles of Differentiated Adipocytes Provide Insights into Adipogenesis Mechanisms of Subcutaneous and Intramuscular Fat Tissues in Pigs.

Subcutaneous fat thickness and intramuscular fat content are closely related to meat production and quality in the pig industry. Adipogenesis in adipocytes from subcutaneous and intramuscular fat tissues involves different genes and regulatory mechanisms. Analyzing the data of mRNA and miRNA transcriptomes during the differentiation of adipocytes from these two sources will help identify the different mechanisms of subcutaneous and intramuscular fat deposition. In this study, RNA sequencing technology was used to analyze the differential expression of genes and miRNAs in subcutaneous and intramuscular adipocytes at days 0, 2, 4, and 8 of differentiation. We mainly attributed the difference between fat depositions of the two types of adipocytes to variations in the expression patterns of related genes. Through combined weighted gene co-expression network analysis and K-MEANS, we identified 30 and 22 genes that mainly regulated the differentiation of subcutaneous adipocytes and intramuscular adipocytes, respectively. A total of 17 important candidate miRNAs were identified. This study provides valuable reference for the study of different mechanisms of adipogenesis among subcutaneous and intramuscular fat and contributes to improving pig breeding.

Genome-scale mRNA and miRNA transcriptomic insights into the regulatory mechanism of cucumber corolla opening

‘Corollas and spines’ is an important trait for fresh market cucumber. In a unique cucumber line, ‘6457’, the super ovary is much larger and corolla opening is delayed by 4–5 days, thus the resulting fruit has a flower that remains on the tip, which has a high commodity value. In this study, to better understand the molecular basis of corolla opening, mRNA and miRNA transcriptome analyses were performed during corolla development of the super and normal ovaries. A total of 234 differentially expressed miRNAs (DEMs) and 291 differentially expressed target genes (DE-target genes) were identified from four developmental stages, and the greatest number of DEMs was found at the yellow bud stage. Thirty of the DE-target genes were regulated by more than five DEMs, among which, CsHD-Zip was regulated by 28 DEMs, followed by DD2X (18). In addition, the expression patterns of miRNA_104, miRNA_157, miRNA_349, miRNA_242, and miRNA_98 were similar during corolla development, and they shared the same target gene, CsCuRX. Moreover, several critical candidate DEMs and DE-target genes were characterized and profiled by a qRT-PCR experiment. Three of the miRNAs, miRNA_157-CsCuRX, miRNA_411-CsGH3.6, and miRNA_161/297/257-CsHD-Zip, might be responsible for corolla opening in the cucumber super ovary. This integrated study on the transcriptional and post-transcriptional profiles can provide insights into the molecular regulatory mechanism underlying corolla opening in the cucumber.

Amphibian-derived peptide homodimer OA-GL17d promotes skin wound regeneration through the miR-663a/TGF-β1/Smad axis.

BackgroundAmphibian-derived peptides exhibit considerable potential in the discovery and development of new therapeutic interventions for clinically challenging chronic skin wounds. MicroRNAs (miRNAs) are also considered promising targets for the development of effective therapies against skin wounds. However, further research in this field is anticipated. This study aims to identify and provide a new peptide drug candidate, as well as to explore the underlying miRNA mechanisms and possible miRNA drug target for skin wound healing.MethodsA combination of Edman degradation, mass spectrometry and cDNA cloning were adopted to determine the amino acid sequence of a peptide that was fractionated from the secretion of Odorrana andersonii frog skin using gel-filtration and reversed-phase high-performance liquid chromatography. The toxicity of the peptide was evaluated by Calcein-AM/propidium iodide (PI) double staining against human keratinocytes (HaCaT cells), hemolytic activity against mice blood cells and acute toxicity against mice. The stability of the peptide in plasma was also evaluated. The prohealing potency of the peptide was determined by MTS, scratch healing and a Transwell experiment against HaCaT cells, full-thickness injury wounds and scald wounds in the dorsal skin of mice. miRNA transcriptome sequencing analysis, enzyme-linked immunosorbent assay, real-time polymerase chain reaction and western blotting were performed to explore the molecular mechanisms.ResultsA novel peptide homodimer (named OA-GL17d) that contains a disulfide bond between the 16th cysteine residue of the peptide monomer and the sequence ‘GLFKWHPRCGEEQSMWT’ was identified. Analysis showed that OA-GL17d exhibited no hemolytic activity or acute toxicity, but effectively promoted keratinocyte proliferation and migration and strongly stimulated the repair of full-thickness injury wounds and scald wounds in the dorsal skin of mice. Mechanistically, OA-GL17d decreased the level of miR-663a to increase the level of transforming growth factor-β1 (TGF-β1) and activate the subsequent TGF-β1/Smad signaling pathway, thereby resulting in accelerated skin wound re-epithelialization and granular tissue formation.ConclusionsOur results suggest that OA-GL17d is a new peptide drug candidate for skin wound repair. This study emphasizes the importance of exogenous peptides as molecular probes for exploring competing endogenous RNA mechanisms and indicates that miR-663a may be an effective target for promoting skin repair.

