Integrated analysis of miRNA and mRNA expression profiles in testes of Duroc and Meishan boars
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ABSTRACT:
SUBMITTER: Ding H
PROVIDER: S-EPMC7531090 | biostudies-literature | 2020 Jan
REPOSITORIES: biostudies-literature
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Project description:Precocious puberty is closely related to testicular development and spermatogenesis, and there is increasing evidence that miRNAs are involved in regulation of testicular development and spermatogenesis. However, little is known about the regulation of microRNAs (miRNAs) during precocious maturation in Hezuo (HZ) boars. In this study, serum Testosterone (T), Estradiol (E2), Follicle-stimulating hormone (FSH) and Luteinizing hormone (LH) levels were detected in HZ and Landrace (LC) boars in the postnatal period at 30, 90, 120, 180, and 240 days, and the testes of HZ and LC boars at 30 and 120 days were used for histological observation. In addition, we performed small RNA-Seq to identify miRNA at sexual immaturity (30-days-old) and maturity (120-days-old) of HZ boar testis (using LC boar as control) to reveal the key miRNA in regulation of precocious puberty. Hormone assay results showed that high levels of T, E2, FSH, and LH may be related to precocious sexual maturity of HZ boars, and that FSH may play an important function before sexual maturity. Histological observation showed that HZ boars developed earlier than LC boars and had reached sexual maturity at 120 days. Small RNA-Seq yielded a total of 359 exist miRNAs, 767 known miRNAs and 322 novel miRNAs in 12 samples; 549, 468, 133, and 247 differentially expressed (DE) miRNAs were identified between Ha vs. Hb, La vs. Lb, Ha vs. La, and Hb vs. Lb (log2 fold change >1 and p < 0.05). Enrichment analysis showed that target genes of these DE miRNAs were enriched in many gene ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathways (such as PI3K-Akt, Hippo and Rap1 signaling pathways) were related to testicular development and spermatogenesis. Further screening, some miRNAs (such as ssc-miR-29b, ssc-miR-199b, ssc-miR-383, ssc-miR-149, ssc-miR-615, and ssc-miR-370) were possibly associated with precocious puberty. These results provide new light on miRNA regulatory mechanisms involved in precocious puberty.
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Project description:Meishan boars are known for their early sexual maturity. However, they exhibit a significantly smaller testicular size and a reduced proportion of Sertoli cells and daily sperm production compared with Duroc boars. The testes of Duroc and Meishan boars at 20, 75 and 270 days of age were used for histological and transcriptome analyses. Haematoxylin-eosin staining was conducted to observe histological structure of the testes in Duroc and Meishan boars at different ages. Although spermatogenesis occurred prior to 75 days in Meishan boars, the number of spermatogonia and Sertoli cells in Meishan boars were less than in Duroc boars at adulthood. The diameters of the seminiferous tubules of the testes differed significantly during the initiation of development of the seminiferous tubules between the two breeds. We obtained differentially expressed functional genes and analysed seven pathways involved in male sexual maturity and spermatogenesis using RNA-seq. We also detected four main alternative splicing events and many single nucleotide polymorphisms from testes. Eight functionally important genes were validated by qPCR, and Neurotrophin 3 was subjected to quantification and cellular localization analysis. Our study provides the first transcriptome evidence for the differences in sexual function development between Meishan and Duroc boars.
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Project description:Small Tail Han Sheep is a highly valued local breed in China because of their precocity, perennial estrus, and high fecundity. The average annual lambing rate of ewes is as high as 180-270%, the semen of ram has characteristics of high yield, high density, and good motility. To reveal the key miRNAs and miRNA-targets underlying testis development and spermatogenesis in male Small Tail Han Sheep, integrated analysis of miRNA and mRNA expression profiles in 2-, 6-, and 12-month-old testes was performed by RNA-seq technology and bioinformatics methods. The results showed that total of 153 known sheep miRNAs and 2712 novel miRNAs were obtained in 2-,6 - and 12-month-old Small Tail Han Sheep testes; 5, 1, and 4 differentially expressed (DE) known sheep miRNAs, and 132, 105, and 24 DE novel miRNAs were identified in 2- vs. 6-, 6- vs. 12-, and 2- vs. 12-month-old testes, respectively. We combined miRNA results of this study and the mRNA results obtained in our previous study to predict the target mRNAs of DE known sheep miRNAs; 131, 10, and 15 target mRNAs of DE known sheep miRNAs and 76, 1, and 11 DE miRNA-targets were identified in the three groups, respectively. GO and KEGG analyses showed that: in 2- vs. 6-month-olds, the target genes of DE known sheep miRNAs were involved in 100 biological processes and 11 signaling pathways; in 6- vs. 12-month-olds, the target genes of DE known sheep miRNAs were involved in 4 biological processes; and in 2- vs. 12-month-olds, the target genes of DE known sheep miRNAs were involved in 17 biological processes and 4 signaling pathways. Three miR-target regulatory networks were constructed based on these DE miRNA-targets. The key miRNA-Targets involved in testis development and spermatogenesis were screened. 6 known sheep miRNAs and 6 novel miRNAs were selected to validate the accuracy of miRNA sequencing data by qRT-PCR. The binding sites of oar-miR-379-5p with WNT8A was validated by a dual luciferase reporter gene detection system.
