With the approval of the Ethics Committee of the Third Affiliated Hospital of Soochow University, the experimental materials and protocols involved in this study were carried out. Informed consent was obtained from, and procedures explained to, all patients in accordance with institutional guidelines and regulations.
Patients and samples
Fifty-five women with BC (age range: 42–83 years) were included in this study. Tumor tissue samples and their corresponding tumor-adjacent tissue samples (not less than 2 cm from the tumor site) were collected during surgery. All samples were quickly frozen in liquid nitrogen after excision and stored at -80°C.
UALCAN and bc-genexminer 4.5 were used to analyse ApoM expression in BC tissue samples. The Kaplan–Meier Plotter database was used to analyse the relationship between ApoM expression and prognosis of BC patients, and the risk ratio, 95% confidence interval (CI) and log-rank P value were used as basic parameters.[19]
Cell culture
The human BC cell lines MCF-7 and MDA-MB-231 were purchased from the Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. MCF-7 was cultured in DMEM (Gibco, Life Technologies, NY, USA) supplemented with 10% foetal bovine serum (FBS) (Gibco, Life Technologies, Melbourne, Australia) and incubated at 37°C with 5% CO2 in a humidified atmosphere. Leibovitz's L-15 medium (Gibco, Life Technologies, NY, USA) containing 10% FBS was used to culture MDA-MB-231 cells at 37°C without CO2. The cells were then digested with trypsin, which was neutralized with complete medium, and the supernatant then discarded by centrifugation. Lastly, the cells were resuspended in fresh complete medium and placed in a suitable medium for further culture.
Lentivirus transfection
Lentivirus carrying the ApoM gene (H14570: pSLenti-SFH-EGFP-P2A- Puro-CMV-ApoM-3Flag, Fig. 2c) and its negative control vector (GL120: pSLenti-SFH-EGFP-P2A-Puro-CMV-MCS-3Flag, Fig. 2b) was synthesized by OBiO Technology (Shanghai, China). BC cells were transfected with lentivirus, and divided into negative control (NC) and ApoM overexpression (ApoM-OE) groups. In order to enhance transfection efficiency, polybrene was added into the cell medium with the lentivirus. After 72 hours of transfection, EGFP-positive cells were observed and photographed under a fluorescence microscope.
Cell proliferation assay
Cell-counting-kit 8 (Dojindo, Kumamoto, Japan) was used to measure cell proliferation. Lentivirus-infected BC cells were plated onto 96-well plates at a density of 1 × 105 cells/mL, with 6 duplicate wells in each group and 100 µl cell suspension in each well. When the cells had adhered to the plate, 10 µL of CCK-8 solution was added to each well and incubated for 2 h, and optical density at 450 nm was used to estimate the number of living cells in each well at 0 h, 24 h, 48 h and 72 h.
Wound healing assay
When the cells were >90% confluent, the culture medium was discarded and several parallel lines were scratched in the cell layer to produce a "wound" on the bottom of the culture plate after washing twice with PBS. The scratch was produced by the tip of a 200 µl sterile pipette oriented perpendicular to the bottom of the culture plate. The cells scraped loose by scratching were decanted with PBS, and 2 mL of medium supplemented with 1% BSA were added after the PBS was discarded. Wounds produced at 0 h, 24 h, 48 h and 72 h was recorded by camera on an inverted microscope, and the percentage of wound healing was recorded.
Cell invasion assay
Before seeding, transwell chambers (Corning, USA) were coated with matrigel (BD Biosciences, San Jose, CA). The cells in the ApoM-OE and NC groups were resuspended in basic culture and seeded into the upper chamber at an appropriate density. The lower chamber was filled with 800 µl complete medium containing 20% FBS. After incubating at 37°C for 48 h, the chambers were soaked in 4% paraformaldehyde for 30 min, followed by staining with crystal violet for 5 min. Six randomly-selected fields of the matrigel were chosen and the number of cells that had invaded were recorded under an inverted microscope.
RNA extraction and reverse transcription
Cells in the logarithmic growth phase were plated at an appropriate density in six-well plates. When the density of cells reached more than 90%, they were lysed and RNA extracted according to the RNA extraction kit (Beyotime, Shanghai, China) instructions. A spectrophotometer was used to measure the absorbance of RNA at 260/280 nm, which reflected the purity and concentration of RNA. A first strand cDNA synthesis kit (Thermo Scientific) was used for reverse transcription of RNA.
Quantitative real-time PCR
Gene expression was detected by the Light Cycler 480II PCR system. The total volume of the amplification system was 25 µL, containing 2.5 µL 10× buffer, 0.5 µL 10 mM dNTPs, 2.5 µL 25 mM MgCl2, 0.4 µL 10 μM primers and probes, 0.25 µL DNA polymerase, and 2 µL sample template. The thermal cycling conditions included initial denaturation at 95°C for 3 min, 5 sec at 95°C and 15 sec at 60°C for 40 cycles. The relative expression of target genes was quantified using 2-∆Ct. [20] The sequences of primers and probes are provided in Table 1.
In vivo tumor xenograft
To establish stable breast tumors in situ, an estrogen-releasing pellet (Innovative Research, America) was implanted into the backs of mice and MCF-7 cells were injected into mammary fat pads of BALBC mice. Tumor volume was calculated as 0.5×length×width2 and recorded twice weekly. When tumor volume reached 90 mm3, 10 µL of adeno-associated virus (AAV) overexpressing ApoM and its negative control AAV was injected into the tumor at each of several sites. Tumor volume and body weight were continuously measured and recorded. When mice were sacrificed on the 30th day, tumors were removed AV, weighed and photographed. The tumor tissues were then divided into two parts, one for measurement of ApoM over-expression and the other for frozen sections.
Western blotting
BC cells were washed by ice-cold PBS and lysed using a total protein extraction kit (Bestbio, China). Protein was quantified by a BCA protein quantitative kit (Bestbio, China). Loading buffer was used to mix with protein samples. After boiling for 10 min, the protein was denatured. Protein was separated under 10% SDS-PAGE and transferred to polyvinylidene fluoride membranes (Merck Millipore, Billerica, MA, USA). 5% skimmed milk powder was used to block the membrane at room temperature, and anti-ApoM (GeneTex 83083, USA) or anti-VDR (ab109234, Abcam) antibodies were incubated with the membrane at 4 ℃ overnight. After washing several times with tris buffered saline with tween, membranes were incubated with horseradish peroxidase-conjugated affinipure anti-mouse IgG (SA00001-1, Proteintech) or anti-rabbit IgG (SA00001-2, Proteintech). ECL Western Blotting Substrate kit (Thermo Scientific, Rockford, IL, USA) was utilized to visualize protein bands using an automatic chemiluminescence image analysis system. β-actin was used to normalize protein loading.
Statistical analysis
GraphPad Prism 8.0 software (Inc, San Diego, CA) was used for statistical analysis. The data are presented by mean ± standard deviation. Wilcoxon matched-pairs signed rank tests, one-way ANOVA and student’s t-tests were used to compare differences among groups. A P value less than 0.05 was considered statistically significant.