Extracellular vesicles derived from hypoxia-preconditioned olfactory mucosa mesenchymal stem cells improve angiogenesis through miR-612 | Journal of Nanobiotechnology

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All experiments have been carried out in accordance with the authorized pointers and have been authorized by Hunan Regular College and the institutional moral and animal care committees. Human nasal mucosa biopsies have been obtained with knowledgeable consent and all experiments have been authorized by the Ethics Committee on the Second Affiliated Hospital of Hunan Regular College (Moral Approval Doc No. 2018-30), and all medical investigations have been performed in keeping with the rules expressed within the Declaration of Helsinki.

Isolation, tradition and identification of human OM-MSCs

OM-MSCs have been obtained from wholesome male volunteers for scientific functions (20–50 years previous) on the Second Affiliated Hospital of Hunan Regular College. The isolation and tradition of OM-MSCs have been carried out utilizing a protocol from our earlier research. Cell floor markers (CD34, CD45, CD44, CD73, CD90, CD105, CD133, CD146) have been used to characterize OM-MSCs by circulate cytometric evaluation.

Isolation and identification of normoxia and hypoxia OM-MSC-EVs

Normoxia and hypoxia OM-MSC-EVs have been purified by differential centrifugation, as described beforehand [19]. Earlier than gathering the supernatant, the circulate apoptosis of hypoxic and normoxic OM-MSCs was decided, and the supernatant might be collected provided that the apoptosis charge was lower than 3%. Hypoxia OM-MSCs was cultured at 3% oxygen focus for 48 h. Briefly, the OM-MSCs have been cultured to 90% confluence in full DMEM; then, the entire medium was changed with DMEM supplemented with 10% sEV-depleted FBS. The sEV-depleted FBS was ready by centrifuging FBS at 120,000g for twenty-four h after which passing it by means of a 0.22-μm filter (Millipore, SLGP033RB). 48 h the conditioned medium was collected and centrifuged at 300 g for 10 min, at 2000g for 10 min, and at 10,000g for 30 min to take away cells and cell particles. The clarified supernatant was then concentrated with a 0.22 μm syringe filter earlier than EVs preparations. Firstly, the supernatant was then transferred to Extremely-Clear tubes and centrifuged at 100,000g for 70 min at 4 °C with a SW32Ti rotor (Beckman Coulter, Netherlands). The exosome-containing pellet was washed with phosphate-buffered saline (PBS) and centrifuged at 100,000g for 70 min. Lastly, the pellet was then rigorously re-suspended in 100 μL PBS. The protein focus was decided by BCA protein assay package (Beyotime, China). The samples have been used instantly or saved at − 80 °C.

The identification of OM-MSC-EVs have been carried out utilizing a protocol from our earlier research.The protein content material of the concentrated EVs was decided utilizing a bicinchoninic acid (BCA) protein assay package (Beyotime, China). The OM-MSC-EVs dimension and focus was assessed by nanoparticle monitoring evaluation (NTA) utilizing a Nanosight NS300 (Malvern, UK). EVs markers CD63, CD81 and TSG101 (1:1000, ProteinTech, China) and OM-MSCs marker Nestin (1:1000, ProteinTech, China) have been decided utilizing Western blot.

miRNA arrays

Complete RNA extracted from the normoxia and hypoxia OM-MSC-EVs was used for miRNA arrays. miRNA profiling was carried out with OE Biotech’s (Shanghai, China) miRNA microarray service based mostly on Affymetrix miRNA 3.0 Array.

Tradition of human mind microvascular endothelial cell and transfection

Human mind microvascular endothelial cells (HBMECs; Cell Financial institution of the Chinese language Academy of Sciences, Shanghai, China) have been cultured in Dulbecco’s modified Eagle’s medium: nutrient combination F12 (DMEM/F12; Invitrogen) with 10% fetal bovine serum (FBS; Invitrogen, United States) at 37℃ in 5% CO2 ambiance. Cells have been incubated at 37 °C, 5% CO2.

