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Characterization of serum small extracellular vesicles and their small RNA contents across humans, rats, and mice.

Zhao, Fengbo, Cheng, Li, Shao, Qian, Chen, Zixing, Lv, Xiufang, Li, Jing, He, Li, Sun, Yufeng, Ji, Qiuhong, Lu, Peng, Ji, Yuhua, Ji, Juling·2020·Scientific Reports
DOI 10.1038/s41598-020-61098-9PMC PMC7060188
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Abstract

Serum small extracellular vesicles (sEVs) have recently drawn considerable interest because of the diagnostic and therapeutic potential of their miRNAs content. However, the characteristics of human, mouse and rat serum sEVs and their differences in small RNA contents are still unknown. In this study, through nanoparticle tracking analysis and small RNA sequencing, we found that human, rat, and mouse serum sEVs exhibited distinct sizes and particle numbers as well as small RNA contents. Serum sEVs contained not only abundant miRNAs but also a large number of tRNA fragments. Most serum miRNAs existed both inside and outside of sEVs but were enriched in sEVs. Common serum sEV miRNAs (188 miRNAs) and species-specific serum sEV miRNAs (265, 58, and 159 miRNAs, respectively) were identified in humans, rats, or mice. The serum sEVs contained miRNAs from tissues and organs throughout the body, with blood cells as the main contributors. In conclusion, our findings confirmed the rationality of exploring serum sEV miRNAs as noninvasive diagnostic markers and revealed great differences in serum sEV small RNAs between humans, rats, and mice. Inadequate attention to these differences and the contribution of blood cells to serum sEV miRNAs could hinder the clinical translation of basic studies.

