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miR-709 exerts an angiogenic effect through a FGF2 upregulation induced by a GSK3B downregulation – Scientific Reports

Animals

All animal procedures were approved by the Institutional Animal Care and Use Committee of Yamaguchi University (No. 31-101). The study was conducted in accordance with the relevant guidelines and carried out in compliance with the ARRIVE guidelines. The methods were carried out in accordance with the approved guidelines and the ARRIVE guidelines. Male C57BL/6 and male BALB/c mice were purchased from Japan SLC, Inc. (Shizuoka, Japan). Five mice were housed in the same cage (mouse polycarbonate cage, #TM-PC-5-I(1), Tokiwa, Tokyo, Japan) Fir wood shavings were used as beddings. Mice were bred in a temperature-, humidity-, and light-controlled sub-SPF room (22 ± 2 °C, 70 ± 20%, and 12 h light/dark cycles, respectively) at Institute of Life Science and Medicine Yamaguchi University. Food and water were provided ad libitum. Mice were randomly divided into each group. Anesthesia was maintained with 1.5–2% isoflurane (MSD Animal Health, Tokyo, Japan).

miRNA extract and array analysis

Mice were anesthetized through the inhalation of 1.5% isoflurane during the surgical procedure. For the first screening, the left femoral artery and vein in 8-week-old C57BL/6 and BALB/c mice was ligated at two points, and was severed with an electric scalpel between the ligated points. In order to collect plasma at 24 h after the ligation of the left femoral artery, blood was drawn from the abdominal vena cava by using a 26-G syringe including 0.1 mL of 1% ethylenediaminetetraacetic acid (EDTA). The collected blood was transferred to a 1.5-mL tube, and was centrifuged at 1,200 g for 20 min, at 4 °C. The supernatant was placed in a new 1.5-mL tube, and was stored at −80 °C. EVs were isolated from the obtained plasma by using the total exosome isolation kit (#4484450; Thermo Fisher Scientific, Waltham, Massachusetts, USA). miRNAs in the EVs were extracted with the use of a total exosome RNA and protein isolation kit (#4478545; Thermo Fisher Scientific). The miRNA expression levels were analyzed in each of the five-sample pools (n = 5 in C57BL/6, and n = 5 in BALB/c) by using 3D-Gene® miRNA oligo chips (Toray Industries, Inc., Kamakura, Kanagawa, Japan) (GEO accession: GSE217938 and Supplementary Data).

Plasmids expressing miRNAs

In order to construct the plasmids expressing the 43 miRNAs shown Supplementary Table 1, the C57BL/6 mouse genome was amplified with Ex Taq (TaKaRa Bio Inc., Kusatsu, Shiga, Japan) and primers (Supplementary Table 1), and the amplified DNA was ligated to a T-vector pMD20 (#3270; TaKaRa Bio Inc). Primers were designed with reference to mice chromosome region (Supplementary Table 1). DNA fragments were subcloned from the T-vector pMD20 into the restriction enzymes’ (Supplementary Table 1) site of the pmR-mCherry vector (#Z2542N; TaKaRa Bio Inc).

Preparation of miRNA-containing EVs for the aortic ring assay

293 T cells (#RCB2202) were provided by the RIKEN BRC through the National Bio-Resource Project of the MEXT/AMED, Japan. The cells were cultured in Dulbecco’s modified Eagle medium (DMEM; #11995-065; Thermo Fisher Scientific) supplemented with 10% exosome-depleted fetal bovine serum (FBS; #A2720801; Thermo Fisher Scientific), and 0.5 mL of 293 T cells (2 × 105 cells/mL) were seeded in 24-well plates. Subsequently, the cells were transfected with 1 μg of the plasmid by using 1 μL of the X-tremeGENE HP DNA transfection reagent (Roche), and were incubated overnight. After removing the culture medium from the wells, 1.2 mL/well of fresh DMEM supplemented with 10% exosome-depleted FBS was applied to the same wells. The culture medium was collected after 3 days. The EVs included in the culture medium were isolated by using the total exosome isolation reagent (from cell culture media; #4478359; Thermo Fisher Scientific). Finally, the pellets were dissolved with 420 μL of Opti-MEM™ I reduced serum medium (Thermo Fisher Scientific).

