Experimental animals and cells
The male BALB/c nude mice weighing 20-25 g at 6-8 weeks of age were purchased from Beijing Huafukang Biotechnology Co., Ltd and were housed in the animal care facilities of China Medical University under specific pathogen-free (SPF) conditions. This research was approved by the Ethics Committee of China Medical University with ethics number CMU2021579. HAMSCs and human Umbilical Vein Endothelial Cells (hUVECs) were obtained from the Stem Cell and Regenerative Medicine Research Laboratory of China Medical University. HAMSCs were isolated, cultured and identified as previously described (He et al. 2020). HUVECs were cultured in RPMI 1640 medium (Gibco, USA) supplemented with fetal bovine serum (FBS, Hyclone, USA) and were placed in a 37 ℃, 5% CO2 incubator.
HAMSC-Exos isolation and extraction
HAMSCs of P3 generation were cultured until the confluence reached 80%, and then the medium was replaced to DMEM/F12 medium (Hyclone, USA). After cells being cultured in a CO2 incubator at 37 ℃, 5% CO2, the medium was collected and centrifuged at 300 g for 5 min, 2000 g for 15 min, and 13000 g for 35 min at a time. After being filtered with 0.22 μ M sterile filter, the medium was transfered into the ultrafiltration tube for ultrafiltration, and discard the lower liquid. The filter was washed with PBS, and the liquid was collected and centrifuged at 150000 g for 3 h at 4 ℃. After the supernatant being discarded, the centrifugal sediment was dissolved in PBS and collected. The concentration of hAMSC-Exos was detected using BCA protein quantification kit.
Transmission electron microscope
The morphology of the extracted exosomes was observed using transmission electron microscopy (Hitachi, Tokyo, Japan). 10 μL purified hAMSC-Exos was added onto the copper mesh. After 5 min, the excess liquid on the copper mesh was aspirated, and then 10 μL phosphotungstic acid was added on the copper mesh. After the copper mesh was drying, The images were displayed on 80KV-120KV.
Nanoparticle tracking analysis (NTA)
The particle size and concentration of Exos was measured using nanoparticle tracking analysis (NTA) with ZetaView PMX 110 (Particle Metrix, Meerbusch, Germany) and software ZetaView 8.04.02. After the detection instrument was calibrating, the sample pool was washed with 1×PBS buffer. After that, the sample was diluted with 1×PBS buffer, and detected.
Western blot
The total protein extraction kit (Takara, China) was used for protein extraction. BCA assay kit (Takara) was used to determine the concentration of extracted protein.
30 μg protein sample was separated by SDS polyacrylamide gel, and transferred to PVDF membrane. The PVDF membrane was incubated in 5% nonfat milk powder blocking solution for 2 h at room temperature. The PVDF membrane was cut according to the molecular weight of the protein, placed in diluted primary antibody solution (CD9 antibody 1:1000, CD63 antibody 1:1000, CD81 antibody 1:1000) at 4 ℃ overnight. Horseradish peroxidase-labeled secondary antibody diluted in 5% BSA (1:10000) was added and incubated for 1 h at room temperature. The PVDF membrane was detected on a Tanon-5200 chemiluminescence detection system (Tanon, Shanghai, China) using ECL kit (Solarbio, China).
Cell viability assay
HUVECs were routinely cultured in hUVECs complete medium (RPMI 1640 medium +10% FBS). 5×103 cells with 200 μL hUVECs complete medium in each well of a 96 well plate were cultured at 37 ℃ in a 5% CO2 incubator for 24, 48, 72 and 96 h, respectively. Each well was added with a final concentration of 0, 50, and 100 μg/mL of hAMSC-Exos. At each time point, each well was added with 20 μL MTS and incubated in the incubator for 2 h. The OD value of each well at 492 nm wavelength was detected.
Transwell migration assay.
To detect the migration ability of hUVECs, 5×104 cells mixed with 100 μL RPMI 1640 medium were placed in the upper chamber of the Transwell chamber, and RPMI 1640 medium supplemented with 10% FBS was added into the lower chamber. 0, 50 and 100 μg/mL of hAMSC-Exos were added to the upper chamber of the Transwell chamber, respectively. After 12 h, the Transwell chamber was removed, and the chamber membrane was removed and stained with hematoxylin and eosin (HE) staining kit. Cell statistics were performed on the lower surface of the Transwell chamber membrane.
