Cell culture
HUVECs were seeded onto cell culture flasks (Corning, Coring, NY, USA) and grown in complete medium (endothelial cell medium (ECM; Sciencell, San Diego, California, USA) supplemented with 5% heat-inactivated fetal bovine serum (FBS; Sciencell), 1% endothelial cell growth supplement (ECGS; Sciencell), and 1% penicillin and streptomycin (Invitrogen, Carlsbad, CA, USA)) at 37°C and 5% CO2 in a humidified incubator.
Cell transfection
Cultured HUVECs were transfected with a chemically synthesized miR-328a-3p mimic, mimic control, miR-328a-3p inhibitor, inhibitor control sequence (RiboBio, Guangzhou, Guangdong, China) using Lipofectamine RNAiMAX transfection reagent (Invitrogen) according to the manufacturer’s instructions. The sequences of miR-328a-3p mimic were 5’-CUGGCCCUCUCUGCCCUUCCGU-3’ and 3’-ACGGAAGGGCAGAGAGGGCCAG-5’, the sequence of miR-328a-3p inhibitor was 5’-ACGGAAGGGCAGAGAGGGCCAG-3’, and the sequences of non-targeting negative controls were random sequences. HUVECs were transfected for 48 hours prior to functional investigations.
Real-time RT-PCR
Total RNA was isolated from cultured HUVECs, reverse transcribed to cDNA using the TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA), and subjected to RNA amplification using the QuantiNova SYBR Green PCR Kit (Qiagen) on an Applied Biosystems StepOne real-time PCR system. Bulge-loopTM miRNA qRT-PCR Primer Sets (one RT primer and a pair of qPCR primers for each set) specific for miR-328a-3p was designed by RiboBio. The abundance of miR-328a-3p in HUVECs was compared with the internal control U6 and the quantification of miR-328a-3p was determined using the ΔΔCt method.
EdU proliferation assay
HUVECs were seeded onto cell culture plates pre-coated with poly-L-lysine, exposed with 5-ethynyl-2’-deoxyuridine (EdU) for 24 hours, and fixed with 4% paraformaldehyde. Cell proliferation was determined by using a Cell-Light EdU DNA Cell Proliferation Kit (RiboBio) following manufacturer’s instructions. Images were taken using a DMR fluorescence microscope (Leica Microsystems, Bensheim, Germany). The numbers of EdU-postive cells and total cells were determined by EdU and Hoechst staining. The proliferation rates of HUVECs were calculated by dividing the numbers of EdU-postive cells to the numbers of total cells.
Wound healing assay
HUVECs were seeded onto a mold chamber with a 1 mm wide insert placed on the bottom of a 6-well plate. After cell grew confluent, placed insert was removed, leaving an blank space. Images were taken using a DMR inverted microscope (Leica Microsystems) at 0 hour and 12 hours after the removal of placed insert. Relative cleaned areas were measured at these time points using Image-Pro Plus (Media Cybernetics, Rockville, MD, USA) from randomly selected image fields.
Transwell migration assay
Transwell chambers with 8 μm pores (Costar, Cambridge, MA, USA) were applied to determine migration abilities. The upper chambers of transwell chambers were filled with HUVECs suspended in ECM medium while the lower chambers were filled with complete cell culture medium. The upper chambers were taken out after 24 hour incubation. After wiping the upper surfaces of the upper chambers and removing cells left on the upper surfaces, the bottom surfaces of these upper chambers were stained with 0.1% crystal violet to label migrated cells. Images were taken using a DMR inverted microscope (Leica Microsystems). Crystal violet was dissolved in 33% acetic acid and the optical densities (O.D.) of crystal violet were measured at 570 nm (Bio-Tek).
Matrigel tubulogenesis assay
HUVECs were seeded onto cell culture plates pre-coated with matrigel (BD Biosciences, Corning) and incubated for 6 hours to allow the formation of tubules. Images were taken using a DMR inverted microscope (Leica Microsystems). The numbers of nodes, meshes, and branches in formed tubules were quantitated using Angiogensis analyzer in the Image J software (National Institutes of Health, Bethesda, MD, USA) (23).
Bioinformatic analysis
Upstream regulatory lncRNAs of miR-328a-3p were predicted using TargetScan. Downstream target mRNAs of miR-328a-3p were predicted using miRWalk 3.0, miRranda, and MicroRNA Target Prediction Database (miRdb). Upstream lncRNAs and downstream mRNAs were linked to construct the miR-328a-3p-centered competing endogenous RNA (ceRNA). Heatmaps of lncRNAs and mRNAs were generated using meV software according to previous obtained sequencing data of sciatic nerve stumps at 0, 1, 4, 7, and 14 days after nerve crush injury (24). Sequencing data were reserved in NCBI database with the accession number PRJNA394957 (SRP113121) (25).
Statistical analysis
A student’s paired two-tailed t-test was performed for statistical analysis. The calculation of p-values and the generation of graphs were made with SigmaPlot (GraphPad Prism 6.0 software, GraphPad Software, La Jolla, CA, USA).