In response to the producer’s directions,when HBMECs at 80% confluency, they have been transfected with 50 nM miRNAs utilizing Lipofectamine 2000 in Opti-MEM (Invitrogen). The artificial miR-612 mimic, miR-612 inhibitor, mimic unfavourable management (NC), inhibitor unfavourable management (NC), have been bought from RiboBio (Guangzhou, China). After transfection for five h, the tradition medium was changed with full medium. Different cells have been cultured in 6-well tradition plate and transfected with miR-612 inhibitor or the unfavourable management inhibitor utilizing Lipofectamine 2000 (Invitrogen), and cultured in full medium containing 100 μg/mL normoxia or hypoxia OM-MSC-EVs (200 μg/effectively) or an equal quantity of PBS. After 24 h of incubation, the downstream experiments have been carried out.

To guage the connection between pulling down or up-regulation of TP53 expression and miR-612 in endothelial angiogenesis, TP53 small interfering RNA (siRNAs) (TP53 siRNA#1, TP53 siRNA#2, TP53 siRNA#3) and pcDNA3.1-CMV-TP53 obtained from RiboBio (Guangzhou, China) have been respectively utilized in HBMECs. Briefly, cells have been transfected with siTP53, pcDNA3.1-CMV-TP53 or the common unfavourable management siRNA (Con siRNA) utilizing Lipofectamine 3000 (Invitrogen) in keeping with the directions of the producers. 24 h later, the effectivity of those siRNAs and pcDNA3.1-CMV-TP53 have been verified by qRT-PCR. The siRNA sequences used on this research have been the next:

  • TP53 siRNA #1: 5′-GTACCACCATCCACTACAA-3′;

  • TP53 siRNA #2: 5′-AGAGAATCTCCGCAAGAAA-3′;

  • TP53 siRNA #3: 5′-GGAGTATTTGGATGACAGA-3′.

EVs uptake assay

OM-MSC-EVs have been labeled with a inexperienced fluorescent dye PKH67 (Sigma Alderich, USA) in keeping with the producer’s directions.Then the cell-labeled suspension was centrifuged at 300g for 15 min and the supernatant was discarded. Cells have been washed twice with PBS and seeded into tradition flasks for 48 h of incubation. Subsequent, EVs have been remoted from the conditioned media of MSCs and incubated with HBMECs at 37 °C for 3 h. Cells have been then washed with PBS and stuck with 4% paraformaldehyde for 15 min. After washing with PBS, nuclei have been stained with DAPI (Invitrogen, USA). The indicators have been analyzed with a fluorescence microscope (Leica DMI6000B, Germany).

In vitro on HBMECs

Proliferation assay

Briefly, cells (5 × 103 cells per effectively; 5 replicates per group) have been seeded into 96-well tradition plates and handled with EVs (100 μg/mL) from totally different teams or VEGF (30 ng/mL) or PBS. A gaggle with out cells served because the clean. On 6 and 12 h, cell counting kit-8 reagent (CCK-8; 10 μL per effectively; DOJINDO, Japan) was added to the tradition medium (100 μL per effectively). After incubation at 37 °C for two h, the absorbance of every effectively was measured at 450 nm by a microplate reader (Bio-Rad 680, Hercules, USA) and cell proliferation was represented by means of the imply absorbance of every particular person effectively minus the clean worth.

Scratch wound therapeutic assay

Cells (5 × 105 cells per effectively; three replicates per group) have been seeded right into a 6-well plate and incubated at 37 °C. After the cells had hooked up, the monolayer was scratched with a p200 pipette tip, washed with PBS to take away floating cells after which uncovered to EVs (100 μg/mL) from totally different teams or an equal quantity of PBS. Mitomycin-C (5 µg/mL; Sigma) was current all through the migration assays to exclude the affect of cell proliferation on wound closure. HBMECs have been photographed at 6 h and 12 h after wounding. The speed of migration space was calculated because the ratio of closure space to preliminary wound as described beforehand [20]:

$${textual content{Migration}},{textual content{space}},{textual content{(% ) = }}{{left( {{textual content{A}}_{0} – {textual content{A}}_{{textual content{n}}} } proper)} mathord{left/ {vphantom {{left( {{textual content{A}}_{0} – {textual content{A}}_{{textual content{n}}} } proper)} {{textual content{A}}_{0} instances 100,}}} proper. kern-nulldelimiterspace} {{textual content{A}}_{0} instances 100,}}$$

the place A0 represents the world of preliminary wound space and An represents the remaining space of wound on the metering level.

