Pcdna3

The design and construction of shRNAs and si-RNA for ACTA2-AS1 and BCL2L11, the synthesis of pcDNA 3.1, miR-4428 mimics vector, and the construction of a lentiviral vector overexpressing ACTA2-AS1 and BCL2L11 were separately conducted by Genechem (Shanghai, China). The transfection was carried out using Lipofectamine 2000 reagent (Invitrogen, USA) according to the guideline of manufacturer. The sequences of genes were as followed:
si-ACTA2-AS1#1: 5′-UAGAUUAUUAUGUCUUCCCAG-3′
si-ACTA2-AS1#2: 5′-UAGUAAAGCAACAUUCUUGGA-3′
si-BCL2L11: 5′-UUAAAUAACGUGAACAUGCUG-3′
miR-4428 mimics: 5′-GUUCCUCUGCCCUUGUACCUCG-3′

Mouse E2F4 gene was cloned into the vector pCDNA3.1 (Genechem Co., Ltd., Shanghai, China). For transfection experiments, the chimeric genes of the Shh promoter plasmid were constructed in a GV148-basic vector (Genechem Co., Ltd., Shanghai, China) by ligating the 5′-flanking regions of the mouse Shh gene upstream of the luciferase gene. Mouse MEF cells were plated into 24-well cell culture plates 1 day before transfection. The transfections included 1 µg each of pCDNA3.1-basic and GV148-basic; E2F4 over-expressing pCDNA3.1 and GV148-basic; E2F4 over-expressing pCDNA3.1 and GV148-mutant; E2F4 over-expressing pCDNA3.1 and GV148-Shh promoter; E2F4 over-expressing pCDNA3.1 and GV148-mutant, E2F4 over-expressing pCDNA3.1 and GV148-Shh promoter. These were respectively co-transfected with Firefly luciferase (Fluc) Renilla luciferase (Rluc) into mouse MEF cells using X-treme GENE HP DNA Transfection Reagent (Cat#6366236001, Roche Diagnostics Corp., Switzerland) according to the manufacturer's protocol. Two days later, the promoter-driven luciferase activity was measured using the Dual-Luciferase® Reporter Assay system (Cat# E1910, Promega Corporation, Madison, WI, USA).

Short hairpin RNA (shRNA) targeting LINC00662 (sh-LINC0062; 5′-GCUGCUGCCACUGUAAUAATT-3′) and nontargeting shRNA negative control (sh-NC) were purchased from Sangon Biotech Co., Ltd. (St. Louis, USA). MiRNA-16-5p mimics and negative control mimics (NC mimics) were synthesized at GenePharma (Shanghai, China). The pcDNA3.1 vector containing ITPR1 (pcDNA3.1/ITPR1) and the empty vector (pcDNA3.1) were obtained from Genechem (Shanghai, China). Cell transfection was performed using Lipofectamine 3000 (Thermo Fisher Scientific, Inc., USA). The transfection efficiency was examined by RT-qPCR 48 h later.

The pcDNA3.1 vector was used to generate MIR503HG-overexpressing vector, and the empty vector, pcDNA3.1, and MIR503HG-overexpressing vector (pcDNA3.1-MIR503HG) were purchased from GeneChem (Shanghai, China). The siRNAs targeting MIR503HG (si-MIR503HG) and the scrambled negative controls (si-NC) were purchased from RiboBio (Guangzhou, China). For plasmid and siRNA transfection, trophoblast cells of a confluent cell monolayer were transiently transfected with plasmids (20 μg) or siRNAs (100 nM) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instruction. At 24 h after transfection, cells were processed for further experimentation.