Lipofectamine 2000

An epigenetic modifier, 5-aza-2'-deoxycytidine (5-aza-dC) and the MTT kit were purchased from Sigma-Aldrich (USA). Two chemically synthesized double-stranded RNA oligonucleotides (miR-34a mimics and mimics NC) were purchased from Ribo Biotech, Ltd (Guangzhou, China). MiRNA mimics can simulate high-level expression of mature miRNA in cells to enhance the regulation of endogenous miRNAs. Transfection reagent Lipofectamine^TM 2000 was purchased from Ribo Biotech, Ltd. (China). The cell count before transfection was 3.0×10⁵ cells/35 mm petri dish. The small RNA molecules labeled with Cy3 dye were diluted with 200 µL of Opti-MEM (Gibco), and then 2 µL of the transfection reagent was well mixed with 200 µL Opti-MEM. The mixture was let stand for 3 to 5 minutes, and the two substances were well mixed and then let stand for 15 minutes, to make a 400 µL mixture (the Cy3-labeled small-molecule RNA mother liquor was 20 µM; 2 µL was used, and the final transfection concentration was 50 nM). The culture medium in the 35-mm Petri dish was discarded, 400 µL of fresh medium was added, and then 400 µL of the above-mentioned mixture was added, to make up 800 µL of mixture. After 24 hours of transfection, it was washed with lead sulfide (PbS), added with 1,500 µL fresh medium, and observed under a microscope.

U-87MG (U87) glioblastoma cell line of unknown origin was obtained from the American Type Culture Collection (cat. no. HTB-14). U87 cells were cultured in Dulbecco's modified Eagle's medium (Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.). Small interfering RNA (siRNA) targeting human GPNMB (5′-GGATAATACTGGCCTGTTT-3′) and a negative control siRNA (5′-GGCTCTAGAAAAGCCTATGC-3′) were purchased from Guangzhou RiboBio, Co., Ltd. A total of 5×10⁵ U87 cells were seeded in 6-well plates overnight and then transfected with 50 nM siRNA using Lipofectamine^® 2000 (cat. no. 11668-019; Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C for 6 h according to the manufacturer's instructions. Subsequent experiments were conducted 24 h post-transfection.

Short hairpin (sh)RNAs synthesized against SNHG9 (sh-SNHG9) and matched negative control sequences (sh-NC) were subcloned into the pGPU6/GFP/Neo vector (Shanghai GenePharma Co., Ltd.). Subsequently, SKOV3 cells were transfected with 100 pmol sh-SNHG9 or sh-NC using Lipofectamine^® 2000 (Thermo Fisher Scientific Inc.), according to the manufacturer's protocol. Stable cell clones were selected using 0.5 mg/ml of G418 (Sigma-Aldrich; Merck KGaA). Thereafter, the cells were transfected with 0.1 nmol miR-214-5p mimics, negative control (NC) mimics (miR-NC), miR-214-5p inhibitors (anti-miR-214-5p) or anti-miR-NC (Guangzhou RiboBio Co., Ltd.) using Lipofectamine^® 2000, according to the manufacturer's protocol. The sequences of miR-214-5p mimics are as follows: Sense, 5′-UGCCUGUCUACACUUGCUGUGC-3′ and antisense, 5′-ACAGCAAGUGUAGACAGGCAUU-3′. The sequences of miR-214-5p inhibitor are as follows: Sense, 5′-GCACAGCAAGUGUAGACAGGCA-3′. Subsequent experimentation was performed 48 h post-transfection.