Transwell migration assay

Boyden chamber assays have been carried out utilizing 24-well transwell inserts (Corning, NY, USA) with 8 μm pore-sized filters and 24-well tradition plates as described beforehand [20]. Cells (4 × 104 cells per effectively; three replicates per group) have been suspended in low serum (5% FBS) medium and plated into the higher chamber. 500 μL full medium (containing 10% FBS) supplemented with EVs (100 μg/mL) from totally different teams or an equal quantity of PBS was added to the decrease chamber. After incubation for 16 h, cells hooked up to the higher floor of the filter membranes have been eliminated by cotton swabs and cells on the underside aspect of the filter (the migrated cells) have been stained with 0.5% crystal violet for a number of minutes. The variety of migrated cells was quantified beneath an optical microscope at a 100× magnification (Leica). The absorbance of every effectively was measured at 550 nm by a microplate reader (Bio-Rad 680, Hercules, USA) and cell migrationn was represented by means of the imply absorbance of every particular person effectively.

Tube formation assay

130 μL chilly Matrigel per effectively was transferred into every effectively of a 24-well plate and incubated at 37 °C for 30 min. Then, HBMECs (2 × 104 cells per effectively; three replicates per group) have been plated into the Matrigel-coated 24-well plates and handled with EVs (100 μg/mL) from totally different teams or PBS. 6–8 h after seeding, tube formation was detected beneath an inverted microscope (Leica). The indications ( complete variety of branches size, junctions, nodes and meshes) revealing the skills to type tubes have been measured through the use of Picture-J software program.

In vivo matrigel plug assay

Athymic‐nude (nu/nu) mice (feminine, 7–8 weeks previous) have been bought from Slac Laboratory Animal Co., Ltd. (Shanghai, China) and have been used for in vivo Matrigel plug research. On the finish of the experiment mice have been euthanized by CO2 inhalation. Mice have been randomly allotted to 12 teams (n = 6 mice per group for the teams): (1) Matrigel solely, (2) Matrigel + N-EVs, (3) Matrigel + H-EVs, (4) Matrigel + HBMECs, (5) Matrigel + HBMECs + N-EVs, (6) Matrigel + HBMECs + H-EVs, (7) Matrigel containing HBMECs transfected with an miR-612 agomir unfavourable management (agomir NC group), (8) miR-612 agomir (miR-612 agomir group), (9) miR-612 antagomir unfavourable management (antagomir NC group), (10) miR-612 antagomir (miR-612 antagomir group), (11) miR-612 antagomir + H-EVs (miR-612 antagomir + H-EVs group), and (12) miR-612 antagomir + N-EVs (miR-612 antagomir + N-EVs group).

To check whether or not normoxia or hypoxia OM-MSC-EVs affected angiogenesis, normoxia and hypoxia OM-MSC-EVs (100 μg/mL) have been resuspended in 500 μL of ice-cold Matrigel respectively, and implanted subcutaneously on the again of nude mice, whereas an equal quantity of Matrigel with out EVs was implanted as unfavourable management. Matrigel plugs have been eliminated for evaluation 14 days later.

To check whether or not normoxia or hypoxia OM-MSC-EVs affected HBMECs angiogenesis, 5 × 106 HBMECs have been blended with 500 μL of Matrigel (BD, USA) at a ratio of 1:1, whereas normoxia and hypoxia OM-MSC-EVs (100 μg/mL) have been resuspended in 500 μL of ice-cold Matrigel respectively, and implanted subcutaneously on the again of nude mice, whereas an equal quantity of Matrigel with 5 × 106 HBMECs was implanted as unfavourable management. Matrigel plugs have been eliminated for evaluation 14 days later.