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Project description:Embryo loss is a major factor affecting profitability in the pig industry. Embryonic mortality occurs during peri-implantation and mid-late gestation in pigs. Previous investigations have shown that the embryo loss rate in Meishan pigs is significantly lower than in commercial breeds. Most studies have focused on embryonic mortality during early gestation, but little is known about losses during mid-late gestation. In this study, we performed a transcriptome analysis of endometrial tissue in mid-late gestation sows (gestation days 49 and 72) sampled from two breeds (Meishan (MS) and Duroc (DU)) that have different embryo loss rates. We identified 411, 1113, 697, and 327 differentially expressed genes, and 14, 36, 57, and 43 differentially expressed miRNAs in four comparisons (DU49 vs. DU72, DU49 vs. MS49, DU72 vs. MS72, and MS49 vs. MS72), respectively. Subsequently; seven differentially expressed mRNAs and miRNAs were validated using qPCR. Functional analysis suggested the differentially expressed genes and miRNAs target genes mainly involved in regulation of hormone levels, blood vessel development, developmental process involved in reproduction, embryonic placenta development, and the immune system. A network analysis of potential miRNA-gene interactions revealed that differentially expressed miRNAs in Meishan pigs are involved in the response to estradiol and oxygen levels, and affect angiogenesis and blood vessel development. The binding site on ssc-miR-503 for epidermal growth factor (EGF) and the binding site on ssc-miR-671-5p for estrogen receptor α (ESR1) were identified using a dual luciferase assay. The results of this study will enable further exploration of miRNA-mRNA interactions important in pig pregnancy and will help to uncover molecular mechanisms affecting embryonic mortality in pigs during mid-late gestation.
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Project description:Increasing amounts of evidence indicate that noncoding RNAs (ncRNAs) have important roles in various biological processes. Here, miRNA, lncRNA, and mRNA expression profiles were analyzed in human HepG2 and L02 cells using high-throughput technologies. An integrative method was developed to identify possible functional relationships between different RNA molecules. The dominant deregulated miRNAs were prone to be downregulated in tumor cells, and the most abnormal mRNAs and lncRNAs were always upregulated. However, the genome-wide analysis of differentially expressed RNA species did not show significant bias between up- and downregulated populations. miRNA-mRNA interaction was performed based on their regulatory relationships, and miRNA-lncRNA and mRNA-lncRNA interactions were thoroughly surveyed and identified based on their locational distributions and sequence correlations. Aberrantly expressed miRNAs were further analyzed based on their multiple isomiRs. IsomiR repertoires and expression patterns were varied across miRNA loci. Several specific miRNA loci showed differences between tumor and normal cells, especially with respect to abnormally expressed miRNA species. These findings suggest that isomiR repertoires and expression patterns might contribute to tumorigenesis through different biological roles. Systematic and integrative analysis of different RNA molecules with potential cross-talk may make great contributions to the unveiling of the complex mechanisms underlying tumorigenesis.