The Emerging Role of MicroRNAs in Bone Diseases and Their Therapeutic Potential.

MicroRNAs (miRNAs) are a class of small (20–24 nucleotides), highly conserved, non-coding RNA molecules whose main function is the post-transcriptional regulation of gene expression through sequence-specific manners, such as mRNA degradation or translational repression. Since these key regulatory molecules are implicated in several biological processes, their altered expression affects the preservation of cellular homeostasis and leads to the development of a wide range of pathologies. Over the last few years, relevant investigations have elucidated that miRNAs participate in different stages of bone growth and development. Moreover, the abnormal expression of these RNA molecules in bone cells and tissues has been significantly associated with the progression of numerous bone diseases, including osteoporosis, osteosarcoma, osteonecrosis and bone metastasis, among others. In fact, miRNAs regulate multiple pathological mechanisms, including altering either osteogenic or osteoblast differentiation, metastasis, osteosarcoma cell proliferation, and bone loss. Therefore, in this present review, aiming to impulse the research arena of the biological implications of miRNA transcriptome in bone diseases and to explore their potentiality as a theragnostic target, we summarize the recent findings associated with the clinical significance of miRNAs in these ailments.

Identification of miRNA-mRNA Regulatory Modules Involved in Lipid Metabolism and Seed Development in a Woody Oil Tree (Camellia oleifera).

Tea oil camellia (Camellia oleifera), an important woody oil tree, is a source of seed oil of high nutritional and medicinal value that is widely planted in southern China. However, there is no report on the identification of the miRNAs involved in lipid metabolism and seed development in the high- and low-oil cultivars of tea oil camellia. Thus, we explored the roles of miRNAs in the key periods of oil formation and accumulation in the seeds of tea oil camellia and identified miRNA–mRNA regulatory modules involved in lipid metabolism and seed development. Sixteen small RNA libraries for four development stages of seed oil biosynthesis in high- and low-oil cultivars were constructed. A total of 196 miRNAs, including 156 known miRNAs from 35 families, and 40 novel miRNAs were identified, and 55 significantly differentially expressed miRNAs were found, which included 34 upregulated miRNAs, and 21 downregulated miRNAs. An integrated analysis of the miRNA and mRNA transcriptome sequence data revealed that 10 miRNA–mRNA regulatory modules were related to lipid metabolism; for example, the regulatory modules of ath-miR858b–MYB82/MYB3/MYB44 repressed seed oil biosynthesis, and a regulation module of csi-miR166e-5p–S-ACP-DES6 was involved in the formation and accumulation of oleic acid. A total of 23 miRNA–mRNA regulatory modules were involved in the regulation of the seed size, such as the regulatory module of hpe-miR162a_L-2–ARF19, involved in early seed development. A total of 12 miRNA–mRNA regulatory modules regulating growth and development were identified, such as the regulatory modules of han-miR156a_L+1–SPL4/SBP2, promoting early seed development. The expression changes of six miRNAs and their target genes were validated using quantitative real-time PCR, and the targeting relationship of the cpa-miR393_R-1–AFB2 regulatory module was verified by luciferase assays. These data provide important theoretical values and a scientific basis for the genetic improvement of new cultivars of tea oil camellia in the future.

Indoleamine 2, 3 Dioxygenase 1 Impairs Chondrogenic Differentiation of Mesenchymal Stem Cells in the Joint of Osteoarthritis Mice Model.

