RNA Interference

The target shRNAs against the human Tph1, 5-HT_2AR and 5-HT_2BR genes (Genbank access numbers: {"type":"entrez-nucleotide","attrs":{"text":"NM_004179","term_id":"1717508376","term_text":"NM_004179"}}NM_004179, {"type":"entrez-nucleotide","attrs":{"text":"NM_000621","term_id":"377520130","term_text":"NM_000621"}}NM_000621, {"type":"entrez-nucleotide","attrs":{"text":"NM_000867","term_id":"1519244514","term_text":"NM_000867"}}NM_000867, respectively) for RNA interference were designed as follows, Tph1: 5'-CCG GCC CAA GAA ATT GGC TTG GCT TCT CGA GAA GCC AAG CCA ATT TCT TGG GTT TTT-3', 5-HT_2AR: 5'-CCG GGC CTA CAA GTC TAG CCA ACT TCT CGA GAA GTT GGC TAG ACT TGT AGG CTT TTT-3', 5-HT_2BR: 5'-CCG GCC GAT ATA TCA CCT GCA ATT ACT CGA GTA ATT GCA GGT GAT ATA TCG GTT TTT-3', and the lentiviral vector GV248-negative (control siRNS): TTC TCC GAA CGT GTC ACG T without interference suppression on the expression of human gene. Target fragment was annealed by 3 'and 5' single strand and digested by AgeI and EcoRI restriction enzymes, then it was connected to the GV248 vector (GeneChem, China). After sequencing identification, except for GV248-vector, GV248-Tph1 alone or in a combination with GV248-5-HT_2AR and GV248-5-HT_2BR were used to transfect 293T packaging cells by Lipofectamine 2000. The supernatant was collected after 48 h and filtered through a 0.45 μm filtrate membrane, then Polybrene was add into it until the final concentration was 5 μg/mL. Supernatant was used to infect target cells for 12 h, and then placed into new medium for another 72 h. After infection, fluorescence of green fluorescent protein (GFP) was detected via fluorescence inversion microscope system (Olympus, Japan), and Tph1, 5-HT_2AR and 5-HT_2BR protein expressions were detected by western blot. Infected HepG2 cells were then transferred to DMEM supplemented with 10% FBS in a six-well plate, further incubation with Dex or sodium palmitate (PA) treatment as same as HepG2 cells culture.