Mimic nc

hVSMC was incubated in a 24-well plate in advance until the cell confluence was up to about 40%. Commercial RNA transfection reagent Entranster^TM-R4000 (Engreen Biosystem, China) was applied to transfect miRNA mimics or sh-RNA. MiR-29b-3p mimics, NC mimics, pcDNA-IGF1 and pcDNA-NC were synthesised by Sangon Biotech. Specifically, the miRNA mimics or pcDNA were diluted in a serum-free medium, then mixed with the diluted transfection reagent, and added to the well. After 48 h, subsequent experiments could be carried out.

For in vitro overexpression studies, HeLa and SiHa cells were selected for they showed the lowest expression of hsa_circ_0107593. For knockdown experiments, CaSki and ME180 were selected for they showed the highest expression of hsa_circ_0107593. A plasmid overexpressing hsa_circ_0107593 was transfected into HeLa and SiHa cells. siRNAs targeting hsa_circ_0107593 were transfected into CaSki and ME180 cells. Three siRNAs (siRNA-1, siRNA-2, and siRNA-3) and corresponding negative control siRNA (si-NC) were purchased from RiboBio (Guangzhou, China). The siRNA with highest knockdown efficiency was selected for all subsequent experiments. The overexpressing plasmid and its corresponding negative control plasmid (OE-NC) were purchased from TSINGKE Biotech Ltd. (Beijing, China). Cells were harvested 48 h post-transfection, then transfection efficiency was checked by qRT-PCR. For dual-luciferase reporter assay, hsa-miR-20a/93/106b-5p mimics, as well as their respective negative control (NC mimics) were purchased from Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. Cell transfection was performed with Lipofectamine 3000 (Invitrogen, Thermo Fisher Scientific, USA) following the manufacturer’s instructions.

Short hairpin RNAs targeting PDZD8 (sh-PDZD8#1/2) or spliced form of hsa_circ_0020123 (sh-circ_0020123#1/2), miR-495 mimics, and their negative controls (sh-NC and NC mimics) were synthesized by Sangon Biotech (Shanghai, China). Empty pcDNA3.1 vectors and pcDNA3.1 vectors overexpressing PDZD8 or circ_0020123 were purchased from GenePharma (Shanghai, China). The above shRNAs (40 nM), mimics (50 nM), and vectors (10 nM) were transfected into A549 and H1975 cells using the Lipofectamine 2000 reagent (Invitrogen) according to the instructions. The transfection efficiency was examined by reverse transcription quantitative polymerase chain reaction (RT-qPCR) after 48 h.

Cells seeded in 25 cm² flasks or 6-well plates (70% confluent) were exposed to different doses of ionizing radiation at a dose rate of 1 Gy/min with an X-ray linear accelerator (LINAC) in our hospital. For transfection, cells were seeded in 6-well plates at a concentration of 3 × 10⁵ cells/well. Twenty-four hours later, the cells were transfected with the lncRNA GAS5 overexpression plasmid or miR-21 mimics as well as NC mimics (Sangon Co., Shanghai) by using Lipofectamine 3000 reagent (Invitrogen).

miR-143-3p mimics and corresponding negative controls (NC mimics) were synthesized by Shanghai Sangon Biotech Co., Ltd. (Shanghai, China). The miR-143-3p mimic sequence was 5′-UGAGAUGAAGCACUGUAGCUCG-3′, and the NC mimic sequence was 5′-UUGUACUACACAAAAGUACUG-3′. The Itga6-3′UTR sequence containing the miR-143-3p binding site was amplified by PCR using cDNA from goat SHFs with a pair of primers, Itga6-WT-UTR-F and Itga6-WT-UTR-R. Then, the amplified fragments were cloned in Dual-Luciferase miRNA Target Expression Vector (Promega, Madison, WI, USA) using Pme I and Xba I enzymes (NEB, Ipswich, MA, USA). The vectors were named Itga6-WT. Next, the Itga6-Mut vectors were generated with a mutagen in the miR-143-3p binding site by two pairs of mutagenic primers, Itga6-Mut-UTR-F and Itga6-Mut-UTR-R, using Mut Express II Fast Mutagenesis Kit V2 (Vazyme, Nanjing, China). All the primers used for vector construction are listed in Table S2.