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Figures

Figure 1 Characterization of sEV isolated from human, rat and mouse serum by Exoquick precipitation. (A) Transmission electron microscopy image of the isolated particles, bar = 200 nm, representative image of sEVs from rat. (B) Representative NTA size distribution profiles of isolated particles and their concentrations. (C) The expression of CD63, CD81 and CD9, as well as calnexin and albumin in the isolated particles was determined by western blotting. Lanes from left to right are serum sEV, de-sEV serum and whole serum, respectively. The images are the representatives of three or more independent experiments. Full-length blots/gels are presented in Supplementary Fig S2. sEV, small extracellular vesicle; de-sEV, small extracellular vesicle depleted.
Figure 1 Characterization of sEV isolated from human, rat and mouse serum by Exoquick precipitation. (A) Transmission electron microscopy image of the isolated particles, bar = 200 nm, representative image of sEVs from rat. (B) Representative NTA size distribution profiles of isolated particles and their concentrations. (C) The expression of CD63, CD81 and CD9, as well as calnexin and albumin in the isolated particles was determined by western blotting. Lanes from left to right are serum sEV, de-sEV serum and whole serum, respectively. The images are the representatives of three or more independent experiments. Full-length blots/gels are presented in Supplementary Fig S2. sEV, small extracellular vesicle; de-sEV, small extracellular vesicle depleted.CC BY · PubMed Central
Figure 2 The length distribution and concentration of small RNAs in serum sEV and de-sEV serum samples from human, rats and mice as detected by Agilent 2100 Bioanalyzer using small RNA chips. (A) Gel-like image of all the samples with biological replicates. (B) For each group, electropherograms for one of the two biological replicates was provided. (C) The concentrations of small RNA (0–296nt) in serum sEV from human, rat and mouse, human vs. rat, ***P = 0.0003; human vs. mouse, **P = 0.0053; rat vs. mouse, *P = 0.0159; de-sEV serum, human vs. rat, P > 0.05, ns; human vs. mouse, **P = 0.0052; rat vs. mouse, *P = 0.0397; the concentrations of small RNA in serum sEV vs. de-sEV serum, human, P > 0.05, ns; rat, P > 0.05, ns; mouse, *P = 0.0134. sEV, small extracellular vesicle; de-sEV, small extracellular vesicle depleted.
Figure 2 The length distribution and concentration of small RNAs in serum sEV and de-sEV serum samples from human, rats and mice as detected by Agilent 2100 Bioanalyzer using small RNA chips. (A) Gel-like image of all the samples with biological replicates. (B) For each group, electropherograms for one of the two biological replicates was provided. (C) The concentrations of small RNA (0–296nt) in serum sEV from human, rat and mouse, human vs. rat, ***P = 0.0003; human vs. mouse, **P = 0.0053; rat vs. mouse, *P = 0.0159; de-sEV serum, human vs. rat, P > 0.05, ns; human vs. mouse, **P = 0.0052; rat vs. mouse, *P = 0.0397; the concentrations of small RNA in serum sEV vs. de-sEV serum, human, P > 0.05, ns; rat, P > 0.05, ns; mouse, *P = 0.0134. sEV, small extracellular vesicle; de-sEV, small extracellular vesicle depleted.CC BY · PubMed Central
Figure 3 The length distribution and frequency of annotated reads of small RNAs in serum sEV samples and de-sEV serum samples from human, rats and mice. (A) Length distribution of the clean reads. (B) Pie charts of annotated small RNA species and their percentages in total clean reads, others including: rRNA, snRNA, Cis-reg and other_Rfam_RNA. For each group, one representative chart of the two replicates was provided. Detailed annotation data for all the samples were provided in Supplementary Table S3. sEV, small extracellular vesicle; de-sEV, small extracellular vesicle depleted.
Figure 3 The length distribution and frequency of annotated reads of small RNAs in serum sEV samples and de-sEV serum samples from human, rats and mice. (A) Length distribution of the clean reads. (B) Pie charts of annotated small RNA species and their percentages in total clean reads, others including: rRNA, snRNA, Cis-reg and other_Rfam_RNA. For each group, one representative chart of the two replicates was provided. Detailed annotation data for all the samples were provided in Supplementary Table S3. sEV, small extracellular vesicle; de-sEV, small extracellular vesicle depleted.CC BY · PubMed Central
Figure 4 Profiling of human, rat, and mouse serum sEV miRNAs. (A) Correlation between normalized miRNAs read counts (Log2TPM) between the biological replications from each group. (B) Correlation between normalized read counts (Log2TPM) obtained by small RNA-seq and adjusted q-RT-PCR CT values for 6 miRNAs (miR-125a-5p, miR-125b-5p, miR-191-5p, miR-27b-3p, miR-486-5p and miR-99a-5p). q-RT-PCR for miRNAs were performed with the same batch of RNAs that prepared for sequencing. The relative expression of the selected miRNAs as determined by qPCR were expressed as (40-Ct) miRNA. TPM, transcripts per million reads; h, human; r, rat; m, mouse; e, serum small extracellular vesicle; d, small extracellular vesicle depleted serum.