Aortic ring assay

An aortic ring assay was carried out based on previous reports36,37. Femoral arteries were removed from both thighs of BALB/c mice, and were immersed in phosphate-buffered saline (PBS) on ice. At first, 50 μL of Matrigel (Cornig) were applied on 44 wells of a 96-well plate that was plated on ice. An aortic ring was placed on the Matrigel of each well. Subsequently, 50 μL of Matrigel were added in each well, and the 96-well plate was incubated at 37 °C, in 5% CO2, overnight. The next day, 50 μL of the EVs’ solution were applied on each well, and the 96-well plate was incubated for 5–7 days at 37 °C, in 5% CO2. Three independent experiments were performed. If neovascularization was observed under the microscope even at a rate of one out of three, the miRNA was considered to have an angiogenic potential.

Preparation of miRNA-containing EVs for screening by using the hindlimb ischemia model

293 T cells were seeded in 6-well plates, and were cultured in DMEM supplemented with 5% exosome-depleted FBS. Cells were transfected with 2 μg of plasmid by using 2 μL of X-tremeGENE HP DNA transfection reagent (Roche), and were incubated overnight. After removing the culture medium from the wells, 2 mL/well of fresh DMEM supplemented with 5% exosome-depleted FBS were applied to the wells. The culture medium was collected after 5 days. The EVs included in the culture medium were isolated by using the total exosome isolation reagent. The pellets were then dissolved with 400 μL of PBS.

Screening of angiogenic miRNAs by using a lower ischemia model

The left femoral artery was ligated in one place in 7-week-old BALB/c mice. The next day, a total of 100 μL of EVs were injected in two places in the femoral muscle. Blood flow was analyzed by using a laser speckle perfusion imaging system (OMEGA ZONE, Omega Wave). Three mice per one group in one experiment were assessed. Two independent experiments were performed. Blood flow was analyzed in six mice per group.

Preparation of miRNA-encapsulating EVs

293 T cells were cultured in HE100 medium (Gmep Incorporated, Kurume, Fukuoka, Japan) with L-glutamine. Scramble, miR-709, and miR-1247-3p mimics were synthesized by Ajinomoto Bio-Pharma Services, GeneDesign Inc. (Osaka, Japan). 293 T cells were seeded in a 10-cm dish (2 × 106 cells/dish) by using 10 mL of HE100 medium supplemented with L-glutamine. The next day, the 293 T cells were transfected with 600 pmol of miRNA mimics by using 25 μL of the RNAiMAX transfection reagent (#13778075; Thermo Fisher Scientific), and were incubated for 24 h after transfection. After removing the culture medium from the dishes, 10 mL/dish of fresh HE100 medium supplemented with L-glutamine was applied to the dishes. The culture medium was collected after 3 days. The culture medium was concentrated with Amicon Ultra-15 (#UFC901024, Merck Millipore Ltd.), and the concentrated solution was applied to a qEV2 70 column (Meiwafosis Co., Ltd., Tokyo Japan) in order to isolate the EVs. The EV concentration was measured by using a CD9/CD63 exosome ELISA kit (#EXH0102EL; Cosmo Bio Co., Ltd., Tokyo, Japan).

Introducing miRNAs directly into EVs

293 T cells were cultured in HE100 medium with L-glutamine. The culture medium was concentrated using an Amicon Ultra-15 and the concentrated solution was applied to a qEV2 70 column to isolate the EVs. The EV concentration was measured using a CD9/CD63 exosome ELISA kit. Scrambled or miR-709 mimics were directly transfceted into EVs using the Exo-Fect siRNA/miRNA Transfection Kit (#EXFT200A-1, SBI). 293 T cells (5 × 104 cells/2 mL/well) were seeded into 6-well plates in DMEM with 5% FBS and cultured overnight. 293 T cells were incubated with scrambled or miR-709-EVs overnight and cultured for 2 days after a medium exchange. Proteins were extracted three days after culture with the addition of scrambled or miR-709-EVs.

Western blotting for EVs

EVs (1,000 pg/ 300 μL) were pelletized by using the total exosome isolation reagent. The pellets were dissolved with 50 μL of 4 × Laemmli sample buffer (#1610747, Bio-Rad). Subsequently, 10 μL of each sample (200 pg EVs) were applied to each well and were subjected to Western blotting by using a CD9 monoclonal antibody (Ts9; 1:500; #10626D; Thermo Fisher Scientific), a CD81 monoclonal antibody (M38; 1:500; #10630D; Thermo Fisher Scientific), a GM130 (D6B1) XP® rabbit monoclonal antibody (1:500; #12480; Cell Signaling Technology), a goat anti-mouse Ig/HRP (affinity isolated) antibody #P0447; Dako, Glostrup, Denmark), and a goat anti-rabbit Ig/HRP (affinity isolated) antibody (1:2,000; #P0448; Dako). Extracted proteins were visualized by using the ECL™ prime Western blotting detection reagent (#RPN2232; Cytiva, Marlborough, MA, USA). The images were detected by using Amersham™ Imager 600 (GE Healthcare). Precision Plus Protein™ Dual Color Standards was used as a marker (#1610374, Bio-Rad).

