Cell culture
Adipose-derived stem cells (ADSCs) and HUVECs were purchased from Procell Life (Wuhan, China). ADSCs were maintained in stem cell culture medium supplemented with 10% fetal bovine serum and antibiotics (100 units/mL penicillin and 100 mg/mL streptomycin). HUVECs were maintained in endothelial cell basal medium supplemented with 1% endothelial cell growth supplement and 10% fetal bovine serum. The glucose concentration of high glucose medium was 30 mmol/L. The ADSCs were transfected with CRISPR lentivirus-carrying miR-423-5p or miR-negative control (miR-NC) (Shanghai GenePharma Co., Ltd, Shanghai, China). ADSCs, miR-NC-ADSCs, and miR-ADSCs were co-cultured with HUVECs, and the supernatant was collected for enzyme-linked immunosorbent assay (ELISA).
Cell apoptosis analysis
Apoptosis was assessed with an Annexin V-FITC/PI Apoptosis Detection Kit (Beyotime Institute of Biotechnology, Shanghai, China) according to the manufacturer’s instructions. Briefly, after treatment and incubation for 48 h, the cells were collected, washed with PBS, and stained with Annexin V and propidium iodide (PtdIns) in the dark using an Annexin V-FITC apoptosis detection kit (Beyotime Biotechnology, Shanghai, China). Cell apoptosis was subsequently analyzed by FACSCalibur flow cytometry (BD Biosciences, San Jose, CA, USA).
Quantitative real-time PCR
Total RNA was extracted from tissue samples or cells using TRIzol following the manufacturer’s instructions. Total RNA was reverse transcribed into cDNA using a PrimeScript RT reagent kit. Reverse transcription polymerase chain reaction (RT-PCR) was performed using the iQ5 Multicolor Real-Time PCR Detection System (Bio-Rad Laboratories, Inc., Hercules, CA, USA) with SYBR Premix Ex Taq II. The PCR primer sequences for miR-423-5p and U6snRNA were as follows: miR-423-5p forward primer 5'-ACACTCCAGCTGGGTGAGGGGCAGAGAGCGA-3', reverse primer 5'-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGAAAGTCTC-3'; U6snRNA forward primer 5'-CTCGCTTCGGCAGCACA-3', and reverse primer 5'-AACGCTTCACGAATTTGCGT-3'. A melting curve analysis of the amplified products was performed at the end of each PCR cycle. U6snRNA was used as internal control, and gene expression was relatively quantified using the 2-∆∆CT method.
Western blotting (WB)
WB was performed as previously described. Briefly, the cells and rat penises were lysed in lysis buffer containing protease inhibitors. Protein concentrations of the lysates were determined by the bicinchoninic acid assay (Beyotime Biotechnology). Equal amounts of protein (20 µg) were separated by 10% SDS-PAGE and subsequently transferred onto PVDF membranes. The membranes were blocked with 5% non-fat dry milk in 0.2% Tween-20 in Tris-buffered saline (TBS-T) for 1 h at room temperature and then hybridized with primary antibodies. The primary antibodies were mouse anti-eNOS (1:400), mouse anti-VEGFa (1:400), and mouse anti-glyceraldehyde phosphate dehydrogenase (GAPDH, 1:10,000). Immunoreactivity was detected after incubation with a horseradish peroxidase-conjugated secondary antibody according to the manufacturer’s instructions (Thermo Scientific, Waltham, MA, USA). GAPDH was used as loading control. The positive bands were analyzed using Gel-pro analyzer software, and integrated optical density (IOD) was measured.
ELISA
The cell culture medium was collected after coculture, the supernatant was collected by centrifugation for 10 min at 1,500 rpm. eNOS and VEGFa expression levels were measured using an ELISA kit (Solarbio Life Sciences, Beijing, China) according to the manufacturer’s instructions (SEA868Ra for eNOS, SEA143Ra for VEGFa). Absorption at a wavelength of 450 nm (A450) was determined using the microplate reader.
Masson trichrome stain
Masson trichrome staining using a Trichrome Stain (Masson) Kit (Sigma-Aldrich Co., St Louis, MO, USA) was performed to visualize fibers in tissues, following the manufacturer’s instructions. Briefly, the tissue slides were deparaffinized, stained in preheated Bouin’s solution, and washed in running tap water to remove the yellow color from sections. Then, the slides were respectively stained in Working Weigert’s Iron Hematoxylin Solution, Biebrich Scarlet-Acid Fucshin, Working Phosphotungstic/Phosphomolybdic Acid Solution, and Aniline Blue Solution. The stained slides were observed under an optical microscope (magnification 40´).
Immunofluorescene staining
For immunofluorescene staining (IF), the primary antibodies were mouse anti-eNOS (1:400), mouse anti-VEGFa (1:400), secondary antibodies included Alexa-488-conjugated antibodies and Alexa-592-conjugated antibodies (1:500), Nuclear staining was accomplished with 4’,6-diamidino-2-phenylindole (DAPI).
Intracavernosal pressure (ICP) measurement
Erectile function was determined by intracavernosal pressure (ICP) and mean arterial pressure (MAP) 4 weeks post-injection. Under 3% pentobarbital sodium, the major pelvic ganglion (MPG) and cavernous nerves (CN) were exposed by midline laparotomy. The penile was exposed by removing overlying skin and ischiocavernosus muscle. One of the 24-gauge needles that were connected to PE-50 tubes with heparinized saline (250 IU ml−1) was inserted into the left carotid to measure MAP. The other one was inserted into corpus cavernosum (CC) to measure ICP. PE-50 tubes were connected to the data acquisition system (MP150, BIOPAC Systems Inc., Goleta, CA, USA). The CNs were stimulated using a stainless steel bipolar hook electrode with the following parameters: 20 Hz, pulse width of 0.2 ms, 1.5 mA, for 50 s. The ratio of maximal ICP (mm Hg) to MAP (mm Hg) was calculated.
Luciferase reporter assay
PmirGLO-NOS3.3UTR and pmirGLO-VEGFA.3UTR were constructed. The miR-423-5p mimics and negative control sequence were as follows: 5'-UGAGGGGCAGAGAGCGAGACUUU-3' and 5'-UUCUCCGAACGUGUCACGUTT-3'. pRL-TK vector (Takara Biotechnology Ltd., Dalian, China) of Renilla luciferase was used as internal reference for adjusting the differences in cell number and transfection efficiency. Approximately 2 × 104 cells were seeded into a 48-well plate individually and co-transfected with 500 ng pmirGLO-NOS3.3UTR and pmirGLO-VEGFA.3UTR. Subsequently, the cells were transfected with mimics negative control and miR-423-5p mimics, respectively. After 48 h of transfection, the luciferase assay was conducted using a dual luciferase reporter assay (Promega, USA) according to the manufacturer’s instructions.
Statistical analysis
All statistical analyses were performed with SPSS 19.0 (SPSS Inc., Chicago, IL, USA). All results are expressed as the mean ± standard deviation (SD). Multiple comparisons between groups were performed using ANOVA followed by post hoc analysis using the Tukey-Kramer test, whereas a comparison between two groups was performed using a t-test. Differences with a P < 0.05 were considered statistically significant.