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Project description:Several studies have investigated miRNA and mRNA co-expression to identify regulatory networks at the transcriptional level. A typical finding of these studies is the presence of both negative and positive miRNA-mRNA correlations. Negative correlations are consistent with the expected, faster degradation of target mRNAs, whereas positive correlations denote the existence of feed-forward regulations mediated by transcription factors. Both mechanisms have been characterized at the molecular level, although comprehensive methods to represent miRNA-mRNA correlations are lacking. At present, genome-wide studies are able to assess the expression of more than 1000 mature miRNAs and more than 35,000 well-characterized human genes. Even if studies are generally restricted to a small subset of genes differentially expressed in specific diseases or experimental conditions, the number of potential correlations remains very high, and needs robust multivariate methods to be conveniently summarized by a small set of data.Nonparametric Kendall correlations were calculated between miRNAs and mRNAs differentially expressed in livers of patients with acute liver failure (ALF) using normal livers as controls. Spurious correlations due to the histopathological composition of samples were removed by partial correlations. Correlations were then transformed into distances and processed by multidimensional scaling (MDS) to map the miRNA and mRNA relationships. These showed: (a) a prominent displacement of miRNA and mRNA clusters in ALF livers, as compared to control livers, indicative of gene expression dysregulation; (b) a clustering of mRNAs consistent with their functional annotations [CYP450, transcription factors, complement, proliferation, HLA class II, monocytes/macrophages, T cells, T-NK cells and B cells], as well as a clustering of miRNAs with the same seed sequence; and (c) a tendency of miRNAs and mRNAs to populate distinct regions of the MDS plot. MDS also allowed to visualize the network of miRNA-mRNA target pairs.Different features of miRNA and mRNA relationships can be represented as thematic maps within the framework of MDS obtained from pairwise correlations. The symmetric distribution of positive and negative correlations between miRNA and mRNA expression suggests that miRNAs are involved in a complex bidirectional molecular network, including, but not limited to, the inhibitory regulation of miRNA targets.
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Project description:p53 is the most frequently mutated gene in human cancers, with over half of all tumors harboring mutation at this locus. R248 and R249 (corresponding to porcine R241 and R242), are among the hotspot mutations frequently mutated in liver, lung, breast, and some other cancers. In this study, p53 gene was knocked out or point-edited (R241 and R242 were converted to 241W and 242S) in porcine fetal fibroblast (PFF) cells via CRISPR-Cas9 technique. High throughput sequencing of miRNA and mRNA uncovered a total of 225 differentially expressed miRNAs (DEMs) and 738 differentially expressed genes (DEGs) in the p53 knockout (p53-KO) cells, and a total of 211 DEMs and 722 DEGs in the point-modified (p53-241W242S) cells. Totally 28 annotated DEMs were found to overlap between p53-KO/p53-WT and p53-241W242S/p53-WT miRNAs datasets, of which miR-34 c, miR-218, miR-205, miR-105-1, miR-105-2, miR-206, miR-224 and miR-429 play important roles in p53 regulatory network. Among the top 10 DEGs in p53-KO and p53-241W242S cells, most genes were reported to be involved in tumors, cell proliferation or cell migration. p53-KO and p53-241W242S cells showed a significantly higher (P < 0.01) proliferation rate compared with p53-WT cells. In conclusion, genetic modifications of p53 gene significantly affect the expression levels of a large number of genes and miRNAs in the PFF cells. The p53-edited PFF cells could be used as non-tumor cell models for investigating the p53 signaling network, and as donor cells for somatic nuclear transfer, with the aim to develop porcine models with the corresponding p53 mutations.Abbreviations: CRISPR-Cas9: Clustered regularly interspaced short palindromic repeats-associated protein 9; PFF: porcine fetal fibroblasts; SCNT: somatic cell nuclear transfer; RNA sequencing: small RNA sequencing and mRNA sequencing; DEGs: differentially expressed mRNAs; DEMs: differentially expressed miRNAs.
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Project description:Donkey milk is consumed by humans for its nutritional and therapeutic properties. MicroRNAs (miRNAs) and messenger RNAs (mRNAs) have been implicated in the regulation of milk component synthesis and mammary gland development. However, the regulatory profile of the miRNAs and mRNAs involved in lactation in donkeys is unclear. We performed mRNA-seq and miRNA-seq and constructed coexpression regulatory networks for the mammary glands during the lactating and nonlactating period of jennies. We identified 3144 differentially expressed (DE) mRNAs (987 upregulated mRNAs and 2157 downregulated mRNAs) and 293 DE miRNAs (231 upregulated miRNAs and 62 downregulated miRNAs) in the lactating group compared to the nonlactating group. The DE miRNA target mRNA were significantly associated with pathways related to RNA polymerase, glycosphingolipid biosynthesis, mRNA surveillance, ribosome biogenesis in eukaryotes, glycerophospholipid metabolism, Ras signaling, and the fly hippo signaling pathway. The mRNA-miRNA coregulation analysis showed that novel-m0032-3p, miR-195, miR-26-5p, miR-23-3p, miR-674-3p, and miR-874-3p are key miRNAs that target mRNAs involved in immunity and milk lipid, protein, and vitamin metabolism in the jenny mammary gland. Our results improve the current knowledge of the molecular mechanisms regulating bioactive milk component metabolism in the mammary glands and could be used to improve milk production in donkeys.