Osteoarthritis (OA) is a serious joint inflammation that leads to cartilage degeneration and joint dysfunction. Mesenchymal stem cells (MSCs) are used as a cell-based therapy that showed promising results in promoting cartilage repair. However, recent studies and clinical trials explored unsatisfied outcomes because of slow chondrogenic differentiation and increased calcification without clear reasons. Here, we report that the overexpression of indoleamine 2,3 dioxygenase 1 (IDO1) in the synovial fluid of OA patients impairs chondrogenic differentiation of MSCs in the joint of the OA mice model. The effect of MSCs mixed with IDO1 inhibitor on the cartilage regeneration was tested compared to MSCs mixed with IDO1 in the OA animal model. Further, the mechanism exploring the effect of IDO1 on chondrogenic differentiation was investigated. Subsequently, miRNA transcriptome sequencing was performed for MSCs cocultured with IDO1, and then TargetScan was used to verify the target of miR-122-5p in the SF-MSCs. Interestingly, we found that MSCs mixed with IDO1 inhibitor showed a significant performance to promote cartilage regeneration in the OA animal model, while MSCs mixed with IDO1 failed to stimulate cartilage regeneration. Importantly, the overexpression of IDO1 showed significant inhibition to Sox9 and Collagen type II (COL2A1) through activating the expression of β-catenin, since inhibiting of IDO1 significantly promoted chondrogenic signaling of MSCs (Sox9, COL2A1, Aggrecan). Further, miRNA transcriptome sequencing of SF-MSCs that treated with IDO1 showed significant downregulation of miR-122-5p which perfectly targets Wnt1. The expression of Wnt1 was noticed high when IDO1 was overexpressed. In summary, our results suggest that IDO1 overexpression in the synovial fluid of OA patients impairs chondrogenic differentiation of MSCs and cartilage regeneration through downregulation of miR-122-5p that activates the Wnt1/β-catenin pathway.

Comprehensive Transcriptome and Pathway Analyses Revealed Central Role for Fascin in Promoting Triple-Negative Breast Cancer Progression.

Recent years have witnessed major progress in development of novel therapeutic agents such as chemotherapy, targeted therapy and immune checkpoint inhibitors for breast cancer. However, cancer-related death remains high especially in triple-negative breast cancer (TNBC) due limited therapeutic options. Development of targeted therapies for TNBC requires better understanding of biology and signaling networks that promote disease progression. Fascin, an actin bundling protein, was identified as a key regulator of many signaling pathways that contribute to breast cancer progression. Herein, fascin ShRNA was used to generate stable fascin knockdown (FSCN1KD) in the MDA-MB-231 TNBC cell line and then were subjected to comprehensive mRNA and miRNA transcriptome analysis. We identified 129 upregulated and 114 downregulated mRNA transcripts, while 14 miRNAs were differentially expressed in FSCN1KD. Ingenuity pathway analysis (IPA) was used to predict the impact of differentially expressed transcripts on signaling pathways and functional categories and to construct miRNA-mRNA regulatory networks in the context of FSCN1 knockdown. Compared to FSCN1KD, fascin-positive (FSCN1CON) breast cancer cells showed enrichment in genes promoting cellular proliferation, migration, survival, DNA replication and repair. Expression of FSCN1high (identified in BRCA dataset from TCGA) in conjunction with elevated expression of the top 10 upregulated or decreased expression of the top 10 downregulated genes (identified in our FSCN1CON vs. FSCN1KD) correlates with worst survival outcome. Taken together, these data confirmed fascin’s role in promoting TNBC progression, and identified a novel opportunity for therapeutic interventions via targeting those FSCN1-related transcripts.

Combined analysis of mRNA and miRNA transcriptomes reveals the regulatory mechanism of PVY resistance in tobacco

Potyvirus (viruses in the genus Potyvirus, family Potyviridae, PVY) is one of the most important pathogens in tobacco (Nicotiana tabacum L.) and other main crops, which cause devastating epidemic and significant production losses of crops worldwide. K326 is an important tobacco cultivar worldwide, but it is seriously plagued by PVY. In this study, an ethyl methanesulfonate (EMS) mutant M867 from K326 was developed, which was highly resistant to PVY. To explore the molecular mechanism of M867 resistance to PVY, RNA-seq and small RNA-seq were applied to analyze the differences between K326 and M867 responds to PVY infection. There are a total of 34 differentially expressed miRNAs and 5083 mRNAs were identified between K326 and M867 following PVY infection. GO and KEGG enrichment analysis showed PVY response process was significantly related with photosynthesis, carbon metabolism, and antioxidant activity. Thereafter, the results of hormone determination showed that the IAA level of K326 was almost zero after PVY infection, but M867 maintained a high IAA content. Simultaneously, the contents of JA and SA in M867 showed a substantial increase. M867 auxin-related genes (e.g., AUX1, IAA4, and ARF9) were significantly down-regulated after PVY infection compared to K326, but genes related to various hormones, such as CTK, GA, ABA, JA, and SA, showed significant up-regulation. Redox-related peroxidase, superoxide dismutase, and catalase were significantly up-regulated by PVY infection. Besides, six pairs of significant mRNA-miRNA interactions were found, and these genes may be closely related to PVY-host interactions. Furthermore, our findings revealed that the differences between M867 and K326 in hormones, redox reactions, and energy metabolism changed the molecular pathway of the PVY-host interaction, which led to PVY resistance in M867. These results provide valuable resources for elucidating the molecular mechanism of the tobacco-PVY interaction.

