Isolation and Identification of Bovine Preadipocytes and Screening of MicroRNAs Associated with Adipogenesis

The elucidation of the mechanisms of preadipocyte differentiation and fat accumulation in adipocytes is a major work in beef cattle breeding. As important post-transcriptional regulators, microRNAs (miRNAs) take part in cell proliferation, differentiation, apoptosis, and fat metabolism through binding seed sites of targeting mRNAs. The aim of this study was to isolate and identify bovine preadipocytes and screen miRNAs associated with adipogenesis. Bovine preadipocytes were isolated from subcutaneous fatty tissue and induced to differentiate into adipocytes. Verification of preadipocytes and adipocytes was performed by qRT-PCR (real-time quantitative reverse transcription PCR), Oil Red O staining, and immunofluorescence staining. Total RNA was extracted for small RNA sequencing. The sequencing data showed that 131 miRNAs were highly expressed in adipocytes, and 119 miRNAs were highly expressed in preadipocytes. Stem–loop qPCR (stem–loop quantitative real-time PCR) results showed that the expression patterns of 11 miRNAs were consistent with the sequencing results (miR-149-5p, miR-24-3p, miR-199a-5p, miR-33a, etc.). According to KEGG pathway and Gene Ontology (GO) analyses, multiple predicted target genes were associated with lipid metabolism. In summary, this study provides a protocol of isolating bovine preadipocytes and screening various differently expressed miRNAs during preadipocyte differentiation.

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Genome-wide analysis of changes in miRNA and target gene expression reveals key roles in heterosis for Chinese cabbage biomass

Horticulture Research ◽

10.1038/s41438-021-00474-6 ◽

2021 ◽

Vol 8 (1) ◽

Author(s):

Peirong Li ◽

Tongbing Su ◽

Deshuang Zhang ◽

Weihong Wang ◽

Xiaoyun Xin ◽

...

Keyword(s):

Chinese Cabbage ◽

Leaf Development ◽

Target Genes ◽

Expression Patterns ◽

Seedling Stage ◽

Differentially Expressed ◽

Small Rna Sequencing ◽

Leaf Morphogenesis ◽

Expression Levels ◽

Parental Lines

AbstractHeterosis is a complex phenomenon in which hybrids show better phenotypic characteristics than their parents do. Chinese cabbage (Brassica rapa L. spp. pekinensis) is a popular leafy crop species, hybrids of which are widely used in commercial production; however, the molecular basis of heterosis for biomass of Chinese cabbage is poorly understood. We characterized heterosis in a Chinese cabbage F1 hybrid cultivar and its parental lines from the seedling stage to the heading stage; marked heterosis of leaf weight and biomass yield were observed. Small RNA sequencing revealed 63 and 50 differentially expressed microRNAs (DEMs) at the seedling and early-heading stages, respectively. The expression levels of the majority of miRNA clusters in the F1 hybrid were lower than the mid-parent values (MPVs). Using degradome sequencing, we identified 1,819 miRNA target genes. Gene ontology (GO) analyses demonstrated that the target genes of the MPV-DEMs and low parental expression level dominance (ELD) miRNAs were significantly enriched in leaf morphogenesis, leaf development, and leaf shaping. Transcriptome analysis revealed that the expression levels of photosynthesis and chlorophyll synthesis-related MPV-DEGs (differentially expressed genes) were significantly different in the F1 hybrid compared to the parental lines, resulting in increased photosynthesis capacity and chlorophyll content in the former. Furthermore, expression of genes known to regulate leaf development was also observed at the seedling stage. Arabidopsis plants overexpressing BrGRF4.2 and bra-miR396 presented increased and decreased leaf sizes, respectively. These results provide new insight into the regulation of target genes and miRNA expression patterns in leaf size and heterosis for biomass of B. rapa.

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Characteristic Dissection of Xanthomonas oryzae pv. oryzae Responsive MicroRNAs in Rice

International Journal of Molecular Sciences ◽

10.3390/ijms21030785 ◽

2020 ◽

Vol 21 (3) ◽

pp. 785

Author(s):

Yanfeng Jia ◽

Chunrong Li ◽

Quanlin Li ◽

Pengcheng Liu ◽

Dongfeng Liu ◽

...

