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MicroRNA signature of murine CD4+ T cell subpopulations under different skewing conditions

ABSTRACT: This SuperSeries is composed of the SubSeries listed below.

ORGANISM(S): Mus musculus

PROVIDER: GSE109995 | GEO | 2021/01/01

REPOSITORIES: GEO

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Project description:The heterogeneity of exosome populations presents a great challenge to their study. The current study was designed to investigate the potential heterogeneity miRNA contents in circulating exosomes purified via different exosomal markers. In this study, exosomes from the serum of C57BL/6 mice after cecum ligation and perforation (CLP) or sham operation were isolated by precipitation using ExoQuick-TC and affinity purified with anti-Rab5b, anti-CD9, anti-CD31, and anti-CD44 antibodies using the Exo-Flow Exosome Capture kit to collect exosome subpopulations. RNA extracted from the exosomes isolated by ExoQuick-TC were profiled by next-generation sequencing (NGS). Real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) was also employed to determine the expression profiles of four representative exosomal miRNAs (mmu-miR-486-5p, mmu-miR-10a-5p, mmu-miR-143-3p, and mmu-miR-25-3p) selected from the NGS analysis. The results revealed that the expression patterns of these miRNAs in exosomes isolated by ExoQuick-TC as determined by RT-qPCR and NGS were similar, showing upregulation of mmu-miR-10a-5p and mmu-miR-143-3p but downregulation of mmu-miR-25-3p and mmu-miR-486-5p following CLP when compared to the levels in exosomes from sham control mice. However, their expression levels in the antibody-captured exosome subpopulations varied. The miRNAs in the exosomes captured by anti-Rab5b or anti-CD9 antibodies were more similar to those isolated by ExoQuick-TC than to those captured by anti-CD44 antibodies. However, there were no significant differences in these four miRNAs in CD31-captured exosomes. This study demonstrated that purification with different exosomal markers allows the collection of different exosome subpopulations with various miRNA contents. The results of this study demonstrate the heterogeneity of circulating exosomes and suggest the importance of stratifying exosome subpopulations when using circulating exosomes as biomarkers or investigating exosome function. In addition, this study also emphasized the necessity of using a consistent exosome marker across different samples as detecting biomarkers.

Project description:Cryptorchidism and scrotal heating result in abnormal spermatogenesis, but the mechanism(s) prescribing this temperature sensitivity are unknown. It was previously reported that the AKR/N or MRL/MpJ-+/+ mouse testis is more heat-resistant than the testis from the C57BL/6 strain. We have attempted to probe into the mechanism(s) involved in heat sensitivity by examining global gene expression profiles of normal and heat-treated testes from C57BL/6, AKR/N, and MRL/MpJ-+/+ mice by microarray analysis. In the normal C57BL/6 testis, 415 and 416 transcripts were differentially expressed (at least 2-fold higher or lower) when compared with the normal AKR/N and MRL/MpJ-+/+ testis, respectively. The AKR/N and MRL/MpJ-+/+ strains revealed 268 differentially expressed transcripts between them. There were 231 transcripts differentially expressed between C57BL/6 and 2 purported heat-resistant strains, AKR/N and MRL/MpJ-+/+. Next, the testes of C57BL/6 and AKR/N mice were exposed to 43 degrees C for 15 minutes and harvested at different time points for terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) studies and microarrays. An increase of TUNEL-positive germ cell numbers was significant 8 hours after heat exposure in the C57BL/6 mouse. However, this increase was not observed in the AKR/N mouse until 10 hours after heat exposure. All tubules showed germ cell loss and disruption in C57BL/6 testis 24 hours after heat shock. In contrast, although a number of seminiferous tubules showed an abnormal morphology 24 hours post-heat shock in the AKR/N mouse, many tubules still retained a normal structure. Numerous transcripts exhibited differential regulation between the 2 strains within 24 hours after heat exposure. The differentially expressed transcripts in the testes 8 hours after heat exposure were targeted to identify the genes involved in the initial response rather than those attributable to germ cell loss. Twenty transcripts were significantly down-regulated and 19 genes were up-regulated by hyperthermia in C57BL/6 and did not show a parallel change in the AKR/N testis. Conversely, heat shock resulted in 30 up-regulated transcripts and 31 down-regulated transcripts in AKR/N that were not similarly regulated in C57BL/6. A number of genes shared similar differential expression patterns and differential regulation by hyperthermia in both strains of mice. Taken together, the results of the present study indicate that the diverse genetic backgrounds in the 3 strains lead to major differences in normal testis gene expression profiles, whereas the differences in heat shock responses involve a significantly smaller number of genes. The data generated may provide insights regarding gene networks and pathways involved in heat stress and their relationship to spermatogenesis.

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MicroRNA signature of murine CD4+ T cell subpopulations under different skewing conditions

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