Lipofectamine 2000 transfection system

The sgRNA targeting LINC00341 has been designed, synthesized and inserted into the downstream of the U6 promoter, while the -CasRx gene was driven by the CMV promoter.
Genepharma Co., Ltd. (Suzhou, China) synthesized the p21 siRNA for the knockdown of gene expression. The p21 siRNA sequence (Sense) 5’- GAU​GGA​ACU​UCG​ACU​UUG​UUU-3’; (Antisense) 5’- ACA​AAG​UCG​AAG​UUC​CAU​CUU-3’. siRNAs targeting Bax along with E-cadherin were supplied by Genepharma (Suzhou, China). The negative control siRNA was also purchased from Genepharma Co., Ltd., Suzhou, China. Transfection of either the specific siRNAs or the siRNA negative control into the cell lines was done using the Lipofectamine 2000 Transfection system (Invitrogen, Carlsbad, CA, United States) as described by the manufacturer.

CELF2 3′‐UTR (positions 379–385: GUGCAAU) was cloned into the pmirGLO vector (Saierbio, Tianjin, China) to create a wild‐type (WT) pmirGLO‐CELF2‐3′‐UTR. The base sequence was mutated from GUGCAAU to CACCGAC to create a mutated (MUT) pmirGLO‐CELF2‐3′‐UTR. U87 cells were co‐transfected with the WT pmirGLO‐CELF2‐3′‐UTR or MUT pmirGLO‐CELF2‐3′‐UTR and miR‐363‐3p mimic or negative control (NC) using the Lipofectamine 2000 transfection system (Invitrogen, USA). The luciferase assays were performed using the Dual‐Luciferase Reporter Assay kit (Beyotime, China). An F200/M200 microplate fluorescence reader (Tecan Infinite) was used to detect luciferase activity under dark conditions. The relative luminescence unit value of Renilla luciferase was used as an internal reference.

The 3’-UTR sequence of Egfr, Cx3cl1, Cdk5r1, Rac2, or Plat was amplified from the genomic DNA and subcloned into the region directly downstream of the stop codon in the luciferase gene in the luciferase reporter vector. Overlap PCR was used to construct 3’-UTR mutant reporter plasmid. HEK 293T cells were cultured in 24-well plates and transfected with a mixture of p-Luc-UTR, miRNA mimics, and Renilla luciferase vector pRL-CMV (Promega, Madison, WI) using the Lipofectamine 2000 transfection system (Invitrogen). After 24 hours of incubation, luciferase activity was analyzed by the Dual-Luciferase Reporter Assay System according to the manufacturer’s protocols (Promega).

Synthetic miRNA mimics were purchased from RiboBio Company (Guangzhou, Guangdong, China). The miRNA mimics were transfected into HEK 293T cells using the Lipofectamine 2000 transfection system (Invitrogen). The transfection of plasmids into HEK 293T cells was performed with the Lipofectamine 2000 transfection reagent according to the manufacturer’s instructions. The wild-type psiCHECK2-circSCIN-WT construct was generated by inserting the circSCIN fragments containing the miRNA binding sequence into the psiCHECK-2 vector (Promega) at the 3′ end of the Renilla luciferase gene. The mutant psiCHECK2-circSCIN-MUT construct was generated by mutating the miRNA-binding sequence to the complementary sequence using overlapping extension PCR. For circSCIN luciferase assays, the HEK 293T cells were transfected with miRNA mimics and either the psiCHECK2-circSCIN-WT or mutated psiCHECK2-circSCIN-Mutreporter plasmid. At 48 h post-transfection, luciferase activity was measured using a dual-luciferase reporter assay system (Promega) according to the manufacturer’s instructions. The relative luciferase activities were calculated by comparing the Firefly/Renilla luciferase activity ratio.

Plasmids were transfected in HeLa cells with the Lipofectamine 2000 Transfection System according to the manufacturers’ protocol (Thermo Fisher Scientific) and in HEK293 cells using the phosphate-calcium method, unless otherwise stated. Cells were collected 36–48 h post-transfection.
Transfection of siRNAs (see sequences in Supplementary Table S13) was performed using Lipofectamine RNAiMAX Reagent (Thermo Fisher Scientific) or JetPRIME (Polyplus) according to the manufacturer's instructions. SiRNA transfections in HUVECs were performed using the GeneTrans 2 (MoBiTec) reagents according to the manufacturer's protocol. Cells were collected 48 or 72 h post-transfection.