Keyword(s):

Rice Variety ◽

Bioinformatic Analysis ◽

Infection Process ◽

Xanthomonas Oryzae ◽

Differentially Expressed ◽

Small Rna Sequencing ◽

Sequencing Data ◽

Stem Loop ◽

Differentially Expressed Mirnas ◽

Plant Pathogen Interaction

MicroRNAs (miRNAs) are crucial player in plant-pathogen interaction. While the evidence has demonstrated that rice miRNAs mediate immune response to pathogens invasion, the roles of miRNAs on Xanthomonas oryzae pv. oryzae (Xoo) attack remain be in place. Herein, we monitored the responsive changes of rice miRNAs at 0, 8, 24 h across Xoo strain PXO86 infection in its compatible rice variety IR24 and incompatible variety IRBB5 by small RNA sequencing, and the genes targeted by miRNAs were also detected via degradome technology. The faithfulness of sequencing data was validated through quantitative real-time stem-loop reverse transcription-polymerase chain reaction assay. Bioinformatic analysis showed that the differentially expressed miRNAs could be divided into three immunity-related clusters, and 80 regulatory units were emerged in infection process, which comprises 29 differentially expressed known miRNAs and 38 cleaved targets. Furthermore, the miRNA presumptive function of separate immunity cluster in rice-Xoo interplay was confirmed through overexpressing osa-miR164a, osa-miR167d and osa-miR159b, and the disruption of regulatory units, osa-miR164a/OsNAC60, osa-miR167d-5p/OsWD40-174 and osa-miR159b/OsMYBGA, OsLRR-RLK2, OsMPK20-4, may reset rice defense response to Xoo infestation in a controllable manner. These findings provide new insights into the complex roles of characteristic miRNAs and their targets in rice-Xoo interactions.

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Distinct miRNA Signatures and Networks Discern Fetal from Adult Erythroid Differentiation and Primary from Immortalized Erythroid Cells

International Journal of Molecular Sciences ◽

10.3390/ijms22073626 ◽

2021 ◽

Vol 22 (7) ◽

pp. 3626

Author(s):

Panayiota L. Papasavva ◽

Nikoletta Y. Papaioannou ◽

Petros Patsali ◽

Ryo Kurita ◽

Yukio Nakamura ◽

...

Keyword(s):

Mirna Expression ◽

Regulatory Networks ◽

Target Genes ◽

Human Peripheral Blood ◽

Expression Patterns ◽

Erythroid Differentiation ◽

Erythroid Cell ◽

Small Rna Sequencing ◽

Erythroid Cells ◽

Mirna Regulation

MicroRNAs (miRNAs) are small non-coding RNAs crucial for post-transcriptional and translational regulation of cellular and developmental pathways. The study of miRNAs in erythropoiesis elucidates underlying regulatory mechanisms and facilitates related diagnostic and therapy development. Here, we used DNA Nanoball (DNB) small RNA sequencing to comprehensively characterize miRNAs in human erythroid cell cultures. Based on primary human peripheral-blood-derived CD34+ (hCD34+) cells and two influential erythroid cell lines with adult and fetal hemoglobin expression patterns, HUDEP-2 and HUDEP-1, respectively, our study links differential miRNA expression to erythroid differentiation, cell type, and hemoglobin expression profile. Sequencing results validated by reverse-transcription quantitative PCR (RT-qPCR) of selected miRNAs indicate shared differentiation signatures in primary and immortalized cells, characterized by reduced overall miRNA expression and reciprocal expression increases for individual lineage-specific miRNAs in late-stage erythropoiesis. Despite the high similarity of same-stage hCD34+ and HUDEP-2 cells, differential expression of several miRNAs highlighted informative discrepancies between both cell types. Moreover, a comparison between HUDEP-2 and HUDEP-1 cells displayed changes in miRNAs, transcription factors (TFs), target genes, and pathways associated with globin switching. In resulting TF-miRNA co-regulatory networks, major therapeutically relevant regulators of globin expression were targeted by many co-expressed miRNAs, outlining intricate combinatorial miRNA regulation of globin expression in erythroid cells.

