Pcd5 cir vector

pGPH1 vector was applied to construct hsa_circ_0002162 knock-down plasmid and circRNA control knock-down plasmid by GenePharma Co., Ltd (China). pCD5-ciR vector was applied to construct hsa_circ_0002162 overexpression plasmid and circRNA control overexpression plasmid by Geneseed Biotech Co., Ltd. (China). Hsa_circ_0002162 knock-down plasmid and circRNA control knock-down plasmid were transfected into CAL-27 cells using HilyMax (Dojindo, Japan), and the cells were divided into Circ(-) cells and NC(-) cells, accordingly. Hsa_circ_0002162 overexpression plasmid and circRNA control overexpression plasmid were transfected into SCC-9 cells using HilyMax (Dojindo), and the cells were divided into Circ(+) cells and NC(+) cells, accordingly. The expression of hsa_circ_0002162 in the four cell lines was evaluated by RT-qPCR at 24 h post transfection.

The pCD5‐ciR vector (Geneseed, Guangzhou, China) was used for circCRKL overexpression. The human p27 cDNA was synthesized and inserted into pcDNA3.1 by Vigene (Shandong, China). The shRNAs against circCRKL and p27 were designed and cloned into pGPU6-GFP-Neo (GenePharma, Shanghai, China). The miR-196a-5p/miR-196b-5p mimics, negative control (miR-NC), and anti-miR-196a-5p/anti-miR-196b-5p inhibitor were purchased from RiboBio (Guangzhou, China). The transfection was performed with lipofectamine 3000 reagent (Invitrogen, CA, USA, Cat no. L3000015) as previously described [17 (link)]. The detailed sequences are provided in Table S2.

The human hsa_circ_0009035 sequence, the hsa_circ_0009035 sequence lacking the mniR-889-3p binding sites, and a nontarget control sequence were synthesized by BGI (Shenzhen, China) and individually inserted into a pCD5-ciR vector (Geneseed, Guangzhou, China) opened with EcoRI and BamHI sites to produce an hsa_circ_0009035 overexpression plasmid, a mutant hsa_circ_0009035 overexpression plasmid (MUT-hsa_circ_0009035), and a negative-control plasmid (pCD5-ciR). Human HOXB7 (accession no. NM_004502.4; synthesized by BGI) was cloned into a pcDNA3.1 vector (Invitrogen, Saint-Aubin, France) with BamHI and XhoI restriction sites to construct HOXB7 overexpressing plasmid, and a nontarget pcDNA3.1 plasmid (pcDNA) was used as the negative-control plasmid. A mature miR-889-3p mimic (5′-UUAAUAUCGGACAACCAUUGU-3′), a mimic negative control (miR-NC mimic; 5′-ACGUGACACGUUCGGAGAATT-3′), an miR-889-3p inhibitor designed for miR-889-3p silencing (anti-miR-889-3p; 5′-ACAAUGGUUGUCCGAUAUUAA-3′), and the scrambled oligonucleotide control (anti-miR-NC; 5′-CAGUACUUUUGUGUAGUACAA-3′) were obtained from Ribobio (Guangzhou, China). HeLa and Siha cells at ∼60% confluence in 24-well plates were transiently transfected with 200 ng of plasmids and 50 nM oligonucleotides using Lipofectamine 3000 as recommended by the manufacturer (Invitrogen). Cells were harvested for further experiments after 48 h.

Goat circRNA8220 full length was obtained by PCR and inserted into pMD™19-T vector. Thereafter, the sequence was subcloned into the pCD5-ciR vector (Geneseed, Guangzhou, China) between EcoRI and BamHI sites. The forward primer of circRNA8220 was (EcoRI) 5′-CGGAATTCTAATACTTTCAGCTGGAGCTGATTGGACACAAT-3′ the reverse primer was (BamHI) 5′-CGGGATCCAGTTGTTCTTACCTCTCATCACAGTAGGTGAAC-3′.

Short hairpin RNAs (shRNAs) against circUHRF2, METTL3, and DDX27 were provided by GenePharma (Shanghai, China). The sequences for shRNAs were shown as follows: sh-circUHRF2-1: 5′-GTATGATGATTGGAAAATGGA-3′; sh-circUHRF2-2: 5′-GATGATTGGAAAATGGATATA-3′; sh-METTL3-1: 5′-GCCTTAACATTGCCCACTGAT-3′; shMETTL3-2: 5′-GCAAGTATGTTCACTATGAAA-3′; sh-DDX27-1: 5′-GCAGAGGAAAGGTCTCAGTTT-3′; sh-DDX27-2: 5′-GCAGGAATTTGACTTGGCCTT-3′; sh- negative control (NC): 5′-TTCTCCGAACGTGTCACGT-3′. The full-length sequences of the circUHRF2 gene (circBase ID: hsa_circ_0002359) were inserted into the pCD5-ciR vector (GENESEED, Guangzhou, China) to establish the circUHRF2 over-expression plasmid. CRC cells were transfected with these segments using Lipofectamine 3000 (Thermo Fisher), according to the user’s guide. For transient transfection assay with shRNAs and plasmids, cells were harvested at 48 h for further experiments. For animal experiments, SW620 cells were stably infected with lentiviruses carrying sh-METTL3 or sh-circUHRF2 (GenePharma) in the presence of 8 μg/mL polybrene (GenePharma). Stably transfected or infected cell clones were chosen by appropriate antibiotics (puromycin, 2–5 μg/mL, Sigma, Saint Louis, MO, USA) for at least one week after virus infection or plasmid transfection.