Pcdna3.1 nc

miR-28-3p-inhibitor, small interfere (si) RNA targeting NORAD (si-NORAD), plasmid overexpressing E2F2 (pcDNA3.1-E2F2), and the corresponding controls (miR-28-3p-negative control (NC), si-NC, and pcDNA3.1-NC) (all from RiboBio Co., Ltd, Guangzhou, Guangdong China) were transfected into cells by Lipofectamine 2000 (Invitrogen Inc., Carlsbad, CA, USA) following the manufacturers’ instructions. After 48 h of transfection, all cells were preserved for further analysis.

Plasmid vector pcDNA3.1-WRN and its negative control (pcDNA3.1-NC) were constructed by RIBOBIO (Shanghai, China) via standard molecular cloning approaches. Small interfering RNA (siRNA) specific targeting WRN (WRN siRNA) and the corresponding NC (Scramble) were purchased from RIBOBIO. These vectors were transfected into SRA01/04 cells separately. The cell transfection assay was performed using Lipofectamine 2000 Transfection Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol.

The cells were divided into the control group, the LPS group, the LPS + mimic-NC group, the LPS + miR-499a-5p group, the LPS + miR-499a-5p + oe-NC group, and the LPS + miR-499a-5p + oe-EIF4E group. The plasmids of mimic NC, miR-499a-5p-mimic, pcDNA3.1-NC, pcDNA3.1-EIF4E were provided by Ribobio Co, Ltd. (Guangdong, China). The AC16 cells were seeded in 6-well plates at a concentration of 5 × 10⁵/cm². After 24 h of culture, the AC16 cells were transfected with the aforementioned plasmids for 24 h using Lipofectamine 2000 (Invitrogen). The total RNA content was extracted using the RNA separation kit (Takara, Dalian, China). The transfection efficiency was estimated by means of reverse transcription quantitative polymerase chain reaction (RT-qPCR).

Human breast cancer cell lines MDA-MB-231, ZR-75-1, MDA-MB-468, BT-474, MCF-7, and a normal breast epithelial cell line MCF-10A were obtained from the American Type Culture Collection. All cell lines were cultured in RPMI 1640 medium (Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco) in a humidified 5% CO₂ atmosphere at 37°C. The small interfering RNAs (siRNAs; si-NC, si-RP11-70C1.3), short hairpin RNA (shRNA; sh-NC, sh-RP11-70C1.3), miR-6736-3p mimics, miR-6736-3p inhibitor, miR-NC, and overexpression plasmids (pcDNA3.1-NC, pcDNA3.1-NRP-1) were synthesized by RiboBio (Guangdong, China). Transfections were conducted with 50 nM oligonucleotides or 100 nM plasmids using lipofectamine RNAiMAX (Invitrogen). Cells were harvested 24 or 48 h after transfection for further study.

Rat PMVECs (r PMVECs) were placed in a hypoxic incubator containing 95% N₂ and 5% CO₂ for 12 h under hypoxic conditions. Next, the culture medium was replaced with DMEM containing glucose, 1% penicillin-streptomycin, 10% FBS, and 4 mM L-glutamine for 4 h to construct the LIRI cell model. The cells were collected for subsequent experiments. For the LIRI + KGF-2, 20 mM KGF-2 (Libang Sciences) was added prior to the establishment of the LIRI cell model. NLRP1 overexpression plasmid (pcDNA3.1-NLRP1) and pcDNA3.1 NC were synthesized by Guangzhou RiboBio Co., Ltd. (Guangzhou, Guangdong, China). Transfection was performed using Lipofectamine® 3000 (50 nM; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol.

The shRNAs (GenePharma, Shanghai, China) specific to LINC01152, MAML2, SRSF1, ADAR, RBPJ, and the NC-shRNAs were transfected into T98G and U343 cells as per protocol of Lipofectamine 3000 (Invitrogen). Besides, miR-466 mimics/inhibitor and NC mimics/inhibitor, together with pcDNA3.1-LINC01152, pcDNA3.1-MAML2, pcDNA3.1-RBPJ and pcDNA3.1-NC were procured from RiboBio (Guangzhou, China). After 48 h of transfection, cells were all reaped.