Alterations of microRNAs expression profiles in small extracellular vesicle after traumatic brain injury in mice

Traumatic brain injury (TBI) is one of the leading causes of mortality and morbidity worldwide. Tools available for diagnosis and therapy are limited. Small extracellular vesicle (sEV) microRNAs (miRNAs) play an important role in TBI disease progression. This study aimed to investigate the alterations in sEV miRNAs expression in the mouse brain extracellular space after TBI. Twenty-four C57BL/6J mice were randomly divided into two groups (12/group). The TBI group was subjected to all surgical procedures and fluid percussion injury (FPI). The sham group only underwent surgery. Brain specimens were collected 3 h after TBI/sham. The brain sEV were isolated. Differentially expressed miRNAs were identified. A total of 50 miRNAs were observed to be differentially expressed (fold change ≥1.5 and P<0.05) after TBI, including 5 upregulated and 45 downregulated. The major enriched Gene Ontology terms were metabolic processes, cell, intracellular, organelle, cytoplasm, axon, binding, protein kinase activity, protein binding, and protein dimerization activity. The KEGG pathway analysis predicted that the pathways affected by the variation of miRNAs in sEVs after TBI included the Wnt signaling pathway and NF-κB signaling pathway. The changes in five miRNAs were confirmed by qRT-PCR. In conclusion, this study demonstrated the differential expression of a series of miRNAs in brain sEV after TBI, which might be correlated with post-TBI physiological and pathological processes. The findings might also provide novel targets for further investigating the molecular mechanisms underlying TBI and potential therapeutic interventions.     

Protein repair in the brain, proteomic analysis of endogenous substrates for protein L-isoaspartyl methyltransferase in mouse brain

Protein L-isoaspartyl methyltransferase (PIMT) catalyzes repair of L-isoaspartyl peptide bonds, a major source of protein damage under physiological conditions. PIMT knock-out (KO) mice exhibit brain enlargement and fatal epileptic seizures. All organs accumulate isoaspartyl proteins, but only the brain manifests an overt pathology. To further explore the role of PIMT in brain function, we undertook a global analysis of endogenous substrates for PIMT in mouse brain. Extracts from PIMT-KO mice were subjected to two-dimensional gel electrophoresis and blotted onto membranes. Isoaspartyl proteins were radiolabeled on-blot using [methyl-(3)H]S-adenosyl-L-methionine and recombinant PIMT. Fluorography of the blot revealed 30-35 (3)H-labeled proteins, 22 of which were identified by peptide mass fingerprinting. These isoaspartate-prone proteins represent a wide range of cellular functions, including neuronal development, synaptic transmission, cytoskeletal structure and dynamics, energy metabolism, nitrogen metabolism, pH homeostasis, and protein folding. The following five proteins, all of which are rich in neurons, accumulated exceptional levels of isoaspartate: collapsin response mediator protein 2 (CRMP2/ULIP2/DRP-2), dynamin 1, synapsin I, synapsin II, and tubulin. Several of the proteins identified here are prone to age-dependent oxidation in vivo, and many have been identified as autoimmune antigens, of particular interest because isoaspartate can greatly enhance the antigenicity of self-peptides. We propose that the PIMT-KO phenotype results from the cumulative effect of isoaspartate-related damage to a number of the neuron-rich proteins detected in this study. Further study of the isoaspartate-prone proteins identified here may help elucidate the molecular basis of one or more developmental and/or age-related neurological diseases.     

Isoaspartate Accumulation in Mouse Brain Is Associated with Altered Patterns of Protein Phosphorylation and Acetylation,Some of Which Are Highly Sex-Dependent

Isoaspartate (isoAsp) formation is a major source of protein damage that is kept in check by the repair function of protein L-isoaspartyl methyltransferase (PIMT). Mice deficient in PIMT accumulate isoAsp-containing proteins, resulting in cognitive deficits, abnormal neuronal physiology and cytoarchitecture, and fatal epileptic seizures 30–60 days after birth. Synapsins I and II, dynamin-1, collapsin response mediator protein 2 (CRMP2), and α/β-tubulin are major targets of PIMT in brain. To investigate links between isoAsp accumulation and the neurological phenotype of the KO mice, we used Western blotting to compare patterns of in vivo phosphorylation or acetylation of the major PIMT targets listed above. Phosphorylations of synapsins I and II at Ser-9 were increased in female KO vs. WT mice, and acetylation of tubulin at Lys-40 was decreased in male KO vs. WT mice. Average levels of dynamin-1 phosphorylation at Ser-778 and Ser-795 were higher in male KO vs. WT mice, but the statistical significance (P>0.1) was low. No changes in phosphorylation were found in synapsins I and II at Ser-603, in CRMP2 at Ser-522 or Thr-514, in DARPP-32 at Thr-34, or in PDK1 at Ser-241. General levels of phosphorylation assessed with Pro-Q Diamond stain, or an anti-phosphotyrosine antibody, appeared similar in the WT and KO mice. We conclude that isoAsp accumulation is associated with altered functional status of several neuronal proteins that are highly susceptible to this type of damage. We also uncovered unexpected differences in how male and female mice respond to isoAsp accumulation in the brain.     

