Plasma sample collection. On July, 27th, 2020, thin camels (n = 5) and normal camels (n = 5) were obtained from Shibatai Township, Zhuozishan Town, Wulanchabu, Inner Mongolia, and the blood (80 mL) were taken from each camel. The information (age, weight, height, and body length) of the all camels was shown in Table 1. The research protocols were approved by the Experimental Animal Welfare and Ethics Committee of Inner Mongolia Agricultural University (No. NND2021034).
Table 1
The information of thin camels and normal camels
Group\Index
|
Age (years)
|
Weight (kg)
|
Height (cm)
|
Body length (cm)
|
Thin camels
|
1
|
10
|
402.6
|
173.8
|
168.7
|
2
|
10
|
408.1
|
175.5
|
169.4
|
3
|
12
|
419.0
|
178.7
|
174.1
|
4
|
11
|
421.2
|
180.9
|
176.2
|
5
|
11
|
410.4
|
176.1
|
170.5
|
Normal camels
|
1
|
11
|
488.3
|
178.2
|
172.2
|
2
|
9
|
452.3
|
171.5
|
166.5
|
3
|
12
|
490.2
|
176.7
|
171.3
|
4
|
10
|
471.7
|
173.9
|
168.5
|
5
|
12
|
492.8
|
181.1
|
175.8
|
Isolation and characterization of exosomes. The exosomes were isolated from the camel plasma samples at 4°C as described previously 12. Briefly, the blood samples were diluted with the isopycnic PBS, and then centrifuged at 300 g for 10 min. After that, the supernatant was transferred to a new tube, and then centrifuged at 2000 g for 10 min, followed by 12000 g for 30 min, and 12000 g for 70 min. The sediments were resuspended with pre-cooling PBS (2500 µL), and the exosomes were obtained and stored at -80°C. The concentrations of the isolated exosomes were quantified using a BCA protein assay kit (Boster Biological Technology Co. Ltd, Wuhan, China) based on the manufacturer’s instructions.
Thereafter, the isolated solution (50 µL) was added to PBS (10 mL), and centrifuged at 120000 g for 70 min at 4°C. Then, the sediments were resuspended with 50 µL PBS, and used for transmission electron microscopy (TEM) and Nano particle tracking analyzer (NTA). A TEM (Tecnai G2 spititi, FEI company, OR, USA) was used to visualize the morphology and ultrastructure of the extracted exosomes based on the previous study 13. Besides, the exosomes size distribution was evaluated using a ZetaView® NTA (ZetaView Particle Metrix, Particle Metrix, Germany) in accordance with the method of Soares Matins et al. 14.
Total RNA extraction and small RNA sequencing. Total RNA was extracted from the plasma-derived exosomes of thin camels and normal camels using mirVANA miRNA Isolation kit (Takara Biomedical Technology Co., Ltd., Beijing, China) according to the manufacturer’s recommendations, and then the quality and concentrations of the total RNA were assessed using a microplate reader (OD260/280). After that, the total RNA was sent to OBiO Technology (Shanghai) Corp., Ltd (China) for small RNA sequencing.
During sequencing, TruSeq small RNA Sample Prep Kit (Illumina, San Diego, USA) was used to construct miRNA library. Thereafter, RNA 3’ and 5’ adaptors were ligated, as well as the enrichment library was amplified by reverse transcription-polymerase chain reaction (RT-PCR), and was purified by gel electrophoresis. After the library preparation, Illumina Hiseq200/2500 was used to sequence the constructed library, and the sequencing read length was 1×50 bp. After that, the samples were also sequenced on this platform.
Raw data by sequencing were submitted to an in-house program, ACGT101-miR (LC Sciences Houston, Texas, USA), to remove the 3’ connectors and junk sequences, and clean data were achieved. The clean data were mapped to databases of mRNA, Rfam and Repbase, and after filtering, valid data were obtained and used for miRNA identification. Subsequently, unique sequences of 18–26 nt in length were mapped to specific species precursors in miRbase 22.0 by BLAST searching, and known or novel miRNAs were annotated. After that, differentially expressed miRNAs (DE-miRNAs) between the plasma-derived exosomes of thin camels and normal camels were identified using Student t test, and the thresholds for DE-miRNAs selection were |log2Fold change (FC)| > 1 and P value ≤ 0.05. Next, the potential target genes of the identified DE-miRNAs were predicted using the TargetScan and miRanda databases, and then were submitted to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. A P value ≤ 0.05 was set as the criterion of the significantly enriched GO terms and KEGG pathways.
