Non targeting sirna

miR-155-5p mimics and inhibitor were bought from RiboBio (Guangzhou, China). MGP siRNAs and nontargeting siRNA were purchased from Gene Pharma (Shanghai, China). According to the manufacturer's protocol, 50 nM of miR-155-5p mimics and 100 nM of miR-155-5p inhibitor were transfected into the HK-2 cells by Lipofectamine 3000 (Invitrogen, China) in a 6-well cell plated. 100 nM of MGP siRNA was used to knockdown endogenous MGP following the manufacturer's instructions.

Gene-specific siRNAs and one non-targeting siRNA were purchased from GenePharma Co. ATG5#1 and ATG5#2 siRNAs target the sequences CCUUUG GCCTAAGAAGAAA and CAUCUGAGCUACCCGG AUA, respectively; ATG7#1 and ATG7#2 siRNAs target the sequences GGAGUCACAGCUCUUCCUU and CAGCUAUUGGAACACUGUA, respectively; Beclin1#1 and Beclin1#2 siRNAs target the sequences GGAA GCUCAGUAUCAGAGA and CAGUUUGGCACAAU CAAUA, respectively; LKB1#1 and LKB1#2 siRNAs target the sequences UGAAAGGGAUGCUUGAGUA and GAAGAAGGAAAUUCAACUA, respectively; AMPKα1 siRNA targets the sequence GAGGAGAGCUAUUUG AUUA; AMPKα2 siRNA targets the sequence GCUGU UUGGUGUAGGUAAA; ULK1 siRNA targets the sequence GCCUGUUCUACGAGAAGAA; while the sequence of the non-targeting control siRNA was 5′-UUCUCCGAACGUGUCACGUTT-3′.

The siRNA targeting human Wnt1 and non-targeting siRNA were purchased from GenePharma Co., Ltd (Shanghai). Transfections were performed using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer’s instructions. For the RNA interference study, we tested three siRNAs designed using the target gene sequences and selected the siRNA that resulted in the greatest inhibition of the target protein.

Human umbilical vein endothelial cells (HUVECs) were purchased from ScienceCell Research Laboratories (USA) and cultured according to the standard guideline. HUVECs were cultured in endothelial cell medium (1001, ScienceCell Research Laboratories) supplemented with 5% foetal bovine serum, endothelial cell growth supplement and antibiotic solution.
Human embryonic kidney 293T cells were purchased from ATCC (Manassas, VA, USA) and maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% foetal bovine serum (Gibco, North America), 100 U/ml penicillin and 20 U/ml streptomycin. Cells were incubated at 37°C in a humidified chamber containing 5% CO₂.
The adenovirus‐encoding IMP3 (ad‐IMP3) was constructed and amplified by Boi‐Link (Shanghai, China). An adenovirus‐encoding red fluorescence protein (ad‐RFP) was used as a negative control. HUVECs were transfected with ad‐IMP3 or ad‐RFP (100 pfu number/cell) for 48 h for the following experiments.
Si‐IMP3 was purchased from GenePharma Company (Shanghai, China); a non‐targeting siRNA (GenePharma, Shanghai, China) was used as a negative control. HUVECs were transfected using Lipofectamine 3000 reagent (Invitrogen, USA) according to the manufacturer's instructions.
Axitinib was resuspended in DMSO and HUVECs treated with 0.5 μg/ml Axitinib.¹⁹