Regulation of WFS1 by miR-142-3p and THC

To determine the impact of miR-142-3p on WFS1, we overexpressed FAM-labeled locked nucleic conjugated miR-142-3p mimics (Qiagen Inc) in primary human HCN2 neuronal cells (ATCC, USA) as WFS1 protein expression was strong and exclusively expressed in BG neurons. HCN2 cells were cultured in CnT Prime medium (CnT-PR, CELLnTEC Advanced Cell Systems AG, Bern, Switzerland) in 8 well chamber slides (Thermo-Fisher Scientific Waltham, MA USA) at 37 °C in a humidified atmosphere with 5% CO₂. At 50–60% confluency, cells were transfected with 30 nM of FAM-LNA- miR-142-3p or FAM-LNA-negative control mimic using the Lipojet transfection reagent (Signagen, DE). Cells were fixed with 2% paraformaldehyde at 96 h post-transfection and immunostained with WFS1 and later with DAPI for nuclear localization.
To determine the impact of THC and the endocannabinoid mechanisms regulating WFS1 expression, miR-142-3p transfected cells (cultured as described in the previous section) were either treated with 3 µM THC or preincubated with the 10 µM cannabinoid receptor 1 inverse agonist AM251 (Tocris Bioscience, Minneapolis, MN) or the cannabinoid receptor 2 antagonist AM630 (Tocris Bioscience, Minneapolis, MN) for 1 h followed by 3 µM THC treatment and incubation for 18 h. At the end of the incubation period, cells were fixed with 2% paraformaldehyde in PBS for WFS1 immunofluorescence staining.