There are altogether 78 pairs of breast cancer tissue samples and adjacent healthy tissue samples which were collected from breast cancer patients in Jinhua central hospital. We have collected informed consents from all patients and the research was supported by the ethics committee of Jinhua central hospital. All samples were collected in liquid nitrogen and frozen in -80°C refrigerator for later experiments. And we classified all samples into Ⅰ/Ⅱ and Ⅲ/Ⅳ stage with TNM stage. The clinical data of patients were as follows: male 8, female 70. About TNM stage, Ⅰ/Ⅱ stage 34, Ⅲ/Ⅳ stage 44.
Normal breast epithelial cell line MCF-10A and four different breast cancer cell lines MCF-7, T47D, SKBR3, MDA-MB-231 were purchased from Shanghai Cell Bank of the Chinese Academy of Sciences (Shanghai, China). All the cells were cultured in an environment of Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Gaithersburg, MD, USA) medium with 10% fetal bovine serum (FBS) (Sigma, St. Louis, MO, USA), containing 1% penicillin/streptomycin in 37 ℃ incubator with 5% CO2.
Small interfering RNA against HCG11 (siHCG11) and the corresponding control siRNA (siNC), overexpression vector pcDNA3.1-HCG11 and the corresponding empty vector (NC) were all constructed and synthesized by QIAGEN (Valencia, CA, USA). The miR-330-3p mimic, miR-330-3p inhibitor and their corresponding mimic control or inhibitor control were synthesized by GenePharma (Shanghai, China). They were transfected into MCF-7 cells with lipofectamine 2000 reagent (Invitrogen, #11668019) for later experiments. RT-PCR was determined to detect the transfection efficiency.
Quantitative real-time polymerase chain reaction (RT-PCR)
Total RNA was extracted from the breast cancer tissues and adjacent healthy tissues, normal breast epithelial cell line MCF-10A and different breast cancer cell lines including MCF-7. Following the manufacturer’s instruction, RNA was collected by using RNAiso Plus reagent (Takara, Code. No. #9109). Complementary DNA (cDNA) was reverse-transcribed in a total of 10 µl reaction system with the reagent PrimeScript™ RT Master Mix (Perfect Real Time) (Takara, Code. No. RR036) (500 ng total RNA, 2 µl Mix, added to 10 µl ddH2O). Then PCR was performed with the reagent kit (Roche, Code. No. 04913914001) in a 10 µl reaction system, consisting of 5 µl FastStart Universal SYBR Green Master, 2 µl cDNA, 1 µl primer and 2 µl ddH2O. Then put the plate in the fluorescence PCR machine (ABI 7500) for 2 h, the procedure set as 50℃ for 2 min, 95℃ for 10 min, then 95℃ for 20 s, 65℃ for 20 s and 72℃ for 30 s for amplification, in a total of 40 cycles. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA was the internal reference of the experiment. The primers used were as follows: HCG11 sense, forward primer: 5’-AGGAGTGGTTGCATTTGGGA-3’ and reverse primer: 5’-CCCACCACGCAGTGAATAGT-3’; GAPDH sense, forward primer: 5’-GTCACCTTCACCGTTCCAGTTTT-3' and reverse primer: 5’-CTTAGTTGCGTTACACCCTTTCTT-3’; miR-330-3p sense, forward primer: 5’-CAACTGCCTCTCTGGGCCTG-3’ and reverse primer: 5’-CTGCAGAGAGGCAGCGCTG-3’; FOXO1 sense, forward primer: 5’-TCGTCATAATCTGTCCCTACACA-3’; and reverse primer: 5’-CGGCTTCGGCTCTTAGCAAA-3’.
