br Luciferase reporter assay br The UTR
2.7. Luciferase reporter assay
The 3’-UTR of human TRIM25 mRNA was constructed with synthetic oligo-nucleotides and cloned in between the Sac I and Xho I sites of the pmirGLO Dual-luciferase miRNA target expression vector (Promega). We constructed pmirGLO-TRIM25 wild-type (WT) and pmirGLO-TRIM25 mutant-type (MUT). 293T cells were seeded into 96-well cul-ture plates (five wells per group). After 24 h, the cells were transfected with the TRIM25-WT or TRIM25-MUT vector along with pre-miR-3614 plasmid or corresponding control pcDNA6.2-GW. The luciferase activity was measured 24 h post-transfection using the Dual-Glo Luciferase Assay System (Promega, Madison, WI, USA) with Renilla (Rluc)
luciferase activity as the reporter gene and firefly luciferase (Luc) as the reference gene.
2.8. RNA immunoprecipitation (RIP) assay
For the RIP assay, the cell lysates were homogenized according to the protocol specified by the Magna RIP™RNA-Binding Protein Immuno-precipitation Kit (MILLIPORE Catalog No. 17-701). MCF-7 and MDA-MB-231 cells were lysed in RIP lysis buffer containing proteinase inhibitors cocktail tablets (Roche, Cat. 04693116001) and RNAse inhib-itor (MILLIPORE, Cat. CS203219) and incubated with anti-IGF2BP3-(Merck, Cat. 03-198), or anti-Ago2 (GeneTex, Cat. GTX60370) or IgG-coupled protein beads for 6 h or overnight at 4 °C. After stringently washing the beads with washing buffer, the bound RNA was purified and subjected to RT–PCR assays.
2.9. Biotin pull-down assay
A biotin pull-down assay was performed using the Pierce™ Mag-netic RNA-Protein Pull-Down Kit (Thermo Scientific, MA, USA) accord-ing to the manufacturer's instructions. Cell lysates were prepared using RIP lysis buffer. The biotin-labeled TRIM25 RNA probes listed in Table S1. These biotin-labeled probes were bound to streptavidin magnetic beads and incubated for 30 min at room temperature with ag-itation. A total of 100 μg of isolated protein lysate was added to the RNA-bound beads and incubated for 60 min at 4 °C with agitation. The bound proteins in the pulled-down material were analyzed by Western blot experiments using monoclonal K 252a recognizing IGF2BP3. After secondary antibody incubations, signals were visualized by enhanced chemiluminescence. The RNA probes sequences are listed in Table 2.
2.10. MTT and colony formation
A total of 3 × 103 BC cells was seeded on 96-well plates and cultured (37 °C, 5% CO2) in DMEM for 12 h. Next, the cells were treated with E2 (100 nM), TAM (100 nM), plasmids (0.1 μg) or siRNA (1.5 pmol) for the MTT cell proliferation assay. Absorbance was measured at a wave-length of 490 nm in a FLUO star OPTIMA microplate spectrophotometer (BMG LABTECH, Offenburg, Germany). Moreover, BC cells (1000 cells) were infected with LV-miR-3614, LV-si-IGF2BP3 and control virus and plated in 6-well plates for colony formation. The cells were grown for 14 days, fixed and stained with crystal violet; colonies with N30 cells were scored.
BC cells were seeded into 12-well plates at the density of 1 × 106 cells per well. 48 h after transfection, cells were collected by trypsinization, washed with PBS and fixed in ice-cold 70% ethanol at 4 °C overnight. Next, the cells were then washed with PBS and incubated with 0.1 mg/mL RNase A for 15 min and 0.05 mg/mL propidium iodide for 30 min at 4 °C. The samples were sorted based on DNA content by using fluorescence activated cell sorting and CellQuest software (FACScalibur; Becton Dickinson) to determine the percentages of cells that were in the G0/G1, S and G2/M-phases of the cell cycle.
We generated BC cells stably overexpressing miR-3614 or with silenced IGF2BP3 and injected them subcutaneously into female nude mice (n = 3/group) of 4–6 weeks of age for tumor engraftment. Tumors were examined every 3 d for a total of 35 d. The tumor volume was cal-culated using the following formula: V = lw2/2. The growth curves were plotted using the mean of the tumor volumes for each treatment group at a given time point. Five weeks after tumor inoculation mice were killed by cervical dislocation under anesthesia. The tumors were
excised and subjected to analysis. All animal experiments were ap-proved by the Institutional Animal Care and Use Committee of Xi'an Jiaotong University (No. 2017-487) and were performed according to the institution's guidelines for the use of laboratory animals.
2.13. Statistical analysis
All experiments were performed in triplicate at a minimum, unless otherwise stated. Data analyses were carried out using the SPSS 22.0 statistics package (IBM, Armonk, NY, USA). Independent sample Student's t-test and ANOVA analysis were used if the quantitative data between groups show normal distribution. If not consistent with the normal distribution, using the Wilcoxon-Mann-Whitney test. The relationship between TRIM25/miR-3614/IGF2BP3 expression level and clinical parameters was calculated by the χ2-test. Data are presented as the mean ± SEM. P values of b0.05 were considered to indicate statis-tical significance.