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Potential role of miR-155-5p in fat deposition and skeletal muscle development of chicken

Bioscience Reports ◽

10.1042/bsr20193796 ◽

2020 ◽

Vol 40 (6) ◽

Author(s):

Sifan Xu ◽

Yang Chang ◽

Guanxian Wu ◽

Wanting Zhang ◽

Chaolai Man

Keyword(s):

Skeletal Muscle ◽

Muscle Development ◽

Target Genes ◽

Expression Profiles ◽

Expression Patterns ◽

Fat Deposition ◽

Skeletal Muscle Development ◽

Stem Loop ◽

Related Target ◽

Kegg Pathways

Abstract miR-155 has multiple functions in many physiological and pathological processes. However, little is known about the expression characteristics of avian miR-155. In the present study, partial pri-miR-155 sequences were cloned from AA+ broiler, Sanhuang broiler and Hy-Line Brown layer, respectively. Stem–loop qRT-PCR was performed to detect the miR-155-5p spatiotemporal expression profiles of each chicken breed, and the target genes of miR-155-5p were predicted in Gene Oncology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The results showed that the partial pri-miR-155 sequences of different breeds of chicken were high conserved. The expression patterns of miR-155-5p between broiler and layer were basically similar, and miR-155-5p was expressed highly in immune related tissues (spleen, thymus and bursa). In the same old chicken (14 days old), miR-155-5p expression activity of fat tissue all had higher level in the three chicken breeds, but the expression activities in skeletal muscle of broilers were significantly lower than that of layer (P<0.05). In different development stages of Hy-Line Brown layer, miR-155-5p expression activities in skeletal muscle of 14-day-old and 10-month-old layers were significantly lower than that of 24-month-old layer (P<0.05). Fat related target genes (ACOX1, ACOT7, FADS1, SCD and HSD17B12) and skeletal muscle related target genes (CCNT2, DMD, CFL2, MAPK14, FLNB, ZBTB18 and CDK5) of miR-155-5p were predicted, respectively. The results indicate that miR-155-5p may be an important factor inhibiting the fat deposition and skeletal muscle development in chicken.

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PSXII-25 Isolation and identification of bovine preadipocytes and screening of miRNAs associated with adipogenesis

Journal of Animal Science ◽

10.1093/jas/skaa278.445 ◽

2020 ◽

Vol 98 (Supplement_4) ◽

pp. 244-245

Author(s):

Xiang Yu ◽

Xibi Fang ◽

Runjun Yang

Keyword(s):

