Dual luciferase reporter kit

The distal promoter region of miR‐223 was amplified from human genomic DNA according to the method described by Fukao et al. (2007). The amplified fragment was digested by SacI and XhoI and was introduced into pGL4.2[lun2/Puro] (Promega, Madison, WI, USA). The 3′‐UTRs of human TGFBR3 and HMGCS1 were cloned in between the SacI and XhoI sites of pmir‐GLO (Promega) using PCR‐generated fragments. The mutant construct was generated by PCR‐directed mutagenesis using Fast Mutagenesis Kit (TransGen, Beijing, China). SiHa and 293T cells were seeded onto 48‐well plates (~ 1 × 10⁵ cells per well) the day before transfections were conducted. pGL4.2‐miR‐223 (100 ng·well⁻¹) and pRL‐TK (50 ng·mL⁻¹) reporters were cotransfected into SiHa/shSTAT3, SiHa/sh16E6, and the corresponding control cells. Forty‐eight hours later, the luciferase activity was determined using a dual‐luciferase reporter kit (Promega). For the 3′‐UTR assay, miR‐223 overexpression, knockdown plasmids, or control plasmids were cotransfected with pmir‐GLO vector or mutant vectors into 293T cells. Forty‐eight hours later, the luciferase activity was determined by using a dual‐luciferase reporter kit (Promega).

HEK293T cells were cultured in DMEM with 10% FBS. HEK293T cells were seeded at 1 × 10⁵ cells per 24-well. Identification of miR-23a targets was performed by transfecting CHK-wt-CXCR4 or CHK-mut-CXCR4 plasmids (50 ng) and miR-23a mimics (80 ng) or inhibitor (50 ng) into HEK293T cells using Lipofectamine 2000 (11668-027, Invitrogen) in a 24-well plate. Cell lysates were prepared and subjected to luciferase assays using the Dual-Luciferase reporter kit (E1910, Promega) at 48 h after transfection according to the manufacturer’s instruction. For luciferase activity test in mammalian two-hybrid, following the manufacturer’s instructions, both or only one of the pGAL4 and pVP16 fusion constructs (pBIND-TXNIP and pACT-CXCR4-C) were co-transfected with the pG5luc Vector into 293T cells using Lipofectamine 2000 (11668-027, Invitrogen) with a molar ratio of 1:1:1 for pACT-CXCR4-C:pBIND-TXNIP:pG5luc Vector. After incubation for 48 h, luciferase activity was measured using the Dual-Luciferase reporter kit (E1910, Promega). Results were expressed as firefly luciferase activity to Renilla luciferase activity ratio (F/R). The F/R ratio was calculated using the following formula: F/R mean of firefly luciferase activity/mean of Renilla luciferase activity.

In the 3′-UTR luciferase reporter assay, the wild-type (WT) luciferase reporter of the PDCD4 gene was generated by annealing the forward and reverse oligonucleotides and then cloned into pmiR-GLO reporter vector (Promega). Oligonucleotides for the PDCD4 3′-UTR were as follows: WT 5′- AAACTGTGCTAATTTAAACTGCCAAATT-3′ (forward) and 5′-CTAGAATTTGGCAGTTTAAATTAGCACAGTTT-3′ (reverse). Anti-miR-182-stable MDA-PCa-2b cells were transfected with a pmiR-GLO vector containing the 3’UTR sequences and luciferase activity was measured with the Promega Dual-Luciferase Reporter kit (Promega) according to the manufacturer’s protocols after transfection. Relative Renilla luciferase activity was normalized to firefly luciferase activity.