Dual luciferase reporter system assay

Dual luciferase reporter (DLR) experiments were performed on the PC12 cells. miR-335 and negative control mimic were purchased from GenePharma Co., Ltd. First, pLUc-FTH1-wild-type (WT) and pLUc-FTH1-mutant (MUT) plasmids (cat. nos. U6885-02 and U6886-02, respectively; Shenzhen Huaan Ping Kang Bio Technology, Inc.) were constructed. Cells were transfected using either WT (200 ng) or MUT (200 ng) constructs, with the miR-335 inhibitor (100 nM), miR-335 mimic (100 nM), mimic negative control (100 nM) or inhibitor negative control (100 nM). At 24 h following transfection, the DLR assay kit (E2920; Promega Corporation) was utilized to detect luminescence, according to the manufacturer's protocol. The absorbance values of sea cucumber luciferase served as an internal reference to perform normalized statistical calculations.