Si zfas1

ZFAS1 pcDNA3.1 vector (ZFAS1), RALY pcDNA3.1 vector (RALY) and empty vector (vector) were subcloned into the vector pcDNA3.1 (Invitrogen, Carlsbad, CA, United States). miR-193a-3p mimic, negative control oligonucleotides (mimic-NC), miR-193a-3p inhibitor, negative control oligonucleotide (NC inhibitor), small interfering RNA of ZFAS1 or RALY (si-ZFAS1, si-RALY) and scramble siRNA of ZFAS1 or RALY (siSCR) were purchased from RiboBio (Guangzhou, China). Cells were transfected using lipofectamine 2000 (Thermo Fisher, CA, United States) following to the manufacturer’s protocols. qRT-PCR was performed at 48–72 h later to determine the transfection efficiency. For further in vivo experiments, sh-ZFAS1 and sh-SCR were obtained from RiboBio (Guangzhou, China) and constructed into HB cell lines.

Cardiomyocytes isolated from 1 to 3-day-old neonatal mice (C57BL/6) were deprived of serum and placed in an anoxic chamber (5% CO₂ and 95% N₂) for 12 h to mimic myocardial ischemia in vitro. The ZFAS1-specific siRNA (siZFAS1), commercially synthesized by Ribobio (Guangzhou, Guangdong, China, sense: GCGUGAACUCCUGAGGCGAdTdT, antisense: UCGCCUCAGGAGUUCACGCdTdT) was transfected into cells for ZFAS1 knockdown according to the manufacturer’s protocol. ZFAS1 transcript cDNA, inserted into the pCDNA3.1 (pCDNA-ZFAS1, ZFAS1-P), were constructed and transfected into cells (2 mg/L) for ZFAS1 overexpression. After transfected with pCDNA-ZFAS1 for 24 h, BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid) (10 μmol/L) was added for Ca²⁺ chelation. ZFAS1-FD, synthesized by Lederer Biological Technology (Guangdong, China, 5′-UGCGUGCCAAGCGCGACAUGGCGCGGAAGCCGAGAAGCCCCGGAGGCCC-3′ was transfected into cells for ZFAS1-FD overexpression. Cyclopiazonic acid (CPA) was added to the NMCMs for SERCA2a inhibition at the concentration of 5 μmol/L. The NMCMs were collected for the following experiments after transfection for 48 h or 72 h.

The siRNA targeting ZFAS1 (si-ZFAS1) was obtained from Ribobio (Guangzhou, PR China), while as control, the mammalian non-targeting siRNA (si-NC) was used. Appropriate siRNA oligos for ZFAS1 were transfected into PC-3 or LNCaP cells by using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer’s protocol. Cells were harvested and examined for PCR analysis to ensure the efficiency of siRNA treatment after 24 h transfection or harvested for transwell assay after 48 h transfection. Detailed information about the sequences of siRNA could be found in Additional file 1: Table S1.