Control mirna

HK2 cells (American Type Culture Collection, Manassas, VA, USA) were cultured in 10% foetal bovine serum (Gibco, USA)-containing Dulbecco’s modified Eagle’s medium/F12 (DMEM/F12) complete medium (Corning, USA). When the cells reached 50% confluence, they were synchronised overnight in serum-free DMEM/F12 medium, which was then replaced with complete medium containing 8 ng/ml recombinant human TGF-β1 (PeproTech Inc., USA), before the cells were cultured for 48 h and 72 h to induce fibrosis. miR-294, miR-133 and control miRNA (GeneCopoeia Inc., Rockville, MD, USA) were transfected into synchronised HK2 cells in the miRNA transfection groups with the jet-PRIME® transfection reagent (Polyplus Transfection, Inc., USA) according to the manufacturer’s instructions. After 24 h of transfection, the medium was replaced with complete medium containing 8 ng/ml recombinant human TGF-β1, and the cells were incubated for an additional 48 h and 72 h.

HPTCs cells (10×10⁵) were seeded in six-well plates and cultured for 18h to 40–50% confluence. 50 nmol/L Small interference RNA (siRNA)-HIF-1α (Santa Cruz, CA, USA) was mixed with 16μL transfection reagent (INTERFERin®) and diluted with 200 μL transfection buffer (OPTi-MEM). While for miRNA Transfection, 50 nmol/L miR-21 mimic or 100 nmol/L antisense miR-21 inhibitor or 50 nmol/L control miRNA (GeneCopoeia, Guangdong, China) was mixed with 8μL transfection reagent (jetPRIME™) and diluted with 200μL transfection buffer (jetPRIME™ buffer). After incubation for 15 min at room temperature, the mixture was added to the 2 mL DMEM/F12 medium, transfection medium replaced by new DMEM/F12 medium 24h after transfection and the cells were cultured for an additional 24 or 48h.

The DNA fragment for miR-135a, control miRNA, and siRNAs were obtained from Gene Copoeia, Inc and inserted into lentiviral expression plasmids. The human renal cancer cell lines ACHN and A498 were cultured in appropriate media with 10% FBS supplementation for 24 hours before transfection. The transfection procedure involved adding miR-135a, miR-135a inhibitor, and control viruses to the culture medium. After transfection, cells were cultured in media supplemented with 10% FBS and 0.5 μg/mL puromycin for selection.