The subjects were 30 breast cancer tissue specimens and matched paracancerous tissue specimens, all of which were confirmed by the case and did not receive chemotherapy or radiotherapy. There were 19 invalids without lymph node metastasis and 11 invalids with lymph node metastasis. All specimens were collected on the morning of the operation. The specimens were instantly soaked in liquid nitrogen and hoarded at -80oC until subsequent study. All human participants provided written informed consent to participate in this research, which was approved by Ethics Committee of The Affiliated Zhuzhou Hospital of Xiangya Medical College CSU.
MCF-7, MDA-MB-231, ZR-75-30, and T47D, as well as normal breast epithelial cell MCF-10A were cultured in Dulbecco's modified eagle medium (DMEM), RPMI-1640 or Leibovitz's Lmur 15 medium containing 10% fetal bovine serum (FBS), 1% streptomycin and penicillin, respectively. All of cells were propagated at 37oC with 5% CO2.
The construction of lentiviral vectors of shRNA (sh)-NUPR1, sh-TFE3 and sh-ATG5, as well as sh-NC, lentivirus packaging and titer determination were completed by GenePharma Co., Ltd (Shanghai, China). ZR-75-30 cells in logarithmic phase were collected and seeded into 24-well plates (5×104 cells/well). The experimental cells were divided into sh-NC and corresponding transfection groups. The sh-NC and the corresponding sh-NUPR1, sh-TFE3 or sh-ATG5 lentivirus infected cells respectively, and the culture medium was changed after 12 h of culture. 0.5 mg/L puromycin solution was used for screening after 72 h of transfection, and the fluorescence expression was observed under fluorescence microscope. Stable transfection cell lines were obtained after 2 weeks of screening.
The tissue specimens were immovable with 10% formaldehyde, embedded in paraffin, sliced up 4 μm slices, baked in 60oC oven for 30 min, dewaxed with xylene for 3 times, 5 min each time, and rehydrated with decreasing gradient ethanol. After rinsed with phosphate buffer solution, the sections were placed in citrate buffer solution at high temperature to repair the exposed antigen, then rinsed with Phosphate buffered saline (PBS). The slices were incubated in 3% H2O2 at constant temperature for 10 min to interdict endogenous peroxidase, and rinsed with distilled water and PBS. The protein were sealed and placed at ambient temperature for 10 min. Sheep anti-human primary antibody was added respectively, and the slices were placed in the 4oC refrigerator overnight. The next day, the slices were rinsed with distilled water and PBS. After that rabbit anti-sheep secondary antibody labeled with horseradish peroxidase (HRP) was added to the slices, and the slices were incubated at 37oC for 30 min. After rinsed with distilled water and PBS, the slices were stained with diaminobenzidine kit for 2~5 min. After the completion of color development, the slices were rinsed with distilled water to stop color development, and then re-stained with hematoxyl for 3~5 min. The slices were differentiated with 1% hydrochloric acid and alcohol for 3~5 s. After fully rinsed, the slices were dehydrated by gradient ethanol incrementing, and the slices were roasted and sealed with neutral gum.
Cell counting kit-8 (CCK-8) assay
ZR-75-30 cells were seeded into the culture plate with 5000 cells per well. After 24, 48 and 72 hrs of culture respectively, appropriate amount of CCK-8 solution was blended and nurtured at 37oC with 5% CO2 for 4 hrs. The OD value was evaluated by automatic enzyme labeling instrument after shaking for 1 min.
The logarithmic growth phase cells were seeded into the 6 cm petri dish with 1000 cells per well. Three-multiple pores were set up, and the cell state was observed every 3 days. After 7 days of culture, the cell colonies were sluiced with PBS twice, fastened with 4% paraformaldehyde, pigmented with Giemsa (Sigma, USA), and then rinsed with ddH2O for 2 times. The number of cell clones was observed under microscope.
After 5 days of lentivirus infection, the cells were prepared into cell suspension, and three-multiple pores were set up in the each group. Centrifuge and discard the supernatant, cell suspension with a cell density of 1×105/mL was collected, sluiced with PBS, and incubated with Annexin V -FITC (ANNEX300F, Bio-Rad) and ReadiDropTM propidium iodide (PI, 1351101, Bio-Rad) for 1 h at 4°C protect from light. Apoptotic cells were detected with Beckman Coulter Gallios Flow Cytometer and analyzed using FlowJo software (FlowJo, Ashland, USA).
