Lipofectamine 2000 reagent

Two human OS cell lines U2OS and MG-63 (ATCC, USA) were used in this study. Cells were cultivated in the mixture of 90% Eagle’s minimum essential medium and 10% FBS. Cell culture conditions were 37°C, 95% humidity and 5% CO₂. U2OS and MG-63 cell were harvested at confluence of 70-80% to perform cell transfections. Negative control (NC) miRNA and miR-522-3p, NC siRNA and GLUT1 siRNA, as well as GLUT1-expression pcDNA3.1 vector and empty pcDNA3.1 vector were all from Sangon (Shanghai, China). Transient transfections were mediated by lipofectamine 2000 reagent (Sangon) to transfect 40 nM miRNA (NC miRNA as NC group), 40 nM siRNA (NC siRNA as NC group), or 10 nM vector (empty vector as NC group) into 106 (link) cells. Subsequent experiments were performed using cells harvested at 24 hrs post-transfection. Control (C) cells for all transfections were untransfected cells.

Saos-2 cells and MG-63 cells were seeded in 24-well plates. SiRNA (si-RUSC1-AS1) or of RUSC1-AS1 overexpression plasmids, Notch1 overexpression plasmids, or their negative controls (100 nM, GenePharma, China) were respectively transfected into Saos-2 cells and MG-63 cells to establish the RUSC1-AS1 knockdown or overexpression model. miR-101-3p mimics and the corresponding negative control (NC) (Guangzhou Ribobio) were used to induce miR-101-3p overexpression model. Lipofectamine 2000 reagent (Sangon Biotech) was applied for transfection. 24-hour after transfection, the culture was exchanged with new fresh medium and the cells were continuously incubated for 24 h. Then qRT-PCR was performed to detect RUSC1-AS1, Notch1 and miR-101-3p to ensure the transfection efficiency.

The DNA sequence of the entire human VEGFA 3′-UTR was amplified by polymerase chain reaction (PCR). The amplified DNA sequences were inserted into the p-MIR-report plasmid (Sangon Biotech, Shanghai, China). For luciferase reporter assays, 293T cells were cotransfected with an individual reporter gene (pmiR-Luc-VEGFA-WT, pmiR-Luc-VEGFA-MUT1, or pmiR-Luc-VEGFA-MUT2), a control, or miR-4306 mimics using TransIT-X2 transfection reagent (Invitrogen).
The pGL4.74(hRluc/TK) plasmid, a control luciferase plasmid, and the pGL4.32(luc2P/NF-κB-RE/Hygro) plasmid (Invitrogen) were transfected using Lipofectamine 2000 reagent (Sangon Biotech). A Renilla plasmid (Invitrogen) was used as an internal control for all transfection assays. After transfection for 48 hours, the activities of luciferase and Renilla were tested using a standard protocol (Sangon Biotech).