Identification of differentially expressed microRNAs and their PKC-isoform specific gene network prediction during hypoxic pre-conditioning and focal cerebral ischemia of mice

We previously reported the involvement of conventional protein kinase C (cPKC) βII, γ, novel PKC (nPKC) ε and their interacting proteins in hypoxic pre-conditioning (HPC)-induced neuroprotection. In this study, the large-scale miRNA microarrays and bioinformatics analysis were used to determine the differentially expressed miRNAs and their PKC-isoform specific gene network in mouse brain after HPC and 6?h middle cerebral artery occlusion (MCAO). We found 4 up-regulated and 13 down-regulated miRNAs in the cortex of HPC mice, 26 increased and 39 decreased gene expressions of miRNAs in the peri-infarct region of 6?h MCAO mice, and 11 up-regulated and 22 down-regulated miRNAs in the peri-infarct region of HPC and 6?h MCAO mice. Based on Diff Score, 19 differentially expressed miRNAs were identified in HPC and 6?h MCAO mouse brain. Then the miRNA-gene-network of 19 specified miRNAs target genes of cPKCβII, γ and nPKCε-interacting protein was predicted by using bioinformatics analysis of genome databases. Furthermore, the down-regulated miR-615-3p during HPC had a detrimental effect on the oxygen-glucose deprivation (OGD)-induced N2A cell injury. These results suggested that the identified 19 miRNAs, notably miR-615-3p, might target these genes of cPKCβII, γ and nPKCε-interacting proteins involved in HPC-induced neuroprotection.     

Novel Biomarkers for Non-functioning Invasive Pituitary Adenomas were Identified by Using Analysis of microRNAs Expression Profile

The microRNAs (miRNAs) are involved in multiple pathological processes among various types of tumors. However, the functions of miRNAs in benign brain tumors are largely unexplored. In order to explore the pathogenesis of the invasiveness in non-functional pituitary adenoma (NFPA), the miRNAs expression profile was analyzed between invasive and non-invasive non-functional pituitary adenoma by miRNAs microarray. Six most significant differentially expressed miRNAs were identified including four upregulated miRNAs hsa-miR-181b-5p, hsa-miR-181d, hsa-miR-191-3p, and hsa-miR-598 and two downregulated miRNAs hsa-miR-3676-5p and hsa-miR-383. The functions and corresponding signaling pathways of differentially expressed miRNAs were investigated by bioinformatics techniques, including Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. The result of GO analysis indicates regulation of voltage-gated potassium channel activity, positive regulation of sodium ion transport, positive regulation of GTPase activity, negative regulation of Notch signaling pathway, etc. KEGG pathway reveals a series of biological processes, including prolactin signaling pathway, endocrine and other factor-regulated calcium reabsorption, fatty acid metabolism, neuroactive ligand-receptor interaction, etc. The miRNAs hsa-miR-181a-5p was verified by quantitative real-time PCR, and the expression level was in accordance with the microarray result. Our result can provide the evidence on featured miRNAs which play a prominent role in pituitary adenoma as effective biomarkers and therapeutic targets in the future.     

Circulating microRNA signature of steroid‐induced osteonecrosis of the femoral head

Objectives

Steroid‐induced osteonecrosis of the femoral head (ONFH) is a common orthopaedic disease of which early detection remains clinically challenging. Accumulating evidences indicated that circulating microRNAs (miRNAs) plays vital roles in the development of several bone diseases. However, the association between circulating miRNAs and steroid‐induced ONFH remains elusive.

Materials and methods

miRNA microarray was performed to identify the differentially abundant miRNAs in the serums of systemic lupus erythematosus (SLE) patients with steroid‐induced ONFH as compared with SLE control and healthy control group. We predicted the potential functions of these differentially abundant miRNAs using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses and reconstructed the regulatory networks of miRNA‐mRNA interactions.

Results

Our data indicated that there were 11 differentially abundant miRNAs (2 upregulated and 9 downregulated) between SLE‐ONFH group and healthy control group and 42 differentially abundant miRNAs (14 upregulated and 28 downregulated) between SLE‐ONFH group and SLE control group. We also predicted the potential functions of these differentially abundant miRNAs using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses and reconstructed the regulatory networks of miRNA‐mRNA interactions.

Conclusions

These findings corroborated the idea that circulating miRNAs play significant roles in the development of ONFH and may serve as diagnostic markers and therapeutic targets.     