To find out whether or not exosomal miR-612 is liable for the proangiogenic impact of normoxia and hypoxia OM-MSC-EVs, HBMECs have been loaded with miR-612 agomiR NC/miR-612 agomiR (1.5 nmol/mouse) and miR-612 antagomiR NC/miR-612 antagomiR (3 nmol/mouse), respectively. Subsequent, 5 × 106 HBMECs have been blended with 500 μL of Matrigel (BD, USA) at a ratio of 1:1, whereas an equal quantity of Matrigel with normoxia and hypoxia OM-MSC-EVs (100 μg/mL) have been implanted as unfavourable management. Then, the cell suspensions have been injected subcutaneously within the dorsal area of nude mice. Lastly, the Matrigel plugs have been eliminated for evaluation on Day 14.

Histology and immunofluorescence

Matrigel plugs have been collected, fastened with 4% PFA, embedded in paraffin, and sectioned. For immunohistochemical analyses, Matrigel plug sections have been stained with major antibodies towards CD31 (1:100, ab222783, abcam) and DAPI (Invitrogen, USA) as beforehand described [21].

Bioinformatics evaluation and luciferase reporter assay

Putative targets of miR-612 have been searched utilizing TargetScan (http://targetscan.org/) and miRDB (http://www.mirdb.org/). TP53, which was predicted as a goal of miR-612, was then assessed by luciferase reporter assay. The three′-UTR of TP53 containing both wild-type (WT) or mutant-type (MT) binding websites of miR-612 have been synthesized by GenePharma Co., Ltd. and inserted into the pmirGLO vector (Promega), with the resultant constructs denoted as WT-TP53 and MT-TP53, respectively. HEK293 cells have been cotransfected with miR-612 mimics or miR-NC and reporter plasmids WT-TP53 or MT-TP53 utilizing Lipofectamine 2000. The actions of Renilla and firefly luciferase have been examined utilizing a Twin-Luciferase® Reporter assay package from Promega based mostly on the producer’s protocols. The exercise of firefly luciferase was normalized to that of Renilla luciferase.

RNA isolation and quantitative real-time PCR

Complete RNA from cells and tissues, and exosome-derived RNA have been extracted utilizing TRIZOL (Tiangen, Beijing, China). The primers used on this research have been synthesized by Sangon Biotech Firm (shanghai, China) (Desk 1). Utilizing GAPDH and U6 as inner reference primers, the relative mRNA expression of goal genes was calculated by the two −Ct technique [22].

Desk 1 Primer sequence for qRT-PCR

Western blotting

EVs, cells and matrigel tissue have been processed for Western blot as described [12, 13]. Immunoblot analyses have been carried out utilizing the next major antibodies towards Calnexin (1:1000, ProteinTech, China), TSG101 (1:1000, ProteinTech, China), CD81 (1:1000, ProteinTech, China), HIF-1α (5 µg/mL, abcam, UK), VEGF (5 µg/mL, abcam, UK), TP53(1:1200, ProteinTech, China), GAPDH (1:3000, ProteinTech, China).The anti-rabbit IgG and anti-mouse IgG secondary antibodies have been obtained from Proteintech. The proteins have been visualized utilizing an enhanced chemiluminescent (ECL) detection package (Advansta Inc., United States).

Statistical evaluation

All experiments have been carried out in at the least three replicates. Knowledge are expressed as imply ± SEM. Variations between teams have been estimated utilizing two-side dunpaired Scholar’s t-test or two-sided ANOVA with the Bonferroni correction for thepost hoc t-test as applicable. Statistical evaluation was performed with GraphPad Prism 6 Software program (La Jolla, CA, United States). Variations with the likelihood of P < 0.05 have been thought of vital.