Patient specimens and cell culture
All of 94 cases of BC cancerous tissues and the non-cancerous normal tissues was retrospectively analyzed from the First Affiliated Hospital of Wenzhou Medical University during April 2013 to Oct 2014. BC Cell lines (BIU–87, 5637 and RT–112) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). 293T cell line were donated by Dr Teng of Shanghai Eighth People’s Hospital (Shanghai, China). Cells were cultured in DMEM/F12 supplemented with 10% FBS (Gibco, Grand Island, NY, USA), penicillin (100 µg/mL, Gibco) and streptomycin (100 µg/mL, Gibco) at 37℃ in 95% humidified air and 5% CO2. All studies were conducted by following the protocol provided by the manufacturer. Studies are performed in accordance with the Declaration of Helsinki and adhere to local ethical regulations. We have acquired the written Informed consent from each participant before the initiation of this experimental study. The protocol of the current investigation was authorized by the Institutional Review Board of the First Affiliated Hospital of Wenzhou Medical University.
We used microarray datasets for identification of candidate circRNAs at platform GPL19978 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc = GSE92675). Four pairs of BC carcinoma tissues and paired para-carcinoma tissues were selected for detection of the expression and potential circRNAs functions.
Total RNA was extracted using TRIzol reagent (Invitrogen, USA). Then, cDNA was reverse-transcribed by the Prime Script RT reagent Kit (New England Biolabs, MA, USA) and subjected to RT-PCR measurement using PowerUp SYBR Green Master Mix (Takara Bio, Dalian, China). The relative expressions of each gene were acquired by using the 2-ΔΔCt method. The primers were listed below: 5′-TATGATCCTGTTTGGTGGTCGGCA–3′(forward), 5′-TGGACCAAGATGGGTAGCTTGTGA–3′(reverse) for circRNA_0058063. 5′-CAATGTACGTTCGCTATCGGC–3′(forward), 5′-CTCTCACGCACTTAATGCGAT–3′(reverse) for miR–486–3p. 5′-CGGCACAGCAGCTGAACTTA–3′(forward), 5′-GCAACACCTGAAAAAGTGTGA–3′(reverse) for FOXP4. 5′-CTCGCTACACCTCAATACATCG–3′(forward), 5′-GCGCCATAAGTCTAGTATTGAGA–3′(reverse) for GAPDH.
siRNA for circRNA_0058063 or scramble siRNA (si-NC) was synthesized by Invitrogen. The miR–486–3p mimic and the negative control (miR-NC) were synthesized by GenePharma (Shanghai, China). Lipofectamine 2000 (Invitrogen, Carlsbad, CA) system was used to conduct cell transfection assay by following the manufacturer’s protocol.
As previously described (H. Liu, Bi, et al., 2018), 2 x 103 cells were split and cultured in 96-well plates at 24 hours before its use for CCK–8 assay. After 48 hours of transfection, 10 μL of CCK–8 solution was then pipetted into the medium. Absorbance was detected at 450 nM on a Spectra Max 250 spectrophotometer (Molecular Devices, USA). After cells were fixed for 15 min in 4% of paraformaldehyde, the colonies were stained by 0.05% crystal violet (20 min) followed by visualized under a microscope.
The rate of apoptosis of the regulatory cells was examined. Both flow cytometry and annexin V/propidium iodide staining were applied to detect cell death rate by using the Annexin V-fluorescein isothiocynate Apoptosis Detection Kit (KeyGen, Nanjing, China).
At the beginning 20% fetal calf serum was put into the lower chamber, and the conditioned cells were suspended in a medium without FBS and inoculated onto an upper chamber. 48 h after adding the cells, get rid of the Matrigel in the upper chamber, at the same time the attached cells of invading ability were fixed, and crystal violet was applied to stain the cells, and then it was subjected for imaging with the aid of a microscope.
RNA immunoprecipitation (RIP) assay
With the aid of Lipofectamine 2000 transfection system, 1×107 cells were incubated in the RIP buffer transfected with MS2bs-circRNA_0058063mt or MS2bs-circRNA_0058063, and control MS2bs-Rluc. After 48 hours of transfection, RIP was conducted by using the Magna RIP RNA-binding protein immunoprecipitation kit (Millipore). For the RIP assay of Ago2, anti-Ago2 antibody (Millipore) was applied to RIP assay after the transfection for 48 h.
Western blot analysis
First, proteins were subjected to electrophoresis with 10% SDS-PAGE and then were transferred onto PVDF membrane (Millipore Corp., MA, USA) for 1 hr. Then, the membranes were blocked with 5% non-fat milk prepared by washing buffer for 1 hr. Next, the membranes were prepared to be incubated with corresponding antibodies against FOXP4 (1:500, Abcam, USA) to detect the relative protein expression of targets. Anti-β-actin antibody (1:1000, Affinity, USA) was applied as loading control. After incubating with primary antibody at 4 ℃ overnight, the membranes were washed with tris-buffered saline for 4 times and each time for 5 minutes. At last, specific corresponding secondary antibodies (1:2000, CST, USA) were further incubated with the membrane at room temperature. Protein bands were visualized and analyzed using Quantity One 4.6.2 software (Bio-Rad, Hercules, CA, USA).
SPSS 21.0 software for Windows (SPSS Inc.) was utilized to conduct statistical analysis. Data sets were expressed as mean ± s.e.m. The differences between two groups or more were compared by two-tail t test and χ2 test, respectively. Kaplan-Meier survival curves were analyzed using log-rank tests. High/low circRNA_0058063 expression groups were divided according to the medium expression values. Statistical differences were taken when P < 0.05. Triplicate experiments were performed in any of the assay unless otherwise stated.