Pcd2.1 cir

Small interfering RNAs (siRNAs) designed to overlap the junction site of circPPP1R13B and miRNA mimic were synthesized by GenePharma Co., Ltd. (Shanghai, China) (Supplementary Table S1). For circPPP1R13B overexpression, the whole linear sequence of circPPP1R13B was synthesized and inserted into pCD2.1-ciR (Geneseed Biotech, Guangzhou, China) according to the manufacturer’s instructions (ov-circPPP1R13B). The empty vector was set as a negative control (ov-NC). For dual-luciferase reporter assay, the wild type (WT) and mutant type (MT) of circPPP1R13B linear fragment which contains miR-9-5p binding site were synthesized and inserted into pmirGLO dual-luciferase reporter vector according to the manufacturer’s instructions. All constructed vectors were verified by sequencing.

The linear sequence of circFAM188B was cloned into pCD2.1-ciR (Geneseed Biotech, Guangzhou, China) using the KpnI and BamHI (Takara, Dalian, China) restriction sites (OV-circFAM188B); the empty vector was used as negative control (OV-NC). The linear sequence of circFAM188B, which includes the sequence encoding Flag tags after the start codon “ATG” (no. 24), was cloned into pCD2.1-ciR, which was designated OV-circFAM188B-Flag. Besides, the start codon “ATG” (no. 24) of the OV-circFAM188B-Flag plasmid was mutated to “CCG” in the OV-circFAM188B-Flag-MT plasmid. Moreover, the linear sequence of flag tagged circFAM188B-103aa was integrated into the HindIII/KpnI restriction sites of the pcDNA3.1 overexpression plasmid (OV-circFAM188B-103aa); the pcDNA3.1 empty vector was used as the corresponding negative control (empty vector). The IRES-like sequence of circFAM188B was cloned into the Luc2-IRES-Reporter plasmid (Geneseed Biotech) using the KpnI and EcoRI (Takara) restriction sites. All sequences were chemically synthesized at Sangon Biotech Co., Ltd. (Shanghai, China). Small interfering RNAs (siRNAs) overlapping circFAM188B junction sites (Supplementary Table S1) were synthesized by GenePharma Co., Ltd. (Shanghai, China).

MiR-221-5p mimics/inhibitors and small interfering RNA (siRNA) overlapping junction sites of circ-XPO4 (si.circ-XPO4, 5′-CCGATGTACCACTTCCTCCTT-3′) were synthesized (GenePharma, Shanghai). For circ-XPO4 overexpression plasmid construction, the linear sequence of circ-XPO4 was synthesized and cloned into pCD2.1-ciR (Geneseed Biotech, Guangzhou, China) using the KpnI and BamHI restriction sites. IPEC-J2 cells were seeded in 12-well plates with 1 × 10⁵ cells per well. After the cells reached 70–80% confluency, they were transfected with miR-221-5p NC/mimics/inhibitors (30 pmol/well) and si.NC/si.circ-XPO4 (40 pmol/well, under the condition of 100 μg/μL PM-sEVs treatment) or OE.NC/OE.circ-XPO4 (1μg/well) using the Lipofectamine 3000 reagent according to the manufacturer’s instructions. The cells were harvested at 24 h, 48 h after transfection for qRT-PCR and Western Blotting analysis, respectively.

The plasmid of pCD2.1-ciR was purchased from Geneseed Biotech (Guangzhou, China) and was combined with circEIF6 sequences to upregulate circEIF6. Short interferon RNAs (si-circ) specific for circEIF6, miR-144-3p mimics, miR-144-3p and control were synthesized by Invitrogen. Cells in the 3^rd-5^th generations were seeded in the 6-well plates at the density of 5×l0⁵ cell/well and then sustained in incubators at 37 ˚C with 5% CO₂. The Lipofectamine 3000 reagent (Invitrogen, USA) was applied for cell transfection. Transfection procedures were implemented according to the guidance of manufacturer.