Lipofectamine 2000

In order to analyze the function of miR-203 in AKI, pre-miR-203 or pre-control (FulenGen Co., Ltd., Guangzhou, China) was transfected into NRK-52E cells using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. The 3′-UTR of Kim-1 (NM_173149.2) containing the Rattus norvegicus (rno)-miR-203 binding sites or its corresponding mutated sequence was cloned into the psi-CHECK™-2 luciferase reporter vector (Promega Corporation, Madison, WI, USA) downstream of the Renilla luciferase, generating Kim-1–3′-UTR and Kim-1-Mut 3′-UTR vectors, respectively. Subsequently, NRK-52E cells were co-transfected with the recombinant reporter constructs and rno-miR-203 mimics, the rno-miR-203 inhibitor, negative control (NC) or NC inhibitor (GeneCopoeia, Guangzhou, China) using Lipofectamine 2000. Luciferase activity was determined after 48 h using the Dual-Glo^® Luciferase Assay system (Promega Corporation) and an LD 400 Luminometer (Beckman Coulter, Inc.). Data are presented as the ratio of experimental (Renilla) luciferase to control (firefly) luciferase.

The BDH2 gene (NM_020139.3) was cloned into the pReceiver-M03 vector backbone for fusion with the GFP reporter gene, which was obtained from Genecopoeia (USA, EX-A4307-M03). The vector also contained the neomycin resistance gene. The HCC cell lines HepG2 and Hep3B were transfected with the BDH2 plasmid (EX-A4307-M03, Genecopoeia, USA) or control vector (EX-NEG-M03, Genecopoeia, USA) by using Lipofectamine 2000 (cat. no. 11668019, Thermo Fisher Scientific). Two clones of the stably transfected cell lines were used for further experiments.

T24 (1×10⁵ cells/plate) were plated in 6-well plates at 37°C overnight and transfected with miR-124 mimics or NC mimics (20 nM; GeneCopoeia, Inc.) using Lipofectamine^® 2000. The miR-124 mimic sequence used was: Forward, 5′-GCTCTAGAGGCCTCTCTCTCCGTGTTCCACAGCGGACCTTGATTTAAATGTCCATACAATTAAGGCACGCGGTTGAATGCCAAGAATGGGGCTG-3′ and reverse, 5′-CGGGATCCCAGCCCCATTCTTGGCATTCACCGCGTGCCTTAATTGTATGGACATTTAAATCAAGGTCCGCTGTGAACACGGAGAGAGAGGCCT-3′. Following transfection for 6 h at 25°C, the Opti-MEM medium (Gibco; Thermo Fisher Scientific, Inc.) without serum was changed with the fresh medium. Then cells were assayed by RT-qPCR and western blot analysis for each group according to the aforementioned protocols following culture for 48 h.

The pGL3 plasmid (Promega Corporation) containing the RAB34 promoter region element was generated by site-directed mutagenesis and subcloned into the pGL3-basic luciferase reporter vector. A mutant type (MUT) and wild-type (WT) RAB34 promoter vector were produced by GeneCopoeia, Inc.. The Lipofectamine^® 2000 transfection reagent was used to co-transfect the HL-60 cells with 400 ng aforementioned plasmids and 100 nM Oe-E2F1 or the Oe-NC plasmids. After 48 h incubation at 37˚C, the luciferase activity was assayed using a Dual-Luciferase Reporter Assay system (Promega Corporation) according to the manufacturer's protocol. The relative luciferase activity was calculated by normalizing the luminescence intensity of the firefly luciferase activity to that of the Renilla luciferase activity.

At approximately 80% confluence, 143B and HOS cells were harvested and resuspended in serum-free DMEM at a density of 2×10⁵ cells/mL. Sufficient six-well plates were prepared and 1 mL cell suspension added to each well. Lentivector-mediated FLVCR-AS1 shRNAs (shFLVCR-AS1-1, shFLVCR-AS1-2, and shFLVCR-AS1-3) and corresponding FLVCR-AS1 shRNA–negative control (NC) were provided by RiboBio (Guangzhou, China). With Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA), these shRNAs were transfected into 143B and HOS cells and grouped: siFLVCR-AS1-1 group, siFLVCR-AS1-2 group, siFLVCR-AS1-3 group, and si-control (Ctrl) group. At the same time, pcDNA3.1 vectors containing miR381-3p mimics and miR381-3p mimics as NCs (GeneCopoeia, Guangzhou, China) were also used to transfect 143B and HOS cells (set as miR381-3p-mimic group and NC-mimic group, respectively) using Lipofectamine 2000. In addition, 143B cells were subjected to transfection with FLVCR-AS1 vector (FLVCR-AS1 group) and cotransfection with FLVCR-AS1 vector and CCND1 siRNA (FLVCR-AS1 + siCCND1 group) or FLVCR-AS1 vector and miR381-3p mimics (FLVCR-AS1 + miR381-3p-mimic group). Those 143B cells without any treatment served as the Ctrl group. Cells of each group were maintained at 37°C, 5% CO₂ for 6 hours, and cells successfully transfected were harvested for continued culture with DMEM (10% FBS).