Lipid Metabolism ◽

Pathway Analysis ◽

Kegg Pathway ◽

Expression Profiles ◽

Marker Gene ◽

Control Group ◽

Small Rna Sequencing ◽

Fatty Tissue ◽

Isolation And Identification ◽

Kegg Pathway Analysis

Abstract Introduction: In the breeding of beef cattle, the adipogenesis is a significant process to fat deposition. Thus isolating and identifying bovine preadipocytes in vitro is a basis for investigating mechanism of adipogenesis. As important transcriptional regulators, miRNAs play a pivotal role in adipogenesis. Materials and methods: Bovine preadipocytes were isolated from subcutaneous fatty tissue and induced to differentiate into adipocytes. The verification of preadipocytes and adipocytes was performed by qPCR, Oil Red O stain and triglycerides detection. Total RNAs were extracted from bovine preadipocytes and adipocytes and proceeded small-RNA sequencing. KEGG pathway analysis and GO analysis were used to screen differently expressed miRNAs associated with adipogenesis. Results: The result showed that more lipid drops in induced group were observed and stained red compared with control group. The expression levels of key genes (pparγ, c/ebpα, fabp4, fasn and leptin) associated with adipogenesis were increased. Dlk1, as a marker gene of preadipocytes, was scarcely expressed in adipocytes. The content of cellar triglycerides detected in adipocytes was significantly upgraded. 131 miRNAs were found to be highly expressed in bovine adipocytes, and 119 miRNAs were highly expressed in bovine preadipocytes. KEGG pathway analysis showed that 4.76% of the differentially expressed genes were enriched in lipid metabolism. Discussion and conclusion: Previous studies have found that some miRNAs could regulate lipid metabolism. But few studies have explored the relationship between miRNAs and adipogenesis. In our study, we characterized the miRNAs associated with adipogenesis in bovine preadipocytes. The expression profiles of 11 miRNAs verified by q-PCR, including miR-149-5p, miR-199a-3p, miR-199a-5p, miR-148a and miR-449a were the same to sequencing data. Based on the KEGG pathway analysis, the predicted target genes were mainly enriched in signal transduction, catabolism and lipid metabolism. This work contributes to existing knowledge by providing an understanding of bovine adipogenesis at the molecular level.

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GCH1 regulated miRNAs are potential targets for microglial activation in neuropathic pain

Bioscience Reports ◽

10.1042/bsr20210051 ◽

2021 ◽

Author(s):

Shu Jia ◽

Guowu Chen ◽

Yanhu Liang ◽

Xiao Liang ◽

Chun yang Meng

Keyword(s):

Neuropathic Pain ◽

Microglial Activation ◽

Target Genes ◽

Expression Profiles ◽

Small Rna Sequencing ◽

Sequencing Analysis ◽

Sequencing Data ◽

Wide Range ◽

Negative Effect ◽

Underlying Mechanisms

Neuropathic pain (NP) is a chronic pain directly caused by injury or disease of the somatosensory nervous system. Previous studies suggest that GTP cyclohydrolase I (GCH1) may play a pivotal role in microglial activation, which has been shown to be essential for NP. However, its underlying mechanisms in microglial activation remain unclear. A wide range of microRNAs (miRNAs) have been found to be involved in microglial activation-induced NP. To identify the miRNAs regulated by GCH1 and predict their functions in the progression of microglial activation, we analyzed the miRNA expression profiles of GCH1-knockdown (KD) BV2 microglial cells. Small RNA sequencing analysis revealed 13 differentially expressed (DE) miRNAs in GCH1-KD cells. The target genes of DE miRNAs mainly participate in PI3K-Akt signaling pathway, peroxisome and ferroptosis. The miRNA-mRNA regulatory network analysis showed that GCH1, MAP4K5 and YWHAB acted as hub genes. qRT-PCR results further verified the expression levels of mmu-miR-1a-3p, mmu-miR-133a-3p, mmu-miR-7a-5p and mmu-miR-10a-5p in GCH1-KD cells, which were consistent with the sequencing data. In addition, our data indicated that overexpression of mmu-miR-133a-3p alleviated the pro-inflammatory cytokines IL-1β and IL-6 production induced by lipopolysaccharide (LPS), indicating that mmu-miR-133a-3p has a negative effect on microglial activation. Taken together, our findings suggest that many miRNAs regulated by GCH1 may be involved in microglial activation, which may provide new potential targets for GCH1 in the pathogenesis of NP.

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Identification of miRNAs and Their Target Genes Involved in Cucumber Fruit Expansion Using Small RNA and Degradome Sequencing

Biomolecules ◽

10.3390/biom9090483 ◽

2019 ◽

Vol 9 (9) ◽

pp. 483 ◽

Cited By ~ 1

Author(s):

Sun ◽

Luo ◽

Chang ◽

Li ◽

Zhou ◽

...

