Mir 144 3p mimic

The miR-144-3p mimics (5'-UACAGUAUAGAUGAUGUACU-3'), mimics negative control (5'-UUCUCCGAACGUGUCACGUTT-3'), miR-144-3p inhibitor (5'-AGUACAUCAUCUAUACUGUA-3'), and inhibitor negative control (5'-CAGUACUUUUGUGUAGUACAA-3') were purchased from GenePharma (Shanghai, China). The ERO1L overexpression plasmid was constructed by introducing the human ERO1L cDNA into the pcDNA3.1 vector (Invitrogen), and was designated as pcDNA3.1-ERO1L. Specific siRNA against ERO1L (5'-GCACTGCTCTGAAGATCTT-3') and a scramble siRNA (5'-TTCTCCGAACGTGTCACGT-3') were synthesized by RiboBio (Guangzhou, China). Cells were seeded in 12-well plates and transfected with the above oligonucleotides or vectors using Lipofectamine 3000 (Invitrogen) following the manufacturer's instructions. Cells were harvested at 72 h after transfection and used in subsequent analyses.

Shanghai GeneChem Co., Ltd. designed and synthesized the GEFT and lnc-FAM59A-1 overexpression plasmids, the GEFT interference plasmid, and the empty vector. The siRNAs against human lncRNAs (lnc-CEACAM19-1, lnc-VWCE-2, lnc-GPX7-1, lnc-PSMA8-1) and mTOR, the miR-144-3p mimic, the antisense miR-144-3p inhibitor, and the negative scramble control RNA oligo were purchased from Shanghai GenePharma Co., Ltd. Lipofectamine 2000 (Thermo Fisher Scientific, Inc.) was used for all transient transfections.

The expressions of SNHG4 or miR-144-3p were regulated by cell transfection techniques in vitro. SNHG4 expression vector was synthesized using pcDNA3.1 vector. MiR-144-3p mimic, miR-144-3p inhibitor and miR-negative control (miR-NC) were all provided by Gene Pharma (Shanghai, China). Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) reagent was used for cell transfection. The specific operation procedure was carried out according to the kits’ instructions. In short, the transfection system is configured, which contains solution A and solution B. Solution A contains 125µL Opti-MEM and 5µL Lipofectamine 3000, and solution B contains 125µL Opti-MEM and 2.5µL plasmid. Solution A and solution B were mixed and added to the cells and continued to culture for 48 h. Subsequent operations were performed after transfection.

To determine the potential function of miR-144-3p, RAW264.7 cells were transiently transfected with 50 nM control or miR-144-3p mimic (GenePharma, Shanghai, China); 50 nM control or miR-144-3p inhibitor (GenePharma, Shanghai, China) by using Lipofectamine 2000 (Invitrogen, USA).

MDA-MB-231 cells and HEK-293T cells were cultured in DMEM medium supplemented with 10% fetal bovine serum in a 5% CO₂ incubator at 37 °C. T47D cells, MCF7 cells were obtained from the ATCC and cultured in RPMI-1640 medium supplemented with 15% fetal bovine serum in a 5% CO₂ incubator at 37 °C. Cells were tested and authenticated in Beijing Microread Genetics Co., Ltd. (Beijing, China) by short tandem repeat (STR) profiling. All cell lines tested negative for mycoplasma contamination. MG132 (C2211) and CHX (C1998) were purchased from Sigma. Recombinant human Wnt-3a was purchased from R&D Systems. The transfection of miR-320a-3p mimic, miR-144-3p mimic, and miR-29a-3p mimic with miRNA control (miR-NC) (GenePharma, China) were performed according to the manufacturer’s instruction using Lipofectamine 3000 reagent (Invitrogen). The final concentration of miRNA was 20 nM. miRNA sequences are listed in Supplementary Table S2.

Human normal breast epithelial cell line MCF 10A (CBP60419) and breast cancer cell lines AU565 (CBP60353), MCF-7 (CBP60380), MDA-MB-157 (CBP60381) and MDA-MB-231 (CBP60382) were all purchased from Cobioer (Nanjing, China). Plasmids including si-NC, si-LINC00461, inhibitor NC, miR-144-3p inhibitor, mimic NC, miR-144-3p mimic, oe-NC, oe-KPNA2 were ordered from GenePharma (Shanghai, China), and the siRNAs were sequenced as below: si-LINC00461: 5′-CTGCAAAGAAGCATAAAATGA-3′; si-NC: 5′-TTCTCCGAACGTG TCACGT-3′.
Before transfection, cells were grown in 6-well plates (3 × 10⁵ cells/well) until the density reached 50%. 250 μL of serum-free Opti-MEM (51985042, Gibco, Gaitherburg, MD, USA) was used to dilute the target plasmids (4 μg) and Lipofectamin 2000 reagent (10 μL; 11668-019, Invitrogen, NY, California, USA), respectively. Thereafter, the dilutions were mixed, and then dripped into cells after 20 min. All cells were maintained in 5% CO₂ at 37 °C for 6 h, and cultured for additional 36–48 h with fresh mediums.

832/3 rat insulinoma cell line (INS-1) purchased from Sigma-Aldrich (MO, USA) were grown in DMEM medium containing 10% FBS and antibiotics at 37 °C in a CO₂ incubator.
Mimic and inhibitor for miR-144-3p (miR-144-3p mimic and miR-144-3p inhibitor) and the corresponding negative controls (NC mimic and NC inhibitor) were supplied by the GenePharma company (Shanghai, China). Gene-specific (USP22 or SIRT1) overexpression plasmids or shRNAs suppling by the GenePharma were transfected into cells to overexpress or knock down the expression level of proteins. According to standard protocols, INS-1 cells were transfected with the stated plasmids using Lipofectamine 2000 (Life Technologies, CA, USA) and harvested at 48 h post transfection for further experiments.