Pcdna meg3

si-MEG3, pcDNA-MEG3, miR-543 mimic, miR-543 inhibitor, si-KLF4, and pcDNA-KLF4 were obtained from Genepharma Company (Shanghai, China). Transfection was performed using Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer’s protocols.

To enhance MEG3 and SOX7 endogenous expressions, the full length of MEG3 and SOX7 sequences was synthesized and inserted into pCDNA3.1 empty plasmid (GenePharma, Shanghai, China), termed as pcDNA-MEG3 (MEG3) and pcDNA-SOX7 (SOX7), respectively. To attenuating MEG3 expression, siRNA against MEG3 (si-MEG3) was synthesized by GenePharma, with nonspecific oligonucleotides (si-NC) as a control. miR-21-5p mimic (miR-21-5p), scrambled oligonucleotides (miR-NC), miR-21-5p antagomirs (anti-miR-21-5p), and antagomirs negative control (anti-miR-NC) were synthesized by GenePharma. Cell transfection with oligonucleotides or plasmids into A549-DDP and H1299 cells was performed using Lipofectamine 2000 (Thermo Fisher Scientific). Cells were collected at 48 h post transfection for further investigations.

The sequences of siRNA targeting lncRNA MEG3 and scrambled negative control siRNA (si-NC) were designed and synthesized by GenePharma (Shanghai GenePharma Co., Ltd., Shanghai, China) according to the previously published protocol (15 (link)). pcDNA-MEG3 and empty vector (pcDNA) were synthesized by GenePharma (Shanghai GenePharma Co., Ltd.). At 60% confluence, HA-VSMCs were cultured in Dulbecco’s modified Eagle’s medium (Gibco; Thermo Fisher Scientific, Inc.) at 37°C with 5% CO₂ for 24 h. HA-VSMCs were transfected with si-NC, si-MEG3 at a final concentration of 20 nmol/l in accordance with the manufacturer’s protocol. Furthermore, HA-VSMCs were transfected with pcDNA-MEG3 at a final concentration of 2 mg/l using. The following sequences were used: si-lncRNA MEG3, 5′-GGGCTTCTGGAATGAGCAT-3′; si-NC, 5′-UUCUCCGAACGUGUCACGUTT-3′ (15 (link)). HA-VSMCs were transfected using Lipofectamine^® 2000 on 6-well plates (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer’s protocol. Cells were harvested for the subsequent experiments 24 h after transfection.