Matrigel tube formation assay
200 μL Matrigel was spread on a 24 well plate and incubated at 37 ℃ for 1 h to solidify. 5×104 hUVECs mixed with 200 μL hUVECs complete medium were placed on the upper layer of Matrigel. 0 ,50 and 100 μg/mL of hAMSC-Exos were added to the medium. After 12 h, the number of tubes were taken under a inverted microscope.
Matrigel plug assays in nude mice
RPMI 1640 medium and 250 μL Matrigel (354262,BD,USA) was mixed at a ratio of 1:1 at 4 ° C. 0, 50 and 100 μg/mL of hAMSC-Exos were added. The male BALB/c nude mice weighing 20-25 g were injected with a total of 500 µL of the mixture subcutaneously in the dorsal region. The plug was restored after 2 weeks. Tissue sections, HE staining and Immunohistochemistry were further used to detect the angiogenesis in vivo.
Bioinformatics analysis
MiRDeep 2.0 was used to analyze candidate new miRNAs in previous next-generation small RNA sequencing data. RNAfold (http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi) was used to analyze the secondary structure, free energy, gene locus and other information of candidate new miRNAs.
RT-qPCR
RNAiso was used to isolate the total RNA of cells and hAMSC-Exos. CDNA was synthesized using a Mir-X miRNA First-Strand Synthesis Kit or PrimeScriptTM RT reagent Kit with gDNA Eraser kit with normal primers or stem loop RT-qPCR primers. The synthesized cDNA was diluted into cDNA working solution by adding 4 volumes of RNase water. TB GreenTM Premix Ex TaqTM II kit was used to detect the expression of mRNAs and miRNAs. The relative levels of genes were calculated by 2–ΔΔCT method, with GAPDH or U6 as the internal reference. Primers were shown as Supplementary table 1 and 2.
Cell transfection
HAMSCs were cultured until the confluence reached 50-60%, and the complete medium of hAMSCs was replaced with serum-free DMEM/F12 medium. Lipo2000 transfection reagent was used to transfect the new miRNAs N-194 mimics, sh-N-194 and their corresponding negative control (NC) into hAMSCs. After 6 h, the medium was changed to complete medium of hAMSCs. HAMSC-Exos or hAMSCs were collected after 24 h for further experiments.
Target gene prediction
It was found that the new miRNA N-194 had the same seed sequence as hsa-miR-4467. TargetScan human 8.0 was applied to predict the target genes of hsa-miR-4467. The binding sites of potential target genes of hsa-mir-4467 and the new miRNA N-194 were analyzed.
Dual luciferase reporter assay
PyrobestTM DNA polymerase PCR kit was used to amplify the wild-type DNA sequence around the predicted binding sites of ING5. The mutant DNA sequence around the binding site was synthesized. The amplified wild-type and mutant DNA sequences and pGL3 control vector were digested by Xba I restriction enzyme, and combined the amplified DNA sequences with pGL3-control vector to construct the corresponding wild-type and mutant firefly luciferase reporter vectors, respectively. Cells were cultured in 24 well plates until the confluence reached 60-70% and serum-free medium was replaced. The experiment was divided into experimental group and control group. In the experimental group, lipo2000 transfection reagent was used to transfect 2 μg firefly luciferase reporter, 0.2 μg Renilla luciferase reporter and 2 μL of the new miRNA N-194 mimics were cotransfected into cells. In the control group, lipo2000 transfection reagent was used to transfect 2 μg firefly luciferase reporter, 0.2 μg Renilla luciferase reporter and 2 μL of NC into cells. After 6 h, the medium was changed to complete medium with FBS. After 24 h, the cells were collected and analyzed by dual luciferase reporter assay system. Renilla luciferase was used as an internal reference to calculate the relative expression of firefly luciferase. Primers and DNA sequences were shown as Supplementary table 3.
Statistical Analysis
Each experiment was repeated for at least three times. GraphPad. Prism v5.0 and Image J were used to analyze data and image results. The experimental results were expressed as mean (standard deviation). T-test was used for statistical analysis of the two samples, and p<0.05 was considered as statistically significantly different.