Seventy-six patients who underwent breast cancer resection in hospital from March 2015 to May 2017 were collected. They were (52.87 ± 6.39) years old on average. Seventy-six breast cancer tissues and 76 paracancerous tissues were obtained during the operation with the consent of the patients and stored in a liquid nitrogen tank. Inclusion criteria were as follows: patients diagnosed with breast cancer by pathological diagnosis; patients diagnosed with breast cancer for the first time. Exclusion criteria were as follows: patients who have received radiotherapy and chemotherapy; patients with other malignant tumors; patients with severe renal dysfunction; patients with serious infectious diseases; patients who refused to provide experimental specimens. This study was reviewed and approved by the Ethics Committee of People’s Hospital of Rizhao. All patients and their families agreed to participate in the experiment and sign an informed consent form.
Cell Culture And Transfection
Human breast cancer cell strains MDA-MB-231, MCF-7, MDA-MB-468, MCF-10A along with human normal breast epithelial cells Hs 278Bst (purchased from ATCC) were placed in DMEM medium containing 10%PBS, 2 mm penicillin as well as streptomycin, and cultured at 37℃, 5% CO2. When the adherent growth and fusion of the cells were observed to reach 85%, 25% pancreatin was added for digestion; after that, the cells were placed into the medium for continuous culture and passage was completed. Subsequently, the drug-resistant cell strain was cultured, and the MDA-MB-231/DDP cell strain resistant to DDP was obtained by the method of Reference . The cell was placed in a medium containing 0.5 µg/mL cisplatin to maintain its drug resistance and cultured at 37℃, 5% CO2. Then SIK2 expression in each cell line was detected. MDA-MB-231, MCF-7, and MDA-MB-231/DDP cells are selected for transfection. SIK2 RNA (si-SIK2) was targetedly inhibited, RNA (NC) was negatively controlled, and SIK2 RNA (sh-SIK2) was targetedly overexpressed; cells were respectively transfected with Lipofectamine™ 2000 kit, and our operation procedures were rigidly carried out on the basis of the kit instructions.
Evaluation Of Cisplatin IC50 In Breast Cancer Cells
Cells were inoculated into 96-well plates at a density of 1 × 105 per well, 100 µL of cisplatin with different concentrations was added respectively, fresh culture medium was changed 48 h after incubation, and 10 µl of CCK-8 solution was added into each well; then, they were put into an incubator to continue culturing for 2 h, the absorbance value of each well was determined at 450 nm wavelength by SpectraMax M5 elisa reader to detect cell proliferation, and this test was duplicated 3 times. Afterwards, the cisplatin IC50 on MDA-MB-231/DDP cells was calculated according to the cell survival rate.
Real-time Quantitative PCR
Firstly, the total RNA in tissues and cells was drawn with Trizol reagent, 5 µg total RNA was taken respectively for transcribing cDNA reversely according to the illustrations of the kit, and 1 µL of synthesized cDNA was taken for amplification after transcription. The PCR reactivation conditions were as follows: pre-denaturation at 95℃ for 10 s, denaturation at 94℃ for 10 s, annealing at 60℃ for 30 s, totaling 40 cycles. We set up three repeated wells for each sample and carried out the test 3 times. SIK2 used GAPDH as internal reference and data were analyzed by 2−△△ct. The primer sequences were shown in Table I.
Western Blot Test
RIPA lysis method was used to lyse cells and extract total protein, and its concentration was detected by the BCA method and then adjusted to 4 µg/µL. The proteins were segregated by 12% SDS-PAGE and then transferred to a PVDF membrane. Then, the membrane was sealed with 5% defatted milk powder for 2 h. SIK2 (1: 500), p-Akt (1: 500), p-PI3K (1: 500), p-mTOR (1:500), Caspase-3 (1:500), Bax (1:500), Bcl-2 (1: 500), Glut1 (1:500), HK2 (1:500), LDH-A (1:500) and β-Actin (1: 1000) primary antibody were added and sealed all night long at 4℃. It was washed to remove primary antibody, horseradish peroxidase labeled goat anti-rabbit secondary antibody (1: 1000) was replenished, and then it was hatched at 37℃ for 1 h, rinsed in PBS 3 times, each time for 5 min. The protein bands were developed in a darkroom via the enhanced chemiluminescence reagent, and the membrane’s excess liquid was removed with a filter paper.
Cell Proliferation Detected By CCK-8
The proliferation ability of cells was evaluated by CCK-8 kit. Cells 48 h after transfection were collected and diluted to 3 × 104 cell/ml. Then, they were inoculated into 96-well plates. Each well was inoculated with 100 µl of cells and cultured at 37℃ with 5% CO2. Each well was added with 10 µl of CCK8 solution at 0 h, 24 h, 48 h and 72 h after the cells adhered to the wall. After adding reagents, they were continuously cultured in an incubator at 37℃, 5% CO2 for 2 h, and then OD values were measured at 450 nm using an enzyme reader to detect cell proliferation and draw a growth curve. The experiment was repeated 3 times.
Transfected cells were digested by 0.25% trypsin, washed two times with PBS after digestion, added with 100 µL of binding buffer, prepared into 1*106/mL suspension, sequentially added with AnnexinV-FITC and PI, incubated at room temperature in dark for 5 min, detected with FACSVerse flow cytometer system, and averaged over 3 repetitions.
Cell Invasion Test
The invasion ability of cells was evaluated by Transwell test. Firstly, we added 200 µL DMEM culture solution containing 1 × 105 cells to the upper chamber, and 500 mL DMEM containing 20% FBS to the lower chamber. After culture at 37℃ for 48 h, the matrix and cells failed to cross over the film surface in the upper chamber were cleaned, washed by PBS 3 times, fastened with paraformaldehyde for 10 min, cleaned by double distilled water 3 times, dyed with 0.1% crystal violet for 10 min after drying, and the cell invasion was observed with a microscope.
Glucose Consumption And Lactic Acid Content
Collected cells were inoculated in a 6-well plate at a density of 3 × 105 per well and cultured at 37℃ with 5% CO2 for 48 h. Then their culture medium was used to measure glucose consumption and lactic acid production. The glucose and lactic acid levels were strictly described in accordance with the operation instructions of the glucose and lactic acid determination kits.
In our research, we used SPSS19.0 software package to analyze the collected data statistically, and adopted GraphPad 7 software package to draw the required pictures; besides, independent-samples T test was used for inter-group comparison, one-way analysis of variance (ANOVA) was used for multi-group comparison, LSD-t test was used for post hoc pairwise comparison, repeated measures ANOVA was used for multi-time point expression, and Bonferroni was used for back testing. A p value lower than 0.05 was regarded as markedly different difference.