MicroRNA profiling of psoriatic skin identifies 11 miRNAs associated with disease severity.

MicroRNAs (miRNAs) are small non-coding RNAs that have emerged as central regulators of gene expression and powerful biomarkers of disease. Much is yet unknown about their role in psoriasis pathology. To globally characterize the miRNAome of psoriatic skin, skin biopsies were collected from psoriatic cases (n=75) and non-psoriatic controls (n=46) and RNA sequenced. Count data were meta-analysed with a previously published dataset (cases, n=24, controls, n=20), increasing the number of psoriatic cases fourfold from previously published studies. Differential gene expression analyses were performed comparing lesional psoriatic (PP), non-lesional psoriatic (PN) and control (NN) skin. Further, functional enrichment and cell-specific analyses were performed. Across all contrasts, we identified 439 significantly differentially expressed miRNAs (DEMs), of which 85 were novel for psoriasis and 11 were related to disease severity. Meta-analyses identified 20 DEMs between PN and NN, suggesting an inherent change in the constitution of all skin in psoriasis. By integrating the miRNA transcriptome with mRNA target interactions, we identified several functionally enriched terms, including "thyroid hormone signalling," "insulin resistance" and various infectious diseases. Cell-specific expression analyses revealed that the upregulated DEMs were enriched in epithelial and immune cells. This study provides the most comprehensive overview of the miRNAome in psoriatic skin to date and identifies a miRNA signature related to psoriasis severity. Our results may represent molecular links between psoriasis and related comorbidities and have outlined potential directions for future functional studies to identify biomarkers and treatment targets.

Mir-671-5p, Mir-193b-5p, Mir-1307-5p Are Useful for Predicting Outcome in Diffuse Large B-Cell Lymphoma

Background: Diffuse large B-cell Lymphoma (DLBCL) is stratified histologically as GCB and ABC phenotypes based on Hans algorithm. Besides this categorization, there is a paucity of molecular markers useful for predicting response to CHOP/R-CHOP chemotherapy and outcome of DLBCL patients. We performed miRNA profiling to identify potential miRNAs for their ability to predict response to chemotherapy and outcome in DLBCL patients.Methods: A total of 41 cases of histologically and immunohistochemically confirmed DLBCL cases who received R-CHOP/CHOP were included in this study after IRB approval. The follow up ranged from 7 to 2273 days with a mean of, 874 days. Cases in sustained complete remission for ≥ 1095 days were identified as “responders” whereas cases with partial response/stable disease or primary progressive disease were identified as“non-responders”. In the first part of the study, miRNA transcriptome profiling was performed from lymph node tissue of 10 cases of DLBCL which included 3 responders and 7 non-responders (3 with refractory disease and 4 with relapse) for identification and selection of differentially expressed miRNAs. Total RNA was isolated from the biopsies, libraries constructed with ION TOTAL RNA-SEQ v2 and sequenced on S5 ION Torrent. The filtered reads were aligned to the reference genome hg19 using Bowtie. Differentially expressed miRNAs were identified by DESeq2. In the second part of the study, validation of the selected miRNAs was performed in a retrospective validation cohort of 41 cases including 17 responders and 24 non-responders by semi-quantitative qRT-PCR. ROC curves were obtained and best cut-off for the delta Ct values was selected for each of the miRNAs to distinguish the two cohorts of patients. In the third part of the study, an additional 38 cases of DLBCL were included prospectively as testing cohort, in whom mi-RNA were isolated from paired pre-treatment biopsy and plasma samples were evaluated for the expression of the selected miRNAs for prediction of response to treatment and outcome.Results: miRNA transcriptome profiling revealed a total of 30 differentially expressed miRNAs among DLBCL responders vs non-responders and the data was submitted to GEO database (GSE179760). The top 4 significantly downregulated miRNAs in non-responders selected for validation included miR-193b-5p, miR-671-5p, miR-1307-5p and miR-326 as shown in the volcano plot and heatmap in Fig.1. In-silico prediction for the interacting genes of the selected miRNAs revealed genes involved in lymphoma progression and chemoresistance . Validation by qRT-PCR revealed significant down-regulation of miR-1307-5p, miR-671-5p and miR-193b-5p in the non-responders compared to responders (Fig.2). There was no significant association of miRNA expression with disease stage, nodal or extranodal origin and the presence of B symptoms. However, the downregulation of miR-671-5p was found to be associated with the presence of B-symptoms. Downregulation of miR-193b-5p, miR-671-5p, miR-1307-5p expression showed a significant association with progression free survival as shown in Fig.3 (Spearman's correlation plot and Kaplan-Meier survival curves).In the prospective cohort of 38 DLBCL patients, all 3 miRNAs were expressed in cell free plasma and tissue but only miRNA-193b-5p showed correlation between them. Using the cut-off Ct values obtained from ROC curves of tissue miRNA levels in the validation cohort , 16/38 patients were predicted to be non-responders. The preliminary data showed that 8 of these 16 cases were correctly predicted (6 died of disease, 2 showed partial response). The remaining cases are still under follow-up. The treatment response could not be predicted on the corresponding plasma levels.Conclusion: Downregulation of miRNA-671-5p, miR-193b-5p and miR-1307-5p in pre-treatment biopsy samples is associated with poor outcome and shorter progression free survival and poor treatment response leading to refractory/relapse DLBCL, which needs confirmation in larger studies. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Integrated transcriptome analysis of immune-related mRNAs and microRNAs in Macrobrachium rosenbergii infected with Spiroplasma eriocheiris

