| Umbilical cord blood is the link between maternal and fetal, which delivering energy, nutrients and metabolic waste during the period of embryonic development, meeting the requirement of fetal growth, development and health condition. MicroRNAs (miRNAs) are widely exist in various kinds of body fluids and tissues, and are selectively packaged inside the exosomes. Exosome is a type of membrane vesicles, secreted by many kinds of cell types. These miRNAs packaged in exosomes could be actively delivered into the recipient cells to regulate the expression of target genes and the function of recipient cells. In this study, exosomes are confirmed to present in pig umbilical cord blood by observating with atomic force microscope (AFM). We constructed 4 umbilical cord blood exosomal small RNA libraries from normal littermate and IUGR littermate umbilical cord artery and vein blood. Utilizing deep sequencing method, the expression profiles of these exosomal miRNAs in porcine umbilical cord blood were analyzed, and verified using fluorescence quantitative method (q-PCR). In addition, we performed functional analysis of the high expression miRNAs in the umbilical vein and artery and the differential expression miRNAs between different libraries. Taken together, we got the following key results:(1) By high speed ultracentrifugation, we isolated abundant of exosome-like vesicles from porcine umbilical cord blood. The morphology and size of these vesicles were observed by AFM at the nanometer-scale. The size of these exosome-like vesicles following normal distribution, and the average width of umbilical vein blood exosome was approximate 120 nm and the height was about 17 nm, the average width of umbilical artery blood exosome was approximate 130nm and the height was about 20 nm. All these morphological characters of these exosome-like vesicles from porcine umbilical cord blood were similar with the reported exosomes in human saliva and breast milk.(2) Average 12.61 M sequencing reads were obtained from four pigs'cord blood exosome small RNA libraries. After filtration,10.58 M,10.78 M and 7.72 M,8.16 M sequence were effectively obtained from the umbilical artery and umbilical vein blood of normal and IUGR pig, respectively. Moreover,83.3%,81.9%,67.57% and 62.74% of counts met the setted criteria and were deemed as mappable reads. The length distribution of these reads peaked at 20-24nt, comply with the basic structure of miRNAs.(3) Bioinformatics analysis detected a total of 554 pre-miRNAs and 636 mature miRNAs and identified 292 mature miRNAs with 260 conservative miRNAs from the 4 libraries.(4)All the 10 universally abundant miRNAs from the 4 libraries were accounted for more than 60% of the total unique miRNAs reads. Functional analysis revealed that the highly expressed miRNAs in Umbilical vein blood were implicated in angiogenesis, erythropoiesis and vascular disease, and the highly expressed miRNAs in Umbilical artery blood were implicated in vascular development, immune and cytokine response.(5) With differential expression miRNAs analysis, there were 122 significant differentially expressed miRNAs between NJ and ND (p<0.001),60 up-regulation and 62 down-regulation; 116 significant differentially expressed miRNAs between NJ and IJ (p<0.001),89 up-regulation and 27 down-regulation; 226 significant differentially expressed miRNAs between NJD and ID (p <0.001),127 up-regulation and 99 down-regulation. Function analysis of these differentially expressed miRNAs showed that hypoxic response, angiogenesis, immune system development and metabolism related miRNAs abnormal present in IUGR pigs, these processes are indispensable condition of embryo growth and development. We speculate that IUGR pig is at least partly regulated by umbilical cord arteriovenous exosome miRNAs during intrauterine embryonic development.(6) Screening differential miRNAs following the standard of Foldchang>5 or<0.2, p< 0.05,to predict target genes by Targetscan and Pictor prediction software and get intersection elements. Bioinformatics functional analysis found 13 up-regulated differentially expressed miRNAs between NJ and ND were enriched in cell proliferation and blood disease,7 down-regulated differentially expressed miRNAs were enriched in growth, nonspecific immunity, response to hypoxic and the innate immune response; 16 up-regulated differentially expressed miRNAs between NJ and IJ were enriched in transcription factor activity, transcription regulator activity, cell proliferation and growth pathways,6 down-regulated differentially expressed miRNAs were enriched in cell part morphogenesis, small GTPase regulator activity. The differentially expressed miRNAs between ND and ID were enriched in death, cell death, negative regulation of gene expression, apoptosis, negative regulation of cell proliferation, programmed cell death, MAPK signaling, p53 signaling pathway. Above showed functions were different between umbilical cord artery and vein. Morever, the differentially expressed miRNAs in umbilical cord blood exosome may lead to the formation of IUGR pig phenotype.(7) Cluster analysis of miRNAs in the four libraries showed womb environment through the umbilical cord blood is less important than IUGR fetus's developmental disorders. Analysis the main differentially expressed miRNAs showed, miRNAs promoting oxygen transport and regulation(miR-210?miR-424-3p?miR-27?miR-26a?miR-24?miR-181a?miR-23b?miR-26b? miR-130b?miR-107) were down-regulated in IJ, inhibited miRNAs (miR-223?miR-24?miR-138) were up-regulated reversely. miRNAs promoting angiogenesis (miR-130a?miR-200c?miR-150? miR-17?92?miR-126?miR-378?miR-221?miR-93?miR-34a) were down-regulated in IJ and ID, inhibited miRNAs (miR-16?miR-34a?miR-222) were up-regulated reversely. Promote immune system development miRNAs (miR-181a?miR-150?miR-155) were down-regulated in IJ and ID, inhibited miRNAs (miR-146a) were up-regulated reversely. Promote metabolism miRNAs(miR-223?miR-210% let-7) were down-regulated in ID, inhibited miRNAs (miR-143? miR-199a-5p) were up-regulated reversely. These processes are necessary for embryonic growth and development. Showed that miRNAs in umbilical cord blood plays an important role in regulating oxygen transmission, angiogenesis, immune system development and energy metabolism. The formation of IUGR pigs during intrauterine embryonic development is regulated by exosome miRNAs from umbilical cord arterial and venous blood.These results in our study provide basic information for further research of the important roles of umbilical cord blood and the mechanism of IUGR in embryo development. In addition, demonstrate pig can be used as an ideal model for studying human development medicine and blood diseases. |
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