Wound healing assay
ZR-75-30 cells were digested with trypsin and transferred to a 6-well plate and cultured for 24 h. The wound was cut open with a 10 μL pipette tip, and then washed off the floating cells with PBS. And take pictures to calculate the initial area. The culture was continued for 48 h, observed under a microscope at the corresponding time points, and photographed.
ZR-75-30 cells (2×105 /mL) at logarithmic growth stage were suspended in 200 μL serum-free RPMI 1640 medium, and the cell suspension was added into the upper chamber of Transwell with matrix glue and the lower chamber with 500 μL FBS. After cultured for 24 h and the cells that did not pass through the stomata were plucked out with swabs. The cells were immovable with 4% paraformaldehyde and pigmented with Giemsa. Five visual points were haphazardly picked under the microscope to take photos and counting. The average number of cells penetrating to the lower chamber was taken as the experimental results.
Quantitative real-time PCR (qPCR)
RNA was extracted from ZR-75-30 cells according to the instructions of RNA extraction kit (Invitrogen, USA). After synthesizing the first strand of cDNA, the SYBR Green method was used for Real-time PCR, and β-actin was used as internal reference to detect the level of mRNA. There were 3 multiple pores in each sample, and the data were analyzed by 2-∆∆Ct method. The primer sequence are used as follows: NUPR1, 5’-AGG ACT TAT TCC CGC TGA CTG A-3’ (Forward) and 5’-TGC CGT GCG TGT CTA TTT ATT G-3’ (Reverse); TFE3, 5’-CCG TGT TCG TGC TGT TGG A-3’ (Forward) and 5’-GCT CGT AGA AGC TGT CAG GAT-3’ (Reverse); β-actin, 5’-CAG CCT CAA GAT CAT CAG CA-3’ (Forward) and 5’-TGT GGT CAT GAG TCC TTC CA-3’ (Reverse).
ZR-75-30 cells were extracted the total protein, and the protein content was determined by BCA kits (Beyotime, Shanghai, China). The same protein content samples were boiled for 10 min, and then subjected to SDS-PAGE, membrane transfer, sealed overnight, membrane washing, primary antibody incubation, membrane washing, secondary antibody incubation, and ECL chemiluminescence development. The results were analyzed by Tanon chemiluminescence imaging analysis system. The primary antibody information is shown below: TFE3 (1:500, ab196681), LC3 (1:1000, ab229327), p62 (1:1000, ab91526), Beclin1 (1:1000, ab62557), ATG5 (1:1000, ab228668), E-cadherin (E-cad, 1:1000, ab231303), N-cadherin (N-cad, 1:1000, ab207608), vimentin (1:1000, ab137321) and β-actin (1:500, ab115777).
Chromatin immunoprecipitation (ChIP)
Breast cancer cells were digested into 200~1000 bp of soluble chromatin by ultrasonic lysis, and DNA/protein immunoprecipitation was carried out. ChIP antibody was added and incubated overnight under the condition of 4oC to obtain DNA/protein precipitation. The DNA was dissociated and purified for qPCR. The primer sequence was synthesized by Sangon Biotech Co., Ltd (Shanghai, China).
In vivo experiment
All animal experimentations were consummated in obedience to stipulations approved by Ethics Committee of The Affiliated Zhuzhou Hospital of Xiangya Medical College CSU. Balb/c female nude mice (6-8 weeks) were obtained from Beijing Experimental Animal Center. Mice were maintained in a 12 h light/dark, 20-25°C and 50-65 humidity specific pathogen free animal facility with adequate food and water ad libitum and were acclimated for 1 week before tumor cell inoculation. ZR-75-30 cells with stably expressing sh-NUPR1 were suspended in phosphate buffer (1×106 /mL). Cell suspension was inoculated subcutaneously into the axilla of nude mice. The dimensions of tumors were measured once a week. On day 49, mice were euthanized before they become moribund. Tumors were isolated for further analysis.
GraphPad Prism 8 software was used to analyze the data, and the measurement data were expressed as Mean ± SD. Student's test and analysis of variance were used for statistical processing. All the data were repeated for at least 3 independent experiments. A value of P < 0.05 is considered significant.