Cell Culture and Grouping

The immortalized RM (Rat microglia) microglial cell line was purchased from the Tongpai Biotechnology (Shanghai, China). It was cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (Gibco, Carlsbad, USA) at 37°C under 95% air and 5% CO₂. In the preliminary experiment, the cells were randomly assigned into the control and the LPS (1 μg/mL) groups. Given previous studies demonstrate that a concentration of 1 μg/mL LPS induces a significant inflammatory response in both immortalized and primary microglia,³⁶ we stimulated RM microglia cells with 1 μg/mL LPS in this study.

In the subsequent cell transfection experiment, the cells were randomly assigned into six groups: control, LPS, LPS + miR-138-5p inhibitor (LPS + inhibitor), LPS + miR-138-5p inhibitor NC (LPS + inhibitor NC), LPS + miR-138-5p mimic (LPS + mimic) and LPS + miR-138-5p mimic NC (LPS + mimic NC). RM cells were seeded in 6-well plates and reached 30–50% confluency before the transfection. According to manufacturers’ instructions (RiboBio Co., Ltd., Guangzhou, China), 100 nM working concentration of miR-138-5p inhibitor, miR-138-5p mimic, miR-138-5p inhibitor NC or miR-138-5p mimic NC was mixed with its riboFECT^tm CP Reagent, respectively; and was added to the 6-well plates. After 72 h post-transfection, the RM cells were stimulated by LPS (1 μg/mL) for 6 h and collected for RNA or protein isolation. The supernatant medium was collected for ELISA.