Patients, tissue specimens and clinical data. The study cohort consisted of 220 female breast cancer patients who underwent radical surgery between 11 August 2015 and 17 May 2016 in Fudan University Shanghai Cancer Center (FUSCC, Shanghai, China). Eligible patients were women who had histologically confirmed invasive breast cancer; had no evidence of distant metastasis; and provided sufficient tissues for further research. Patients were ineligible if they had received neoadjuvant chemotherapy or radiation therapy. Clinical data of the patients was retrieved from the Outcome unit. We completed the follow-up on 26 June 2018 and the medium follow-up period was 29.2 months (range 0.50–34.25). Recurrence free survival (RFS) time was calculated from the date of radical surgery to the date of breast cancer recurrence (ipsilateral breast, local-regional, or distant), death or the last time of follow-up.
Immunohistochemistry (IHC). Paraffin-embedded tissues were made into tissue microarray. After deparaffinized and rehydrated, the sections were heated in an autoclave at 120℃ in sodium citrate buffer (pH 6.0) for 10 min for antigen retrieval. The sections were then incubated with 3% hydrogen peroxide for 15 min. After blocking of nonspecific binding with QuickBlock™ Blocking Buffer (Beyotime, P0260, China) for 15 min, the sections were incubated at 4℃ with ME1 antibody (Abcam, ab97445, 1:1000, USA) overnight. The IHC Kit (GTVision, GK500705, China) including second antibody and DAB substrate was used. After washing with PBS, the sections were incubated with the second antibody for 30 min at room temperature. Color was developed with DAB (1:100) for 2 min. The sections were counterstained with hematoxylin (Servicebio, G1004, China), and finally dehydrated.
Immunohistochemical staining score. The standards for IHC staining scoring was previously described (12). Herein the intensity range was 0 = negative; 1 = low; 2 = medium and 3 = high. The quantity 0 = no positivity; 1 = positivity in 0–10%; 2 = positivity in 11–50%; 3 = positivity in 51–80%; 4 = positivity in > 80%. The final immunoreactive score (IRS, ranging from 0–12) was obtained by multiplying the intensity score and the quantity score. Two pathologists blinded to the patients’ information scored the immunohistochemical staining. In discrepant cases, they further reviewed the cases and reached a consensus. For ME1 low and high expression was defined as IRS ≤ 6 and IRS > 6, respectively.
Cell lines and cell culture. Cell lines including MCF–7, MDA-MB–468, SKBR3, MDA-MB–231, ZR–75–1 and 293FT were obtained from the Cell Research Institute (Shanghai, China). Cells were routinely cultured in high-glucose DMEM (MCF–7, SKBR3, ZR–75–1 and 293FT) (Hyclone, USA) or L–15 (MDA-MB–468 and MDA-MB–231) (Hyclone, USA), supplemented with 10% (v/v) fetal bovine serum (Gibco, USA) at 37℃ in a humidified 5% CO2 atmosphere (MCF–7, SKBR3, ZR–75–1 and 293FT) or 100% atmosphere (MDA-MB–468 and MDA-MB–231).
Protein extraction and Western blotting analysis. Cells were washed twice with pre-chilled PBS, pelleted by centrifugation and lysed in RIPA (Beyotime, PC102, China). After incubation for 30 min on ice, lysates were centrifuged (12,000g, 10 min, 4℃). Supernatants were collected and the protein concentration was measured using a BCA protein assay reagent (Enzyme, ZJ101, China). Total protein (20ug) was separated on 10% SDS-PAGE and then transferred to nitrocellulose membranes for 2h at room temperature. The membranes were incubated with the appropriate primary antibodies (anti-ME1, Abcam, ab97445, 1:1000, USA; anti-β-actin, Proteintech, 60008–1-Ig, 1:1000, USA) overnight at 4℃, washed three times with TBST, and then incubated with the corresponding secondary antibody (goat anti rabbit IgG, SA00001–2, 1:1000; goat anti mouse IgG, SA00001–1, 1:1000, Proteintech, USA) for 1h at room temperature. The bands were visualized by Immobilon Western Chemiluminescent HRP Substrate (Millipore, WBKLS0100, USA) and detected with a luminescent image analyzer (GE Image Quant LAS4000 mini, USA).
Plasmids and lentivirus infection. The shRNA plasmids for negative control and ME1 were purchased from Genechem Company (Shanghai, China). The ectopic overexpression plasmids for negative control and ME1 were purchased from XY biotech Company (Shanghai, China). Lentivirus carrying ME1 cDNA or shRNA targeting ME1 was produced from 293FT cells. MCF–7 and MDA-MB–468 cells were infected with lentivirus and then selected with puromycin. The overexpression and knockdown efficacy were evaluated by Western blotting.
Cell viability assays. Cells were seeded in 96-well plates (2000 cells/well) in triplicate and cell viability was examined by Cell Counting Kit–8 (CCK–8) assay (Dojindo Laboratories, Japan). Cells were seeded in 6-well plates (1000 cells/well) in triplicate and cultured for two weeks in colony-formation assay. Colonies were washed three times in PBS, fixed with 4% formaldehyde and stained with Crystal violet for 5 min.
Cell motility assays. The migration and invasion assays were done in a 24-well Chemotaxis chamber with 8-μm pores (Corning, USA). For migration assays, 5×104 cells were seeded into the Matrigel-uncoated upper insert at 24-well in medium without serum. Medium containing 20% serum was added to the well as a chemoattractant. Following a culture of 36 h, non-invading cells were removed from the upper surface by wiping with a cotton swab. For invasion assays, 1×106 cells were seeded into the Matrigel-coated upper insert at 24 wells in medium without serum. Medium containing 20% serum was added to the well as a chemoattractant. Following a culture of 48 h, non-invading cells were removed from the upper surface by wiping with a cotton swab. Then, the membranes were fixed with 4% formaldehyde for 15 min. The invading cells were stained with Crystal violet for 5 min.
Detection of ROS level. Cells in 6-well plates were washed with PBS three times and incubated with Dihydroethidium (DHE, Beyotime, S0063, China). or 2’,7’-Dichlorodihydrofluorescein diacetate (DCFH-DA, Beyotime, S0033, China) for 30 min at room temperature. Afterwards, cells were collected, washed with PBS three times and resuspended in PBS. DHE or DCF fluorescence was measured by FACScan Flow Cytometer (Beckman-Coulter, USA) within 30 min.
Statistical analyses. We evaluated the correlations between ME1 expression and clinicopathological parameters by Pearson Chi-square test or Fisher’s exact test. Recurrence free survival was plotted and calculated using Kaplan-Meier (KM) curve while differences between groups were compared by log-rank test. Univariate Cox proportional hazard model was used to estimate the influence of each variable on RFS. Variables with P values < 0.1 were further included in multivariate Cox proportional hazard models. In laboratory experiments, quantitative variables were illustrated as means ± SD and analyzed with the Student’s t test. Two-sided P values<0.05 were considered statistically significant. All analyses were performed by SPSS 22.0.