For protein sample preparation, the protein samples were taken from different treated MCF-7 cells with a mixed lysate consisting of protease inhibitors. Then cell lysates fully lysed 30 min in ice, centrifuged at 12000 g for 5 min at 4℃. After discarding the precipitate, the supernatant was collected and diluted with 5 × loading buffer in a ratio of 1:4, then heated for 5 min at 100 ℃ for fully protein denaturation. For electrophoresis, the samples were electrophoretically separated on 10% SDS-PAGE at 60V for 30 min and 100V for 90 min conventionally, then electrotransferred to polyvinylidene difluoride (PVDF) (Millipore, Cat. No. OPVH00010) membranes. After blocking in western blocking fluid (Beyotime, Cat. No. P0023B) for 2 h at a shaker. Washing the residual liquid, the membranes were incubated well at 4℃ for overnight with primary antibody which was pre-diluted with western primary antibody dilution buffer (Beyotime, Cat. No. P0023A). After washing the membranes in 1 × TBST for 3 times, the corresponding secondary antibody conjugated with goat anti-rabbit (Beyotime, Cat. No. A0208) or goat anti-mouse HRP (Beyotime, Cat. No. A0216) in a dilution of 1:1000 with secondary antibody dilution buffer were incubated for 2 h. After washing the membranes for 3 times, detection of proteins was performed with BeyoECL Plus kit (Beyotime, Cat. No. P0018). The primary antibodies used in the article were FOXO1 (CST, #2880, rabbit, 1:1000), GAPDH (CST, #5174, rabbit, 1:1000).
Cells suspension were evenly cultured in a 96-well plate in a concentration of 5000 cells in 100 µl per well at 37℃ incubator for 24 h. Then cells were treated and continue cultured for 48 h. For detecting cell viability, every well was added with 10 µl CCK-8 solution softly and continued full response at incubator for 2 h. The absorbance of the 96-well plate in 450 nm wave length in the microplate reader. The result was recorded in an excel table.
Colony formation assay
As a density of 1 × 103 cells per well, we seeded MCF-7 cells to six-well plate and cultured in 37℃ incubator for 2 weeks. Cells culture were terminated when the colonies were formed, then discarded the supernatant, washed twice with phophate-buffered saline (PBS). The colonies were fixed with 4% paraformaldehyde for 15 min, stained by crystal violet (Sinopharm Chemical Reagent, Shanghai, China) for 20 min. Recorded the number of colonies by using a microscope.
Wound scratch assay
MCF-7 cells were seeded in six-well plates at 5 × 105 for 24 h for 70–80% confluence. After treated cells, continue cultured cells for 48 h. Then scratch the cell monolayer in a straight line with a sharp pipette tip. For creating scratches of similar size to minimize variations due to width differences, the pipette tip should be perpendicular to the bottom of the well point to the straightedge. After scratch, wash away residual cells gently. And cells were treated with Mitomycin C (10 µg/ml) (a proliferation inhibitor) to avoid the effect of cell proliferation. Then cultured cells in incubator for 48 h and acquired image under a phasecontrast microscope.
Cell invasion assay
100 µl cell suspension with a density of 5 × 105 was added to transwell chambers with 8-µm pore membranes, and 24-well plates were added with 600 µl DMEM containing of 20% FBS, incubated at 37℃ in 5% CO2 for 48 h. After culture terminated, taken out transwell chambers, and discard cell medium, fixed with 4% paraformaldehyde for 30 min. Then transwell chambers were stained with 0.1% crystal violet for 20 min, and wiped off the upper layer of non-invasioned cells with a cotton swab. Imaged and counted under a microscope.
Luciferase reporter assay
The HCG11, miR-330-3p, and FOXO1 3'- UTR containing the binding sites were amplified and then inserted into the pmirGLO vector of luciferase expression (Promega, Madison, WI, USA) to obtain wild‐type HUCG11/FOXO1 (HCG11-WT / FOXO1-WT) and the same way to generate mutant HCG11/FOXO1 (HCG11-MUT / FOXO1-MUT) vectors. Cells were grown in 96-well plates and pmirGLO, HCG11/FOXO1 wild-type vector, HCG11/FOXO1 mutant vector, miR-330-3p mimics and the corresponding empty vector mixed with lipofectamine 2000 (Invitrogen, #11668019), respectively. And then transfected into MCF-7 cells. After transfection for 48 h, luciferase activity was measured by dual luciferase assay (Promega) and normalized to Renilla luciferase activity.
The data were obtained from at least three independent experiments and were shown as the mean ± SD. The difference of two groups were analyzed by Student’s t test. Multiple groups comparisons were compared by one-way analysis of variance (ANOVA), followed by Bonferroni’s post hoc test. Comparison of normal breast epithelial cell line and different breast cancer cells, was analyzed by ANOVA, followed by Dunnett’s post hoc test. P value < 0.05 was considered statistically significant. The data were analyzed by Graphpad Prism 8 (GraphPad Software, Inc.).