Dual luciferase reporter kit

miRNA target prediction tools, including miRanda (https://miranda.org.uk/) and TargetScan Release 7.0 (http://targetscan.org/) were used to search for the putative targets of miR-93. The dual-luciferase reporter assay was performed as described previously (32 (link)). Briefly, partial sequences of TLR4 3'-UTR containing miR-93 binding sites was amplified by PCR and cloned into the dual-luciferase reporter vector (pmirGLO; Promega Corporation) (wild-type pmirGLO-TLR4-3'-UTR, wt). Mutation of the predicted binding site (mutation pmirGLO-TLR4-mut-3'-UTR) was generated using the QuikChange Site-Directed Mutagenesis kit (Stratagene; Agilent Technologies, Inc.) according to the manufacturer's instructions. RAW264.7 cells were co-transfected with miR-93 mimics, miR-93 inhibitor and the luciferase reporter plasmids using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). At 48 h post-transfection, luciferase activities were detected using the dual luciferase reporter kit (Beyotime Institute of Biotechnology) with Renilla luciferase activity as an internal control.

TargetScan 7.0 (http://www.targetscan.org) and PicTar (https://pictar.mdc-berlin.de/; release 2007) were used to search for the putative targets of miR-199a-3p. The dual-luciferase reporter assay was performed as described previously (19 (link)). HK-2 cells were transfected with 20 nM miR-199a-3p or inhibitor and the luciferase reporter plasmids using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). After 48 h, luciferase activity was assessed using the dual luciferase reporter kit (Beyotime Institute of Biotechnology). Renilla activity was used to normalize firefly luciferase activity.

Luciferase reporters were generated based on the Firefly luciferase expressing vector pGL3-control (Promega Corporation). The 3′-UTR fragment of the IGF1R gene and its mutant of the theoretical miR-153 binding site were cloned into the pGL3 control vector (Promega Corporation) to form the reporter vector, wild-type (wt) and mutant-type (mut) of IGF1R, respectively. To construct pGL3-IGF1R-3′UTR, a partial 3′UTR of the IGF1R segment of human IGF1R mRNA containing the putative miR-153 binding sites was amplified and cloned into the vector pGL3-control. Mutations within potential miR-153 binding sites were introduced using the QuikChange Site-Directed Mutagenesis kit (Life Technologies; Thermo Fisher Scientific, Inc.). When the Y79 cells grew to 60-70% confluency, the cells were co-transfected with 100 ng luciferase plasmid and 50 ng Renilla luciferase plasmid along with 100 ng miR-153 mimics/inhibitor or miR-NC using Lipofectamine 2000^® (Invitrogen; Thermo Fisher Scientific, Inc.). Following incubation for 48 h at 37°C, the luciferase activity was assessed using the dual luciferase reporter kit (Beyotime Institute of Biotechnology). Renilla activity was used to normalize Firefly luciferase activity.

Lung adenocarcinoma cells (A549 and H1299) were seeded in 48-well plates at the number of 3×10⁴ cells/well and cultured under normal conditions for 24 h, then transfected with 0.2 µg pSTAT3-TA-luc (Beyotime Institute of Biotechnology) and 0.01 µg pRL-TK Renilla (Beyotime Institute of Biotechnology) using Lipofectamine 2000. Twenty-four hours after transfection, the cells were treated with shikonin and IL-6 (50 ng/ml) for 24 h. After treatment, the cells were lysed using passive lysis buffer, and the luciferase activity was detected using a Dual Luciferase Reporter kit (Beyotime Institute of Biotechnology). The experiment was carried out in triplicate and repeated three times independently.