Figure 4 Profiling of human, rat, and mouse serum sEV miRNAs. (A) Correlation between normalized miRNAs read counts (Log2TPM) between the biological replications from each group. (B) Correlation between normalized read counts (Log2TPM) obtained by small RNA-seq and adjusted q-RT-PCR CT values for 6 miRNAs (miR-125a-5p, miR-125b-5p, miR-191-5p, miR-27b-3p, miR-486-5p and miR-99a-5p). q-RT-PCR for miRNAs were performed with the same batch of RNAs that prepared for sequencing. The relative expression of the selected miRNAs as determined by qPCR were expressed as (40-Ct) miRNA. TPM, transcripts per million reads; h, human; r, rat; m, mouse; e, serum small extracellular vesicle; d, small extracellular vesicle depleted serum.CC BY · PubMed Central
Figure 5 The frequency of known miRNAs among total clean reads of small RNAs. (A) The frequency of known miRNAs in serum sEVs from human, rat and mouse, human vs. rat, **P = 0.0035; human vs. mouse, ***P = 0.0002; rat vs. mouse, *P = 0.0458; de-sEV serum, human vs. rat, **P = 0.0019; human vs. mouse, **P = 0.0039; rat vs. mouse, ns, P = 0.2693. (B) The frequency of known miRNAs in serum sEVs vs. de-sEV serum, human, *P = 0.014; rat, *P = 0.0103; mouse, *P = 0.0399. sEV, small extracellular vesicle; de-sEV, small extracellular vesicle depleted.
Figure 5 The frequency of known miRNAs among total clean reads of small RNAs. (A) The frequency of known miRNAs in serum sEVs from human, rat and mouse, human vs. rat, **P = 0.0035; human vs. mouse, ***P = 0.0002; rat vs. mouse, *P = 0.0458; de-sEV serum, human vs. rat, **P = 0.0019; human vs. mouse, **P = 0.0039; rat vs. mouse, ns, P = 0.2693. (B) The frequency of known miRNAs in serum sEVs vs. de-sEV serum, human, *P = 0.014; rat, *P = 0.0103; mouse, *P = 0.0399. sEV, small extracellular vesicle; de-sEV, small extracellular vesicle depleted.CC BY · PubMed Central
Figure 6 The expression of miRNAs in serum sEV vs. de-sEV serum, and the expression of miRNAs in serum sEV between species. (A) The venny diagram depicts the common and unique miRNAs identified in serum sEV miRNAs and de-sEV serum miRNAs from human, rat and mouse, respectively. (B) The composition of miRNAs based on the relative expression of serum sEVs vs. sEV-depleted serum, only those miRNAs existed both inside and outside of serum sEV were included. (C) The venny diagram depicts the common and unique miRNAs identified in serum sEV miRNAs from human, rat and mouse. (D) Heat map of hierarchical clustering of the 188 common serum sEV miRNAs between species based on log2 transformed TMP (transcripts per million reads). Each column represents an individual sample, each row represents an individual miRNA. miRNAs were ordered by Pearson correlation and complete linkage. TPM, transcripts per million reads; sEV, small extracellular vesicle; de-sEV, small extracellular vesicle depleted; e, serum small extracellular vesicle; d, small extracellular vesicle depleted serum.
Figure 6 The expression of miRNAs in serum sEV vs. de-sEV serum, and the expression of miRNAs in serum sEV between species. (A) The venny diagram depicts the common and unique miRNAs identified in serum sEV miRNAs and de-sEV serum miRNAs from human, rat and mouse, respectively. (B) The composition of miRNAs based on the relative expression of serum sEVs vs. sEV-depleted serum, only those miRNAs existed both inside and outside of serum sEV were included. (C) The venny diagram depicts the common and unique miRNAs identified in serum sEV miRNAs from human, rat and mouse. (D) Heat map of hierarchical clustering of the 188 common serum sEV miRNAs between species based on log2 transformed TMP (transcripts per million reads). Each column represents an individual sample, each row represents an individual miRNA. miRNAs were ordered by Pearson correlation and complete linkage. TPM, transcripts per million reads; sEV, small extracellular vesicle; de-sEV, small extracellular vesicle depleted; e, serum small extracellular vesicle; d, small extracellular vesicle depleted serum.CC BY · PubMed Central
Figure 7 Pie charts of the top ten most abundant serum sEV miRNAs in human, rat and mice, and venny diagram depicts the common and unique miRNAs identified in the top ten most abundant serum sEV miRNAs from human, rat and mouse.
Figure 7 Pie charts of the top ten most abundant serum sEV miRNAs in human, rat and mice, and venny diagram depicts the common and unique miRNAs identified in the top ten most abundant serum sEV miRNAs from human, rat and mouse.CC BY · PubMed Central

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Metadata

Authors
Zhao, F., Cheng, L., Shao, Q., Chen, Z., Lv, X., Li, J., He, L., Sun, Y., Ji, Q., Lu, P., Ji, Y., & Ji, J.
Venue
Scientific Reports
Publisher
Springer Science and Business Media LLC
Published
2020-03-06
Volume
10
Issue
1
ISSN
2045-2322
DOI
10.1038/s41598-020-61098-9
PMC
PMC7060188
Cited by
45
References
4
Language
en
Licence
Funders
  • National Natural Science Foundation of China (81201016, 81272027, 81761128018)
  • Six Talents Peak Project of Jiangsu Province (2017-WSN-094)
  • Natural Science Foundation of Jiangsu Province (BK20151277)

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Zhao, F., Cheng, L., Shao, Q., Chen, Z., Lv, X., Li, J., He, L., Sun, Y., Ji, Q., Lu, P., Ji, Y., & Ji, J. (2020). Characterization of serum small extracellular vesicles and their small RNA contents across humans, rats, and mice. Scientific Reports. https://doi.org/10.1038/s41598-020-61098-9