Hif 1α sirna

For siRNA transfection, Hs578T, MDA-MB-231, and HCC1937 cells were seeded in six-well plates and allowed to grow to 50–70% confluence. Then, the cells were transfected with HIF-1α siRNA or negative control (GenePharma Inc., Shanghai, China) at a concentration of 50 nM using Lipofectamine 2000 reagent (Invitrogen, USA) according to the manufacturer’s instructions. To generate stable MIR210HG-depleted TNBC cells, the pCDH-CMV-MCS-EF1-copRFP lentiviral vector was used. The shRNA sequences were shown as follows: sh-MIR210HG-1, 5′-GCATTAGTACAGGCACCAGCCTA-3′; sh-MIR210HG-2, 5′-UUUAGACCCAUUCUCGUAUGGAGGU-3′. A non-silencing shRNA (sh-Ctrl) oligonucleotide was used as a negative control.

Transient transfection of 4T1 cells was carried out using Lipofectamine 2000 (Invitrogen) as recommended by the manufacturer. HIF‐1α siRNA, overexpression pcDNA3.1 + HIF‐1α vector and their respective negative control oligonucleotides were purchased from GenePharma.

HIF-1α siRNA was purchased from GenePharma Co., Ltd. (Shanghai, China) with the following sequences: sense 5’-CCAUGUGACCAUGAGGAAATT-3’, antisense 5’-UUUCCUCAUGGUCACAUGGTT-3’; negative control (NC): sense 5’-UUCUCCGAACGUGUCACGUTT-3’, antisense 5’-ACGUGACACGUUCGGAGAATT-3’. siRNA transfection was performed with Lipofectamine 2000 Transfection Reagent (Invitrogen) according to manufacturer’s instructions. PC12 cells were transfected with 30 pM siRNA for 48 h, followed by Rox treatment for another 24 h. Then, knockdown efficiency was validated by immunoblot analysis.

The HIF‐1α siRNA, miR‐210 mimics, miR‐210 inhibitors, RUNX3 siRNA and scrambled control sequences were purchased from GenePharma (Shanghai, China). RUNX3 plasmid was generated by GeneChem (Shanghai, China) using the GV208 vector. Cells were transiently transfected with the HIF‐1α siRNA, miR‐210 mimics, miR‐210 inhibitors, RUNX3 siRNA, RUNX3 plasmids or scrambled control sequences using Lipofectamine 2000 according to the manufacturer's protocol, with slight modifications. Specifically, both cells were cultured in six‐well culture plates 24 hrs prior to transfection. Then, 4 μl of Lipofectamine 2000 were incubated with 100 pmol of siRNA/mimics/inhibitors (2 μg plasmids) or negative control sequences in 500 μl of Opti‐MEM for 20 min. at room temperature. Cells were transfected by replacing the medium with 2 ml of Opti‐MEM containing the siRNA/mimics/inhibitors (plasmids) or negative control sequences and Lipofectamine 2000, and incubated at 37°C in a humidified atmosphere of 5% CO₂ for 6 hrs. Then, the Opti‐MEM was replaced with 2 ml of fresh culture medium. 24 or 48 hrs, the cells were incubated with PQ for 24 hrs, collected, and real‐time PCR and Western blotting assays were performed.

sh-MAPKAPK5-AS1 (shRNA#1,2,3) and sh-negative control (sh-control) lentiviruses were purchased from GeneCreate Biological Engineering Co., Ltd. (Wuhan, China). The expression vector pcDNA3.1(+) (MAPKAPK5-AS1) and the empty plasmid pcDNA3.1(+) (Vector) were obtained from Invitrogen Corporation (Carlsbad, CA, USA). Hsa-miR-154-5p mimic (miR10000452–1-5) and hsa-miR-154-5p inhibitor (miR20000452–1-5) were purchased from RiboBio (Guangzhou, China). PLAGL2 Human cDNA ORF Clone (PLAGL2) and PLAGL2 siRNA (si-PLAGL2) were purchased from Technologies, Inc. (USA). The HIF-1α siRNA was purchased from Shanghai GenePharma Co., Ltd. (Shanghai, China). Cell transfection was performed according to the manufactures’ instructions.