Keyword(s):

Small Rna ◽

High Throughput Sequencing ◽

Target Genes ◽

Expression Patterns ◽

Differentially Expressed ◽

Sequencing Data ◽

Degradome Sequencing ◽

Complex Biological Process ◽

Cucumber Fruit ◽

Differentially Expressed Mirnas

Fruit expansion is an essential and very complex biological process. Regulatory roles of microRNAs (miRNAs) and miRNA–mRNA modules in the cucumber fruit expansion are not yet to be investigated. In this work, 1253 known and 1269 novel miRNAs were identified from nine cucumber fruit small RNA (sRNA) libraries through high-throughput sequencing. A total of 105 highly differentially expressed miRNAs were recognized in the fruit on five days post anthesis with pollination (EXP_5d) sRNA library. Further, expression patterns of 11 differentially expressed miRNAs were validated by quantitative real-time PCR (qRT-PCR). The expression patterns were similar to sRNAs sequencing data. Transcripts of 1155 sequences were predicted as target genes of differentially expressed miRNAs by degradome sequencing. Gene Ontology (GO) enrichment showed that these target genes were involved in 24 biological processes, 15 cell components and nine molecular functions. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis demonstrated that these target genes were significantly enriched in 19 pathways and the enriched KEGG pathways were associated with environmental adaptation, signal transduction and translation. Based on the functional prediction of miRNAs and target genes, our findings suggest that miRNAs have a potential regulatory role in cucumber fruit expansion by targeting their target genes, which provide important data for understanding the miRNA-mediated regulatory networks controlling fruit expansion in cucumber. Specific miRNAs could be selected for further functional research and molecular breeding in cucumber.

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Gene Module Analysis Reveals Cell-Type Specificity and Potential Target Genes in Autism’s Pathogenesis

Biomedicines ◽

10.3390/biomedicines9040410 ◽

2021 ◽

Vol 9 (4) ◽

pp. 410

Author(s):

Guoli Ji ◽

Shuchao Li ◽

Lishan Ye ◽

Jinting Guan

Keyword(s):

Target Genes ◽

Neurodevelopmental Disorder ◽

Expression Patterns ◽

Autism Spectrum ◽

Anterior Cingulate ◽

Specific Gene ◽

Cell Type ◽

Sequencing Data ◽

Cell Type Specific ◽

Gene Modules

Multiple genetic factors contribute to the pathogenesis of autism spectrum disorder (ASD), a kind of neurodevelopmental disorder. Genes were usually studied separately for their associations with ASD. However, genes associated with ASD do not act alone but interact with each other in a network module. The identification of these modules is the basis for the systematic understanding of the pathogenesis of ASD. Moreover, ASD is characterized by highly pathogenic heterogeneity, and gene modules associated with ASD are cell-type-specific. In this study, based on the single-nucleus RNA sequencing data of 41 post-mortem tissue samples from the prefrontal cortex and anterior cingulate cortex of 19 ASD patients and 16 control individuals, we applied sparse module activity factorization, a matrix decomposition method consistent with the multi-factor and heterogeneous characteristics of ASD pathogenesis, to identify cell-type-specific gene modules. Then, statistical procedures were performed to detect highly reproducible cell-type-specific ASD-associated gene modules. Through the enrichment analysis of cell markers, 31 cell-type-specific gene modules related to ASD were further screened out. These 31 gene modules are all enriched with curated ASD risk genes. Finally, we utilized the expression patterns of these cell-type-specific ASD-associated gene modules to build predictive models for ASD. The excellent predictive performance also proved the associations between these gene modules and ASD. Our study confirmed the multifactorial and cell-type-specific characteristics of ASD pathogeneses. The results showed that excitatory neurons such as L2/3, L4, and L5/6-CC play essential roles in ASD’s pathogenic processes. We identified the potential ASD target genes that act together in cell-type-specific modules, such as NRG3, KCNIP4, BAI3, PTPRD, LRRTM4, and LINGO2 in the L2/3 gene modules. Our study offers new potential genomic targets for ASD and provides a novel method to study gene modules involved in the pathogenesis of ASD.