Proteomics analysis of exosomes. The exosomes from the plasma of thin and normal camels were further sent to OBiO Technology (Shanghai) Corp., Ltd for proteomics analysis. Briefly, SDT lysate (4% SDS, 100mM Tris-HCl, pH 7.6) was added in the plasma-derived exosomes of thin and normal camels, and then bathed in boiling water for 15min. After centrifuged at 14000 g for 15 min, the supernatant was collected, and then FASP ultrafiltration method was adopted to remove the SDT solution in the supernatant 15. Then, 100µg peptide of each sample was labeled using a TMT labeling kit (Thermo) according to the manufacturer’s protocols. The labeled peptides in each group were mixed, and graded by Agilent 1260 Infinity II HPLC system. Thereafter, each sample was separated using Easy nLC system, and then analyzed with a Q Exactive plus mass spectrometer.
The raw data generated by Q Exactive plus were converted to the .mgf format using Proteome Discoverer 2.2 (Thermo Fisher Scientific) software, and then submitted to MASCOT2.6 for protein annotation. Differentially expressed proteins (DEPs) between the plasma-derived exosomes of thin and normal camels were screened based on the thresholds of FC > 1.2/FC < 0.85 and P < 0.05. Afterwards, the screened DEPs were subjected to GO terms, KEGG pathways and subcellular localization analyses.
Cell culture and construction of MHCC-97H-LUC cells. MHCC-97H cells were purchased from Cell Bank, Chinese Academy of Sciences (Shanghai, China), and cultured in Dulbecco’s modified Eagle’s medium (DMEM, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scientific). Then, the cells were maintained in an incubator with 5% carbon dioxide at 37°C. The MHCC-97H cells were passaged upon reaching 80%-90% confluence.
The MHCC-97H cells with LUC fluorescence (MHCC-97H-LUC cells) were constructed using lentivirus (H7656 pLenti-CBh-3xFLAG-Luc2-tCMV-mNeonGreen-F2A-Puro-WPRE, OBiO Technology (Shanghai) Corp., Ltd) package. Briefly, The MHCC-97H cells were seeded into a 6-well plate at a density of 3×105 cells/well, and cultured overnight. After that, the cells were transfected with H7656 lentivirus, and then 10 µL polybrene (1 mg/mL) was added to each well at a final concentration of 5 µg/mL. After 24 h of transfection, the medium was changed to fresh medium, and after 72 of culture, final concentration of 2 µg/mL puromycin was added to select the stable transfection cell line (MHCC-97H-LUC cells).
Animal experiments. A total of 21 SPF female Balb/c-nude mice aged 5–6 weeks were purchased from Jiangsu GemPharmatech Biotechnology Co. Ltd (Jiangsu, Chian). During the experiments, all the mice were free access to food and water, and maintained under controlled temperature (24 ± 2°C) and humidity (50 ± 10%) conditions, with a 12 h light/dark cycle. After acclimatization for 7 days, all the mice were randomly and equally divided into three groups as follow (n = 7 for each group): control group, N-Exo and T-Exo groups. All the mice were used to perform in situ tumor of the liver as previously described 16. Briefly, the mice were anesthetized using an isoflurane air hemp machine. After anesthesia, the liver lobe was gently extruded, and 40 µL MHCC-97H-LUC cell suspension (5×106 cells/mouse) was injected to the liver. After stopping bleeding, the liver lobe was slowly returned to the abdominal cavity, sutured, and the wound was disinfected with iodophor. The animals were kept in a state of gas anesthesia all the time. After seven days, the mice in the N-Exo and T-Exo groups were respectively injected with 200 µg normal camel plasma-derived exosomes and thin camel plasma-derived exosomes through a tail vein once a week for three weeks. The mice in the control group were treated with equal amount of PBS.
At the end of the experiment, all the mice were used for in vivo imaging using in vivo imaging system (PE LuminaLT series III, USA) 17. After that, the mice were sacrificed by cervical dislocation, and the liver tissues were collected for hematoxylin eosin (HE) staining to observe the effects of normal and thin camel plasma-derived exosomes on tumor growth. All animal experiments were conducted in compliance with the ARRIVE guidelines, and in accordance with the National Medical Advisory Committee (NMAC) guidelines using approved procedures of the Experimental Animal Welfare and Ethics Committee of Inner Mongolia Agricultural University (No. NND2021034).