Plcdh cir vector

All the cells were purchased from the American Type Culture Collection (Manassas, VA, USA). MCF-7, MDA-MB-231, MDA-MB-468, and HEK293T cells were cultured in DMEM. T47D, SKBR3, and ZR-75-1 cells were cultured in RPMI 1640 medium. The medium was supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin. Cells in the medium were incubated in a humidified atmosphere containing 5% CO₂ at 37°C.
We constructed circBMPR2-overexpressed vector as previously reported.⁴⁸ (link) Briefly, the sequences of exons 2 and 3 of BMPR2 with a full length of 342 bp was subcloned into a pLCDH-ciR vector (RiboBio, Guangzhou, China) to generate pLCDH-circBMPR2 constructs. The subcloned sequence containing a front circular frame (SA), back circular frame (SD) of circRNA biogenesis and full length of circBMPR2, and 5′-TGAAATATGCTATCTTACAG-circBMPR2-GTGAATATATTTTTTCTTGA-3′ was directly synthesized. The pLCDH-ciR empty vector was used as an NC. For the knockdown experiments, the small interfering RNA (siRNA) target sequences for circBMPR2 were as follows: sense: 5′-CACCACUCACUUCGCAGAATT-3′; antisense: 5′-UUCUGCGAAGUGAGUGGUGTT-3′. The sequences for NC siRNA were as follows: sense: 5′-UUCUCCGAACGUGUCACGUTT-3′; antisense: 5′-ACGUGACACGUUCGGAGAATT-3′. The procedure of transfection was performed using Lipofectamine 2000 (Invitrogen) as previously reported.⁴⁸ (link)

The sequence of circ_0018414 was inserted into the PLCDH-cir vector (Ribobio, Guangzhou, China) to produce lentivirus for circ_0018414 overexpression. Short hairpin RNA (shRNA) targeting circ_0018414 (sh-circ_0018414) was applied for downregulating circ_0018414. In order to upregulate DKK1, the full-length of the DKK1 cDNA sequence was cloned into the pcDNA3.1 vector (GenePharma, Shanghai, China). miR-6807-3p mimics/inhibitor were gained from GenePharma, with negative control (NC) mimics/inhibitor as corresponding NCs. All of the plasmids were transfected into LUAD cells by the use of Lipofectamine 3000 (Invitrogen).