EV size distribution and morphology

The EV size distribution was analyzed by using LM10 NanoSight (Malvern Instruments, UK). The relevant results are shown by standard error (n = 5). The morphology of the EVs was observed with a transmission electron micrograph.

Analysis of miRNA expression levels in miRNA-encapsulating EVs and miRNA directly -transfected EVs

miRNAs were extracted from cells and EVs with the use of miRNeasy mini kits (#217004; Qiagen) and the total exosome isolation reagent. cDNAs were synthesized from miRNAs with the use of the TaqMan™ advanced miRNA cDNA synthesis kit (#A28007; Thermo Fisher Scientific), and qPCR was performed by using the cDNAs with a TaqMan™ fast advanced master mix (Thermo Fisher Scientific) and the following TaqMan advanced miRNA assays (Thermo Fisher Scientific): hsa-miR-186-5p (477940_mir), mmu-miR-709 (mmu482967_mir), and mmu-miR-1247-3p (mmu480903_mir). Quantitative PCR was performed on a StepOnePlus instrument (Thermo Fisher Scientific), and the employed quantitative PCR parameters for cycling were as follows: 95 °C for 20 s, 40 cycles of PCR at 95 °C for 1 s, and 60 °C for 20 s. All reactions were undertaken in 20-μL reaction volumes, in triplicate. The miRNA expression levels were determined by using the 2–ΔCT method.

Analysis of the levels of the FGF2 mRNA after injecting miR-709-containing EVs in hindlimb ischemia model mice

The left femoral artery in 14-month-old BALB/c mice was ligated in one place. At 4 days after the ligation of the left femoral artery, a total of 100 μL of EVs (100 pg) were injected in two places in the femoral muscle. Two days after injecting the EVs, the femoral muscles were removed and were stored at 4 °C, overnight, after being immersed in the RNAlater™ stabilization solution (#AM7020; Thermo Fisher Scientific). Total RNAs were extracted from the femoral muscles with the use of the RNeasy mini kit (#74104; Qiagen). The extracted total RNA was then reverse-transcribed into single-stranded cDNA by using the PrimeScript™ RT reagent kits (Perfect Real Time; TaKaRa Bio Inc.), and qPCR was performed by using cDNAs with the SYBR™ select master mix (#4472918; Thermo Fisher Scientific). The primer sequences used are summarized in Supplementary Table 2. Quantitative PCR was performed on a StepOnePlus instrument (Thermo Fisher Scientific). Quantitative PCR parameters for cycling were as follows: 50 °C for 2 min followed by 95 °C for 2 min, 40 cycles of PCR at 95 °C for 3 s, and 60 °C for 30 s. All reactions were performed in 10-μL reaction volumes, in triplicate. The mRNA expression levels were determined by using the 2–ΔCT method.

FGF2 measurement using ELISA in miRNA-transfected Human aortic endothelial cells (HAoEC)

Human aortic endothelial cells (HAoECs; #C-12271; TaKaRa Bio Inc.) were seeded to 6-well plates (1 × 105 cells in 2 mL/well), and were cultured with (ready-to-use) endothelial cell growth medium MV 2 (#C-22022; TaKaRa Bio Inc) overnight. Subsequently, 100 pmol of miRNA mimics were transfected to the HAoECs, and were left to incubate for 3 days. A total of 300 μL of cold buffer containing 10 mM of Tris–HCl (pH 7.4), 0.5 mM of egtazic acid, 0.5 mM of EDTA, 1% Triton X-100, and cOmplete™ mini protease inhibitor cocktail (Sigma, St Louis, MO, USA) were applied to each well, and the cells were incubated for 30 min on ice and then centrifuged at 13,000 rpm for 20 min, at 4 °C. The supernatants were collected, and their protein concentration was measured by using a Pierce™ BCA protein assay kit (#23227; Thermo Fisher Scientific). After that, 1 μg of protein was applied to each well and the FGF2 concentration was measured by using a human FGF basic/FGF2/bFGF Quantikine HS ELISA kit (#HSFB00D; R&D Systems, Inc., Minneapolis, Minnesota, USA).