Macrobrachium rosenbergii (M. rosenbergii), is a major aquaculture species in China and Southeast Asia. However, infection with Spiroplasma eriocheiris (S. eriocheiris) has caused huge economic losses to the cultivation of M. rosenbergii. Currently, there are few reports on the immune response mechanism of M. rosenbergii that are infected with S. eriocheiris. To clarify the immune response mechanism of M. rosenbergii infected with S. eriocheiris, the key immune genes which respond to the infection with the pathogen and the regulation of related microRNAs (miRNAs) on them were identified. In this study, the mRNA and miRNA transcriptome of hepatopancreas of M. rosenbergii at different infection stages were analyzed using high-throughput sequencing and qRT-PCR. In the mRNA transcriptome, 27,703 and 33,402 genes were expressed in healthy and susceptible M. rosenbergii, respectively. By digital gene-expression profiling analysis, 23,929 and 24,325 genes were expressed, and 223 and 373 genes were significantly up-regulated and down-regulated, respectively. A total of 145 key genes related to Toll, IMD, JAK/STAT and MAPK were excavated from the transcriptome. In the miRNA transcriptome, 549 miRNAs (Conserved: 41, PN-type: 83, PC-type: 425) were sequenced, of which 87 were significantly up-regulated and 23 were significantly down-regulated. Among the related immune pathways, there are 259 miRNAs involved in the regulation of target genes in the Toll and IMD pathways, 231 JAK/STAT pathways and 122 MAPK pathways. qRT-PCR differential detection of immune-related miRNAs and mRNAs showed that 22 miRNAs with significant differences (P < 0.05) such as mro-miR-100, PC-mro-3p-27 and PN-mro-miR-316 had corresponding regulatory relationships with 22 important immune genes such as TLR2, TLR3, TLR4, TLR5, MyD88, Pelle and Relish in different stages after infection. In this study, the immune genes and related regulatory miRNAs of M. rosenbergii in response to S. eriocheiris infection were obtained. The results can provide basic data to further reveal the immune defense mechanism of M. rosenbergii against S. eriocheiris infection.

A blueprint from nature: miRNome comparison of plasma cells and CHO cells to optimize therapeutic antibody production

Chinese Hamster Ovary (CHO) cells are the most frequently used biopharmaceutical production hosts, although industry is presently suffering from their variable recombinant product quality, insufficient long-term stability and low productivity. Here, we present an effort to address overall cell line engineering by a novel bottom-up microRNA (miRNA) screening approach. miRNAs are small non-coding RNAs known to regulate global gene expression at the post-transcriptional level and have proved to serve as promising tools for cell line engineering for over a decade. Here the miRNome of plasma cells (PCs) has been analyzed as the natural blueprint for optimized production and secretion of antibodies. Performing comparative miRNome cross-species expression analysis of four murine/human PC-derived (PCD) and two CHO cell lines showed 147 conserved miRNAs to be differentially expressed between PCDs and CHOs. Conducting a targeted miRNA screen of this PC-specific miRNA subset revealed 14 miRNAs to improve bioprocess relevant parameters in CHO cells, among them the PC-characteristic miR-183 cluster. Finally, miRNA target prediction tools and transcriptome analysis were combined to elucidate differentially regulated lysine degradation and fatty acid metabolism pathways in monoclonal antibody (mAb) expressing CHO-DG44 and CHO-K1 cells, respectively. Thus, substantial new insights into molecular and cellular mechanisms of biopharmaceutical production cell lines can be gained by targeted bottom-up miRNA screenings.