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Comparative profiling of microRNA expression in the developing seeds of high- and low-oil tea oil camellia (Camellia oleifera) cultivars

10.21203/rs.2.11530/v1 ◽

2019 ◽

Author(s):

Bo Wu ◽

Chengjiang Ruan ◽

Wanchen Zhang ◽

Asad Hussain Shah ◽

Sihei Liu

Keyword(s):

Lipid Metabolism ◽

Target Genes ◽

Sequence Data ◽

Southern China ◽

Expression Patterns ◽

Integrated Analysis ◽

Small Rna Sequencing ◽

Camellia Oleifera ◽

Illumina Platform ◽

Function Modules

Abstract Background Tea oil camellia (Camellia oleifera), an important woody oil tree, is a source of seed oil of high nutritional and medicinal values and has been widely planted in southern China. However, there are few reports on the identification of miRNAs involved in seed lipid metabolism in high- and low-oil cultivars of tea oil camellia. Results An miRNA sequencing database was constructed for an Illumina platform, which was used to perform high-throughput small RNA sequencing of seeds of high- and low-oil cultivars of tea oil camellia at four different developmental stages, and the important relevant miRNAs and their target genes were identified. 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. An integrated analysis of miRNA and mRNA transcriptome sequence data and qRT-PCR-based information was performed and revealed that 10 miRNA-mRNA function modules were related to lipid metabolism and 23 miRNA-mRNA function modules were involved in the regulation of seed size. Conclusion Mining and studying the expression patterns and functions of miRNAs and their regulatory target genes can not only promote the development of miRNAs related to tea oil camellia in public resource databases but also provide important theoretical value and a scientific basis for the genetic improvement of new varieties of tea oil camellia in the future.

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Integrative MicroRNAome Analysis of Skeletal Muscle of Colossoma Macropomum (Tambaqui), Piaractus Mesopotamicus (Pacu), and the Hybrid Tambacu, based on Next-generation Sequencing Data.

10.21203/rs.3.rs-51788/v1 ◽

2020 ◽

Author(s):

Bruno EA Fantinatti ◽

Erika S Perez ◽

Bruna TT Zanella ◽

Jéssica S Valente ◽

Tassiana G de Paula ◽

...

Keyword(s):

Skeletal Muscle ◽

Target Genes ◽

Good Choice ◽

Next Generation Sequencing Data ◽

Small Rna Sequencing ◽

Sequencing Data ◽

Fish Farms ◽

Colossoma Macropomum ◽

Piaractus Mesopotamicus ◽

Specific Regulation

Abstract Background: Colossoma macropomum (tambaqui) and Piaractus mesopotamicus (pacu) are good fish species for aquaculture. The tambacu, individuals originating from the induced hybridization of the female tambaqui with the male pacu, present rapid growth and robustness, characteristics which have made the tambacu a good choice for Brazilian fish farms. Here, we used small RNA sequencing to examine global miRNA expression in the genotypes pacu (PC), tambaqui (TQ), and hybrid tambacu (TC), (Juveniles, n=5 per genotype), in order to better understand the relationship between tambacu and its parental species, and also to clarify the mechanisms involved in tambacu muscle growth and maintenance based on miRNAs expression. Results: Regarding differentially expressed (DE) miRNAs between the three genotypes, we observed 8 upregulated and 7 downregulated miRNAs considering TC vs. PC; 14 miRNAs were upregulated and 10 were downregulated considering TC vs. TQ, and 15 miRNAs upregulated and 9 were downregulated considering PC vs. TQ. The majority of the miRNAs showed specific regulation for each genotype pair, and no miRNA were shared between the 3 genotype pairs, in both up- and down-regulated miRNAs. Considering only the miRNAs with validated target genes, we observed the miRNAs miR-144-3p, miR-138-5p, miR-206-3p, and miR-499-5p. GO enrichment analysis showed that the main target genes for these miRNAs were grouped in pathways related to oxygen homeostasis, blood vessel modulation, and oxidative metabolism.Conclusions: Our global miRNA analysis provided interesting DE miRNAs in the skeletal muscle of pacu, tambaqui, and the hybrid tambacu. In addition, in the hybrid tambacu, we identified some miRNAs controlling important molecular muscle markers that could be relevant for the farming maximization.

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