| S-EPMC9498803 | biostudies-literature
Project description:Neuroinflammation contributes to many neurologic disorders including Alzheimer's disease, multiple sclerosis, and stroke. Microglia is brain resident myeloid cells and have emerged as a key driver of the neuroinflammatory responses. MicroRNAs (miRNAs) provide a novel layer of gene regulation and play a critical role in regulating the inflammatory response of peripheral macrophages. However, little is known about the miRNA in inflammatory activation of microglia. To elucidate the role that miRNAs have on microglial phenotypes under classical (M1) or alternative (M2) activation under lipopolysaccharide ('M1'-skewing) and interleukin-4 ('M2a'-skewing) stimulation conditions, we performed microarray expression profiling and bioinformatics analysis of both mRNA and miRNA using primary cultured murine microglia. miR-689, miR-124, and miR-155 were the most strongly associated miRNAs predicted to mediate pro-inflammatory pathways and M1-like activation phenotype. miR-155, the most strongly up-regulated miRNA, regulates the signal transducer and activator of transcription 3 signaling pathway enabling the late phase response to M1-skewing stimulation. Reduced expression in miR-689 and miR-124 are associated with dis-inhibition of many canonical inflammatory pathways. miR-124, miR-711, miR-145 are the strongly associated miRNAs predicted to mediate anti-inflammatory pathways and M2-like activation phenotype. Reductions in miR-711 and miR-124 may regulate inflammatory signaling pathways and peroxisome proliferator-activated receptor-gamma pathway. miR-145 potentially regulate peripheral monocyte/macrophage differentiation and faciliate the M2-skewing phenotype. Overall, through combined miRNA and mRNA expression profiling and bioinformatics analysis we have identified six miRNAs and their putative roles in M1 and M2-skewing of microglial activation through different signaling pathways.
| S-EPMC3823621 | biostudies-literature
Project description:BackgroundIndependent transcriptome profile analyses of miRNAs or mRNAs under conditions of cadmium (Cd) stress have been widely reported in plants. However, a combined analysis of sRNA sequencing expression data with miRNA target expression data to infer the relative activities of miRNAs that regulate gene expression changes resulting from Cd stress has not been reported in rice. To elucidate the roles played by miRNAs in the regulation of changes in gene expression in response to Cd stress in rice (Oryza sativa L.), we simultaneously characterized changes in the miRNA and mRNA profiles following treatment with Cd.ResultsA total of 163 miRNAs and 2,574 mRNAs were identified to be differentially expressed under Cd stress, and the changes in the gene expression profile in the shoot were distinct from those in the root. At the miRNA level, 141 known miRNAs belonging to 48 families, and 39 known miRNAs in 23 families were identified to be differentially expressed in the root and shoot, respectively. In addition, we identified eight new miRNA candidates from the root and five from the shoot that were differentially expressed in response to Cd treatment. For the mRNAs, we identified 1,044 genes in the root and 448 genes in the shoot that were up-regulated, while 572 and 645 genes were down-regulated in the root and shoot, respectively. GO and KEGG enrichment analyses showed that genes encoding secondary, metabolite synthases, signaling molecules, and ABC transporters were significantly enriched in the root, while only ribosomal protein and carotenoid biosynthesis genes were significantly enriched in the shoot. Then 10 known miRNA-mRNA interaction pairs and six new candidate ones, that showed the opposite expression patterns, were identified by aligning our two datasets against online databases and by using the UEA sRNA toolkit respectively.ConclusionsThis study is the first to use high throughput DNA sequencing to simultaneously detect changes in miRNA and mRNA expression patterns in the root and shoot in response to Cd treatment. These integrated high-throughput expression data provide a valuable resource to examine global genome expression changes in response to Cd treatment and how these are regulated by miRNAs.
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