FGF2 mRNA expression after the addition of miR-709-EVs in HAoECs

HAoECs were seeded in 6-well plates (6 × 104 cells in 2 mL/well) and were incubated overnight. Subsequently, 1,000 pg of scramble- or miR-709-EVs were added in each well, and were incubated for 8, 24, and 48 h. The expression levels of miR-709, FGF2, and BMX were analyzed by the method described above.

3′UTR luciferase reporter assay

A pmirGLO dual-luciferase miRNA target expression vector (#E1330; Promega) was used for the 3′UTR luciferase assays. The 3′UTR of the human and the mouse GSK3B sequences targeted by miR-709 were predicted based on the RNA22 v2 miRNA target detection software (https://cm.jefferson.edu/rna22/Interactive/). Supplementary Table 3 summarizes the oligo sequences used for constructing the 3′UTR plasmids for the human GSK3B. The plasmids for the 3′UTR luciferase assays were constructed as described previously38. For the 3′UTR luciferase assay, 293 T cells were seeded to 48-well plates (2 × 104 cells in 0.2 mL/well) and were cultured at 37 °C, in 5% CO2, overnight. 293 T cells were then transfected with 40 pmol of the miR-709 mimic and 200 ng of pmirGLO dual-luciferase miRNA target expression vectors with wild-type or mutated target sequences by using 0.67 μL of lipofectamine 2000 (Thermo Fisher Scientific) for each well of a 48-well plate. The luciferase assay was performed at 2 days after transfection by using the dual-luciferase® reporter assay system (#E1910; Promega). The recombinant DNA research was approved by the Genetic Modification Safety Committee of Yamaguchi University (#J20003).

FGF2 and GSK3B protein expression and cell proliferation assay in GSK3B-knockdown HAoECs and miR-709-EVs-treated HAoECs

For GSK3B-knockdown HAoECs, a pSIH1-H1-Puro shRNA cloning and expression vector (#SI500A-1; SBI) was used for the target gene knockdown. Target sequences for knockdown were predicted based on siDirect version 2.0 (http://sidirect2.rnai.jp/) data. Supplementary Table 4 presents the target sequences and the oligo sequences used for the construction of the plasmids. 293 T cells were seeded in 10-cm dishes and were transfected with 6 μg of the pSIH1-H1-Puro shRNA cloning and expression vector and with 4 μg of the pPACKH1 lentivector packaging kit (#LV500A-1; SBI) by using 10 μL of the X-tremeGENE HP DNA transfection reagent (Roche) for constructing pseudoviral particles. HAoECs were incubated with the pseudoviral particles and the TransDux MAX lentivirus transduction enhancer (#LV860A-1; SBI) for 3 days, and were cultured under puromycin dihydrochloride (#A1113803; Thermo Fisher Scientific) for 2 days followed by a further culturing of the cells for the undertaking of the proliferation assay. For miR-709-EVs-treated HAoECs, cells (1 × 104 cells in 2 mL/well) were seeded in a 6-well plate and incubated overnight. Subsequently, 500 pg of scramble- or miR-709-EVs were added in each well and incubated for 3 days. For the determination of the FGF2 and GSK3B protein expression, the protein was extracted by the method described above. Subsequently, 30 μg of the sample were applied to each well and were subjected to Western blotting by using a β-actin (13E5) rabbit monoclonal antibody (1:1,000; #4970; Cell Signaling Technology, Inc.), a GSK-3β (27C10) rabbit monoclonal antibody (1:1,000; #9315; Cell Signaling Technology, Inc.), an anti-FGF2 (EPR20145-219) antibody (1:1,000; #ab208687; Abcam), and a goat anti-rabbit Ig/HRP (affinity isolated) antibody (1:2,000; Dako). For the undertaking of the cell proliferation assay, the cells (5 × 103 cells in 0.2 mL/well) were seeded into a 96-well plate and were incubated for 7 days. Cell proliferation was evaluated by absorbance measurements at 490 nm, through the use of a 2030 ARVO X4 (PerkinElmer, Boston, MA, USA) after using the CellTiter 96® AQueous One Solution Cell Proliferation Assay kit (#G3580, Promega) according to the manufacturer’s instructions.

Statistical analysis

All statistical analyses were performed by using the GraphPad Prism 8 software (GraphPad Software, USA). A p-value of <0.05 was considered as indicative of statistically significant differences.

Ethical approval and consent to participate

All animal procedures were approved by the Institutional Animal Care and Use Committee of Yamaguchi University (#31-101). The methods were carried out in accordance with the approved guidelines.