Analysis of microRNA expression profile in specific pathogen-free chickens in response to reticuloendotheliosis virus infection

Reticuloendotheliosis virus (REV) is an avian retrovirus that causes immunosuppression, growth retardation, and oncogenesis in a variety of birds. REV infection is epidemic in many countries. In this study, we used high-throughput sequencing to identify microRNAs (miRNAs) associated with REV infection. A total of 88 differentially expressed miRNAs were identified in samples collected on days 21 and 28 post-REV infection. Possible target genes of the differentially expressed miRNAs were analyzed. We observed that expression of proapoptotic, proto-oncogene, and carcinogenic cytokine mRNAs was highly upregulated, whereas expression of antiapoptotic cytokine mRNAs was significantly downregulated. Our findings provide a potential link between miRNA expression and the pathogenesis of REV infection.

     

Identification and comparative analysis of the ovary and testis microRNAome of mud crab Scylla paramamosain

The role of microRNA (miRNA) in reproductive regulation is attracting increasingly more attention. In this study, we obtained 9,643,114 and 15,498,999 raw reads from the ovary and testis library of important farmed mud crab Scylla paramamosain, respectively. After data mining, a total of 4,096,464 and 11,737,973 mappable small RNA sequences remained for analysis. By mapping to the reference genome and expressed sequence tag (EST) of Daphnia pulex and other crabs, a total of 1,417 miRNAs were identified. On the basis of 1,417 miRNAs, 514 (36.3%) unique miRNAs coexpressed in the gonad of female and male libraries, and 336 (23.7%) and 567 (40%) expressed preferentially in female and male libraries, respectively. Analysis of library sequencing data resulted in the identi?cation of 108 miRNAs (out of 1,417; 7.6%) that showed signi?cant differential expression between the two samples. Of these, 13 miRNAs were expressed only in the testis, two miRNAs were expressed only in the ovary, and 93 miRNAs were coexpressed: 57 (61.3%) were upregulated (ovary/testis) and 36 (38.7%) were downregulated (ovary/testis). To confirm the expression patterns of the predicted miRNAs, we randomly selected 14 candidate miRNAs from 108 differentially expressed miRNAs and performed stem–loop real time quantitative PCR (RT‐qPCR) assays in five ovary developing stages. Five miRNAs showed similar expression patterns in almost every stage as those revealed by identification of differentially expressed genes (IDEG6) analysis. The above five miRNAs were predicted to match the 3′‐untranslated region of the published S. paramamosain gene. Four out of five miRNA had a regulation effect on many genes, especially the genes related to gonadal development.     

Identification of candidate susceptibility and resistance genes of mice infected with Streptococcus suis type 2

Streptococcus suis type 2 (SS2) is an important swine pathogen and zoonosis agent. A/J mice are significantly more susceptible than C57BL/6 (B6) mice to SS2 infection, but the genetic basis is largely unknown. Here, alterations in gene expression in SS2 (strain HA9801)-infected mice were identified using Illumina mouse BeadChips. Microarray analysis revealed 3,692 genes differentially expressed in peritoneal macrophages between A/J and B6 mice due to SS2 infection. Between SS2-infected A/J and control A/J mice, 2646 genes were differentially expressed (1469 upregulated; 1177 downregulated). Between SS2-infected B6 and control B6 mice, 1449 genes were differentially expressed (778 upregulated; 671 downregulated). These genes were analyzed for significant Gene Ontology (GO) categories and signaling pathways using the Kyoto Encylopedia of Genes and Genomes (KEGG) database to generate a signaling network. Upregulated genes in A/J and B6 mice were related to response to bacteria, immune response, positive regulation of B cell receptor signaling pathway, type I interferon biosynthesis, defense and inflammatory responses. Additionally, upregulated genes in SS2-infected B6 mice were involved in antigen processing and presentation of exogenous peptides, peptide antigen stabilization, lymphocyte differentiation regulation, positive regulation of monocyte differentiation, antigen receptor-mediated signaling pathway and positive regulation of phagocytosis. Downregulated genes in SS2-infected B6 mice played roles in glycolysis, carbohydrate metabolic process, amino acid metabolism, behavior and muscle regulation. Microarray results were verified by quantitative real-time PCR (qRT-PCR) of 14 representative deregulated genes. Four genes differentially expressed between SS2-infected A/J and B6 mice, toll-like receptor 2 (Tlr2), tumor necrosis factor (Tnf), matrix metalloproteinase 9 (Mmp9) and pentraxin 3 (Ptx3), were previously implicated in the response to S. suis infection. This study identified candidate genes that may influence susceptibility or resistance to SS2 infection in A/J and B6 mice, providing further validation of these models and contributing to understanding of S. suis pathogenic mechanisms.     

