Tissue specimens
All human breast cancer tissue specimens included in this study were obtained from 59 patients between July 2018 and July 2019 at Taizhou Hospital of Zhejiang Province. All patients were female and aged between 18 and 70 years old. The specimens were pathologically diagnosed as invasive ductal carcinoma of the breast according to the eighth edition of the American Joint Committee on Cancer (AJCC) standard. Patients who underwent any neoadjuvant treatments before surgery were not included in this study. In addition, this project received approval from the Ethics Committee of Taizhou Hospital of Zhejiang Province. We collected the specimens immediately after surgery and stored them in liquid nitrogen. The following clinicopathological features of these patients were collected: age (≥ 50 years or < 50 years), menopausal status (premenopausal or postmenopausal), surgical approach (modified radical mastectomy or breast-conserving surgery), histological grade (I, II or III), pTNM stage (based on the eighth edition of the pTNM pathological staging system published by AJCC in 2018) (I, II or III), ER/PR/HER2/EGFR status (positive or negative), Ki-67 status (high expression: Ki-67 ≥ 14 or low expression: Ki-67 <13), tumour size (≤ 2 cm or > 2 cm), and lymph node status (positive: N1-N3 or negative: N0).
Immunohistochemical detection of AQP3 and c-Met protein expression in cancer tissue specimens
Breast cancer tissue specimens were embedded in paraffin after being fixed in phosphate-buffered formalin. Then, we cut the paraffin blocks into sections approximately 0.5 µm thick, and the sections were detected by immunohistochemistry using standard ABC peroxidase techniques. The following antibodies were used: AQP3 Rabbit Polyclonal Antibody (1:1,000; Abcam, Shanghai, China) and c-Met Rabbit Polyclonal Antibody (1:1000, Abcam). Immunohistochemical staining results were analysed by two senior pathologists independently. The scores for the AQP3 and c-Met proteins consisted of scores for the immunostaining intensity and stained area for the protein. The cell staining intensity was divided into four grades with a score range of 0-3: 0 (no colouration); 1 (weak colour intensity); 2 (moderate colour intensity); and 3 (strong colour intensity). The stained area of AQP3-positive cells was divided into five grades, with scores of 0-4: 0, 0%; 1, 1%-10%; 2, 11%-50%; 3, 51%-80%; and 4, > 80% (13). The product of the final staining intensity score and the positive cell staining area score was considered the immunohistochemical staining score for the AQP3 protein. A score > 4 was defined as high expression, and a score ≤ 4 was defined as low expression. The stained area of c-Met-positive cells was also divided into five grades, with scores of 0-4: 0, 0%; 1, 1%-24%; 2, 25%-49%; 3, 50%-74%; and 4, ≥75%. The sum of the final staining intensity score and the positive cell staining area score was considered the immunohistochemical staining score for the protein; a score > 4 was defined as high expression, and a score ≤ 4 was defined as low expression.
Cell culture
We routinely cultured MCF-7 (Bioleaf Biotech, Shanghai, China) and MDA-MB-231 (Bioleaf Biotech) cells in high-glucose DMEM (Gibco, New York, USA) supplemented with 10% foetal bovine serum (Beyotime, Shanghai, China). The cells were cultured in a 5% CO2 incubator at 37°C.
RNA interference and hHGF treatment
Cells were seeded in 6-well plates with 2-3×105 cells per well and treated overnight. For an RNA interference assay, 20 µM c-Met/AQP3-specific silencing small interfering (si)RNA or a negative control (NC) siRNA (Genepharma Biotech, Shanghai, China) was transfected into breast cancer cells using LipofectamineTM 3000 (Invitrogen, Carlsbad, CA, USA), and the final siRNA concentration was 100 nM. The siRNA sequences used were shown in Table 1. For an hHGF (Yuduobio, Shanghai, China) treatment assay, different concentrations of hHGF (5, 25, or 50 ng/ml) were added to cultures.
Real-time Quantitative polymerase chain reaction
Total RNA was extracted from breast cells with TRIzol (Beyotime). cDNAs were synthesized using the 2 Step Real Time RT-PCR Kit (TAKARA, Dalian, China). Then, quantitative real-time polymerase chain reaction (RT-qPCR) was performed with the TB Green Premix Ex Taq Kit (TAKARA) using the Applied Biosystems 7500 Real-time PCR System. All primers used were synthesized by Sangon Biotech (Shanghai, China), and their sequences were shown in Table 2. This process was repeated more than three times for each sample in each experiment.
Western blotting
Ice-cold radioimmune precipitation assay (RIPA) buffer (Beyotime) mixed with phenylmethanesulfonyl fluoride (PMSF) (Beyotime) and phosphatase inhibitors (Applygen, Beijing, China) was used to lyse cells, and the cell lysates were evaluated with a BCA protein detection kit (Beyotime) to obtain the protein concentration. For immunoblotting, 10% SDS-PAGE (Beyotime) was used to separate samples, and then we transferred sample strips to polyvinylidene fluoride (PVDF) membranes (Beyotime). After skim milk powder (Beyotime) was used for blocking for 1.5 hours, the membranes were incubated with primary and secondary antibodies in sequence. Then, the protein bands were visualized with an enhanced chemiluminescence (ECL) kit (Beyotime) and imaged with the ImageQuant LAS 500 Ultrasensitive Chemiluminescence Imager (GE Healthcare, Shanghai, China). The following antibodies were included: AQP3 Rabbit Polyclonal Antibody (1:1,000; Abcam), c-Met Rabbit Polyclonal Antibody (1:1000, Abcam), β-Actin Mouse Antibody (1:1,000; Beyotime), HRP Goat Anti-Rabbit IgG Antibody (1:4,000; Biosharp, Beijing, China), HRP Goat Anti-Mouse IgG Antibody (1:4,000; Biosharp).
Wound scratch assay
Breast cancer cells were seeded in 24-well plates and then given different treatments. After the cells became confluent, a straight scratch was created in the centre of each well using a 200-µl pipette tip. Then, the cells were treated with low-serum medium and put back into the incubator for an incubation. Then, the scratch repopulation gap width was recorded by imaging (Optika XDS, Ponteranica, Italy) at 0 and 24 hours after treatment. The results of the wound scratch test are represented as the size of the cell-free area, which was quantified as the "average gap", and measured with ImageJ version 1.52a (Rawak Software, Stuttgart, Germany). The cell-free area at 0 hours was defined as 100%, and the results of the assay at 24 hours were compared with the results at 0 hours.
Statistical analysis
All the statistical data were analysed by SPSS 24.0 statistical software (IBM Corp., Armonk, USA). Values are representative of more than three independent experiments and presented as the mean ± standard error (SE). Clinicopathological results are expressed as percentages, and unpaired t-tests or the Pearson ꭓ2 test was used for comparisons between groups. The correlation between AQP3 and c-Met protein expression was analysed by Pearson correlation analysis. Data were compared among group by analysis of variance (ANOVA). p < 0.05 was deemed to be statistically significant.