Pcdna3.1 malat1

The pcDNA3.1-MALAT1, MALAT1 siRNA (si-MALAT1) and their corresponding controls were purchased from Shanghai GenePharma Co., Ltd. H9C2 cells (2×10⁵ cells/ml) were transfected at 37°C for 24 h with Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) as previously described (20 (link)). Cells were divided into the following groups: i) NG, cells were cultured in normal glucose (5.5 mM) for 24 h; ii) HG, cells were cultured in high glucose (30 mM) for 24 h; iii) HG + MALAT1 overexpression negative control (NC1), cells were transfected with 50 nM control non-targeting adenovirus vector for 24 h, then cultured in HG medium for 24 h; iv) HG + MALAT1 overexpression (MALAT1), cells were transfected with 50 nM pcDNA3.1-MALAT1 for 24 h, then cultured in HG medium for 24 h; v) HG + MALAT1 siRNA scramble control (NC2) cells were transfected with 50 nM MALAT1 siRNA scramble control (5′-UUCUCCGAACGUGUCACGUTT-3′) for 24 h then cultured in HG medium for 24 h; and vi) HG + MALAT1 siRNA (si-MALAT1), cells were transfected with 50 nM MALAT1 siRNA (5′-GATCCATAATCGGTTTCAA-3′) for 24 h then cultured in HG medium for 24 h. Transfection efficiency was assessed via RT-qPCR.

Cell transfection was performed before cultured cells were grouped into four groups: pcDNA3.1 (empty vector pcDNA3.1), pcDNA3.1-MALAT1 (to induce MALAT1 overexpression), si-MALAT1 (to induce MALAT1 knockdown), and si-NC (control for MALAT1 knockdown).
The following sequence of si-RNA oligonucleotides (si-MALAT1) was used to knockdown MALAT1 expression: 5ʹ-CACAGGGAAAGCGAGUGGUUGGUA-3ʹ. The sequence of the noncoding control siRNA (si-NC) was 5ʹ-UUCUCCGAACGUGUCACGU-3ʹ. si-NC and si-MALAT1 were synthesized by Shanghai Sangon Biotechnology Co., Ltd. (Shanghai, China). pcDNA3.1 and pcDNA3.1-MALAT1 were purchased from Shanghai GenePharma Co., Ltd. (Shanghai, China).Cell transfection was performed by introducing 100 nM of miR-503-5p mimic, 100 nM of miR-503-5p inhibitor, mimics negative control (NC), inhibitor negative control, si-MALAT1, si-NC, pcDNA3.1, or pcDNA3.1-MALAT1 into cells for incubation at 37°C in a humidified chamber with 5% CO₂ for correspondently 24, 48, and 72 hrs. Lipofectamine^® 2000 transfection reagent (Invitrogen; Thermo Fischer Scientific, Inc.) was used according to the manufacturer’s protocol. After transfection for 48 hrs, RT-qPCR was performed to assess the transfection efficiency of MALAT1 knockdown and overexpression.