Circulating microRNA signatures serve as potential diagnostic biomarkers for Helicobacter pylori infection

Helicobacter pylor (H pylori), a Gram-negative, microaerobic human pathogen, has been found to be involved in many gastroduodenal diseases. Accurate diagnosis of H pylori infection is a vital part of the effective management of gastroduodenal diseases. Circulating microRNAs (miRNAs) have shown the potential to be used as noninvasive biomarkers for the diagnosis of infectious diseases. The aim of this study was to explore plasma miRNAs as noninvasive biomarkers for H pylori infection. We performed a plasma miRNA expression profile using Illumina high-throughput sequencing and validated the levels of differentially expressed miRNAs in the plasma of 63 H pylori-infected patients and 41 healthy volunteers by quantitative real-time polymerase chain reaction (qRT-PCR). The sequencing results showed that 37 miRNAs were upregulated in the H pylori-infected patients compared with that in the healthy volunteers, while six miRNAs were downregulated. qRT-PCR and receiver operator characteristic analysis suggested that the expression of miR-28-3p, miR-143-3p, miR-151a-3p, and miR-148a-3p were closely associated with H pylori infection. Therefore, the four plasma miRNA panels mentioned above could serve as promising noninvasive biomarkers of H pylori infection.     

Gene and protein expression in the epididymis of infertile c-ros receptor tyrosine kinase-deficient mice

Transgenic male mice bearing inactive mutations of the receptor tyrosine kinase c-ros lack the initial segment of the epididymis and are infertile. Several techniques were applied to determine differences in gene expression in the epididymal caput of heterozygous fertile (HET) and infertile homozygous knockout (KO) males that may explain the infertility. Complementary DNA arrays, gene chips, Northern and Western blots, and immunohistochemistry indicated that some proteins were downregulated, including the initial segment/proximal caput-specific genes c-ros, cystatin-related epididymal-spermatogenic (CRES), and lipocalin mouse epididymal protein 17 (MEP17), whereas other caput-enriched genes (glutathione peroxidase 5, a disintegrin and metalloproteinase [ADAM7], bone morphogenetic proteins 7 and 8a, A-raf, CCAAT/enhancer binding protein beta, PEA3) were unchanged. Genes normally absent from the initial segment (gamma-glutamyltranspeptidase, prostaglandin D2 synthetase, alkaline phosphatase) were expressed in the undifferentiated proximal caput of the KO. More distally, lipocalin 2 (24p3), CRISP1 (formerly MEP7), PEBP (MEP9), and mE-RABP (MEP10) were unchanged in expression. Immunohistochemistry and Western blots confirmed the absence of CRES in epididymal tissue and fluid and the continued presence of CRES in spermatozoa of the KO mouse. The glutamate transporters EAAC1 (EAAT3) and EAAT5 were downregulated and upregulated, respectively. The genes of over 70 transporters, channels, and pores were detected in the caput epididymidis, but in the KO, only three were downregulated and six upregulated. The changes in these genes could affect sperm function by modifying the composition of epididymal fluid and explain the infertility of the KO males. These genes may be targets for a posttesticular contraceptive.     

Expression of exosomal microRNAs during chondrogenic differentiation of human bone mesenchymal stem cells

The aim of the current study was to compare the expression of microRNAs (miRNAs) in exosomes derived from human bone mesenchymal stem cells (hBMSCs) with and without chondrogenic induction. Exosomes derived from hBMSCs were isolated and identified. Microarray analysis was performed to compare miRNA expression between exosomes derived from hBMSCs with and without chondrogenic induction, and quantitative real-time polymerase chain reaction (qRT-PCR) was used to verify the differentially expressed miRNAs. hBMSCs were transfected with miRNA mimic to extract miRNA-overexpressed exosomes. The results showed that most exosomes exhibited a cup-shaped or round-shaped morphology with a diameter of approximately 50-200 nm and expressed CD9 and CD63. We detected 141 miRNAs that were differentially expressed with and without chondrogenic induction by over a twofold change, including 35 upregulated miRNAs, such as miR-1246, miR-1290, miR-193a-5p, miR-320c, and miR-92a, and 106 downregulated miRNAs, such as miR-377-3p and miR-6891-5p. qRT-PCR analysis validated these results. Exosomes derived from hBMSCs overexpressing miR-320c were more efficient than normal exosomes derived from control hBMSCs at promoting osteoarthritis chondrocyte proliferation, down-regulated matrix metallopeptidase 13 and up-regulated (sex determining region Y)-box 9 expression during hBMSC chondrogenic differentiation. In conclusion, we identified a group of upregulated miRNAs in exosomes derived from hBMSCs with chondrogenic induction that may play an important role in mesenchymal stem cell-derived exosomes in cartilage regeneration and, ultimately, the treatment of arthritis. We demonstrated the potential of these modified exosomes in the development of novel therapeutic strategies.