HUVEC Transfection and Gene Expression

HUVECs in the logarithmic growth phase were digested, passed, and inoculated into 6-well plates with a density of 5 × 10⁶/well. The cells were transfected when they achieved steady growth. Ribobio Biotechnology Co., Ltd (Guangzhou) took on the design and synthesis of miR-301a-3p mimics, inhibitors, and corresponding negative controls (miR-NC, in-NC). IGF1 overexpression plasmids (IGF1) and the negative control (NC) were purchased from GenePharma (Shanghai, China). IGF1-overexpression plasmids or NC were transfected into HUVECs using Lipofectamine® 3000 (Invitrogen; ThermoFisherScientific, Inc.) as instructed by the supplier. The cells were cultured in an incubator with 5% CO₂ at 37°C. Following 48 hours’ transfection, the original medium was replaced by a fresh and complete one for another 24 hours’ culture. With total RNA extracted from the cells, quantitative real-time polymerase chain reaction (qRT-PCR) was implemented to check alterations in the profile of each molecule in the transfected cells [27 (link)].