Mir 130a 3p mimic

The miR‐130a‐3p mimic and negative control (NC) mimic were purchased from RiboBio (Guangzhou, China). RiboBio (Guangzhou, China) provided SMAD4 siRNA (Si‐SMAD4) and control (Si‐NC). We subcloned the full‐length SMAD4 into the pcDNA3.1 plasmid to increase the expression level of SMAD4, and the control plasmid was pcDNA3.1 empty vector. EC‐1 cells were seeded in 6‐well plates (1.5 × 10⁹ cells per well) under the action of Lipofectamine 2000 (Invitrogen, Carlsbad, CA) conferring to the manufacturer's protocol for 6 hours. After transfection for 48 hours, the cells were collected for western blot analysis or qPCR assays.

To overexpress circGRAMD1B, a 285 bp fragment of circGRAMD1B was cloned into the pHBLV-CMV-crRNA-EF1-T2A vector, and the plasmid or lentivirus was constructed with the assistance of Hanbio Biotech (Shanghai, China). The procedure for GC cells transfected with a lentivirus was conducted according to the manufacturer’s instructions, and after 48 h post-transfection, cells were selected with puromycin (2 μg/mL) for 2 weeks to construct cell lines with stable circGRAMD1B overexpression. Two individual circGRAMD1B siRNAs (siRNA1# and siRNA2#), a miR-130a-3p mimic, a miR-130a-3p inhibitor, and a scrambled negative control siRNA (si-NC) or miRNA negative control (miR-NC) were purchased from Ribobio (Guangzhou, China). Additionally, GC cells were transfected with the abovementioned oligonucleotides and plasmids using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The nucleotide sequences are listed in Supplementary Table 2. After transfection for 48–72 h, the GC cells were harvested for further experiments.

NSCLC cells were plated at 50% confluence in 35-mm Petri dishes. RiboFECTTM (RiboBio, China) was used as a transfection reagent. The concentration of both the miR-130a-3p mimic and si-H19 (RiboBio, China) was 50 nM, and that of si-WNK3 (RiboBio, China) was 100 nM. After a further 24 h, cells were harvested. The transfection efficiency of the siRNA and mimic was measured relative to the expression of a housekeeping gene by qRT–PCR.
lncRNAH19: siRNA: CCTCTAGCTTGGAAATGAA.
WNK3: siRNA: GACCGACAGTTGTTTCACA.