Mir 148a 3p mimic

For transient transfection of miRNAs, Cells were seeded at 1 × 10⁵ per well in 12‐well plates and cultured for 24 h. Cells were transfected with miR‐148a‐3p mimics (GenePharma, Shanghai, China), or the negative controls at a final dose of 25, 50 nM, using Lipofectamine RNAiMAX transfection reagent (Invitrogen, USA) and in serum‐free Opti‐MEM medium (GIBCO, USA). After 6 h, the medium was replaced with RPMI containing 1% penicillin/streptomycin and 10% FBS.
For silencing specific gene expression, the synthesis of siRNAs was completed by GenePharma company, and transfection of siRNAs targeting ATP7A, ALCAM, NRP1, SMAD2, and SPIN1, as well as nontargeting control was carried out according to the previous protocol of miR‐148a‐3p. Total RNAs and proteins were extracted after 48 h of transfection. Knockdown efficiency of ATP7A was measured using Q‐PCR and western blot. While, the knockdown efficiency of other genes was assessed by PCR.
For enforced expression of ATP7A, ATP7A open reading frame was amplified to the pcDNA3.1(+) expression vector, and the empty plasmid pcDNA3.1(+) was used as a control. Lipofectamine 2000 (Invitrogen) was utilized to transfect the above plasmids into cells. The sequences of siRNA or detection primers are listed in Supporting Information Materials and Figure legends.

RAW264.7 cells were randomly divided into NC (miR mimic) group, miR mimics group, NC (miR inhibitor) group, miR inhibitor group, LPS + NC (miR mimic) group, LPS + NC (miR inhibitor) group, LPS+ miR inhibitor group and LPS + miR mimics. The MiR-148a-3p mimic, miR-148a-3p inhibitor and their negative controls were transfected into RAW264.7 macrophages using Lipofectamine 2000 Transfection Reagent (Invitrogen, USA) according to the manufacturer's instructions. Twenty-four hours after transfection, cells were used for further experiments. MiR-148a-3p mimic and its negative control miR-NC, miR-148a-3p inhibitor and its negative control miR-NC were purchased from Shanghai GenePharma Co. The cells were collected 4 h later for LPS (1 μg/mL) stimulation for RT-PCR and 24 h for western blotting.