Human PC cell lines Panc-1, BxPC-3, AsPC-1, and SW1900 were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and cultured in RPMI-1640 medium (GIBCO, New York, USA) containing 10% fetal bovine serum (FBS) and 100 U/mL penicillin/streptomycin (Invitrogen, Carlsbad, USA). All cells were maintained in a humidified atmosphere containing 5% CO2 in air at 37°C.
After washed with phosphate-buffered saline (PBS), PC cells were lysed on ice with RIPA buffer containing 1 mM phenylmethylsulfonyl fluoride (PMSF) (PMSF:RIPA = 1:100) for 15 min. After centrifugation, the supernatant was collected. The BCA kit was used to measure the total protein concentration. Equal amounts of protein samples were separated by 10% SDS-PAGE and transferred to the polyvinylidene fluoride membrane. After blocked with 10% skimmed milk in Tris-buffered saline Tween-20 at room temperature for 2 h, the membrane was incubated with the following primary antibodies (dilution 1:1000; Santa Cruz Biotechnology, Santa Cruz, USA) overnight at 4°C: anti-MAPK, anti-pMAPK, anti-p53, anti-cyclin D1, anti-Cyclin-dependent kinase 2 (CDK2), anti-HSP70, anti-KCNH2, anti-E-cadherin, anti-Vimentin, anti-MMP9, anti-RhoA, anti-GAPDH, anti-β-actin. After washed with PBS, the membrane was incubated with horseradish peroxidase-linked secondary antibody (dilution: 1:5000) at room temperature for 2 h. The signal was detected using the ECL detection reagent (Beyotime, Haimen, China).
RNA extraction and real-time quantitative PCR (qRT-PCR)
Total RNA was extracted from cells with Trizol reagent (Invitrogen, Shanghai, China) and converted into cDNA using the MMLV Reverse Transcriptase Kit (Promega, USA). The cDNA aliquots were then analyzed by qRT-PCR using the Step One Plus Real-Time PCR System (Applied Biosystems, USA). The mRNA level was normalized to the level of GAPDH or β-actin. The primers used in this experiment were as follows: KCNH2 (157 bp), forward: 5’-CAATGGCGACCGTCACTC-3’; reverse: 5’-TCCTCTGCCCGCTTCTTC-3’; GAPDH (121 bp), forward: 5’-TGACTTCAACAGCGACACCCA-3’; reverse: 5’-CACCCTGTTGCTGTAGCCAAAC-3’. β-actin forward: 5’-CCCA TCTATGAGGGTTACGC-3’ and reverse: 5’-TTTAATGT CACGCACGATTTC-3’. The 2-ΔΔCt method was used to analyze the data.
Construction of lentiviral vectors
To construct lentiviral vectors expressing short hairpin RNA (shRNA) targeting KCNH2, the RNA interference (RNA1) sequence of human KCNH2 (CTC CCA ATA TCC ATC TGC T) was designed using the RNAi Designer Program (GeneChem, Shanghai, China). The negative control vector (control RNAi) was constructed using a scrambled sequence (TTC TCC GAA CGT GTC ACG T) with no homology to the human genome. The DNA oligonucleotide containing the target sequence was synthesized and inserted into the vector by double digestion with Age I and EcoR I, and subsequent ligation with T4 DNA ligase. The constructed plasmids were then transformed into E. coli DH5α cells. DNA sequencing analysis was performed using restriction endonucleases. The sequence was cloned then cloned into the lentiviral vector pGCSIL-Green fluorescent protein (GFP; GeneChem, Shanghai, China) to generate a lentiviral vector expressing short hairpin RNA (shRNA) targeting KCNH2 (pGCSIL-KCNH2-shRNA-LV) or a control vector (pGCSIL-neg-shRNA-LV). The vectors were then transfected into 293 T cells using Lipofectamine 2000. After 48 h, the supernatant containing the lentiviral vectors was harvested. After purified by ultracentrifugation, and the titer of the lentiviral vector was determined.
PC cells in the logarithmic growth phase were seeded in a 96-well plate (5 × 103/well) and cultured overnight. The lentiviral vectors were diluted in 0.2 mL complete culture medium containing 10 µg/mL of polybrene and incubated at 37°C for 12 h. Next, vector-containing medium was replaced with fresh culture medium. A fluorescence microscope (TE2000, Nikon, Tokyo) was used to detect the percentage of GFP-positive cells, which indicated transfection efficiency. Five days after infection, the expression of KCNH2, proliferation, migration, and invasion of transfected cells were analyzed.
Transwell migration and invasion assay
The migration and invasion assessment was performed using a Boyden chamber composed of Transwell membrane filters (#3422, Corning Costar, Cambridge, USA). PC cells (5 × 104/well) were plated to 24-well Transwell plates (pore size 8 µm) for migration assay or to the Matrigel-coated plates for invasion assay. Plates were filled with complete culture medium containing 10% FBS. The periods of migration and invasion assay were 24 h and 48 h, respectively. At the end of the experiment, cells that had not penetrated the filter membrane were wiped off. Cells on the lower surface of the filter membrane were stained with 0.4% crystal violet. The number of migrating or invading cells in a single chamber was counted from five fields under an optical microscope. This experiment was performed in triplicate and the average values were shown as mean ± standard error.
MMT assay was performed to detect the proliferation rate of cells in the Scrambled, shKCNH2_6, and shKCNH2_7 groups, each in triplicate. Cells were seeded in 96-well plates (1×104 cells/well). At 1, 2, and 3 days of culture, cells were incubated with 2 µL of MTT reagent (5 mg/mL; Sigma, St. Louis, USA) for 4 h. Then, the original medium was aspirated and 150 mL of DMSO was added to cells. Finally, the absorbance of each sample was measured at 492 nm by a microplate spectrophotometer (Thermo, Spectronic, Madison, USA).
Colony formation assay
The colony formation assay was performed to investigate the effect of KCNH2 silencing on the colony formation ability of PANC-1 cells. In this assay, 8 × 102 cells were seeded in a 6-cm petri dish and cultured in RPMI-1640 medium supplemented with 10% FBS. Cells were maintained in an atmosphere of 5% CO2, 95% humidity, and 37°C for 2 weeks. Then, cell colonies were washed twice with PBS and fixed with 4% paraformaldehyde for 15 min. The fixed colonies were subsequently stained with Giemsa for 20 min and washed twice with ddH2O. The colonies consisting of ≥ 50 cells were counted.
PC cells were transfected with designated sequences for 48 h. Then, cells were harvested, centrifuged, and resuspended with 500 µL of 1X binding buffer (BD Biosciences, Franklin Lakes, NJ, USA). After incubation with 5 µL V-FITC and 5 µL propidium iodide (BD Biosciences, Franklin Lakes, NJ, USA) for 5 min at room temperature in the dark, the apoptosis rate was analyzed by flow cytometry using a Cytomics FC 500 flow cytometer system (Beckman Coulter, Brea, CA, USA) as previously described.
PC tumor growth and metastasis in vivo
Six-week-old male BALB/c nude mice were purchased from the Shanghai Institute of Biological Sciences (Shanghai, China). Mice were anesthetized by inhaling a 1:1 mixture of isoflurane gas and oxygen. shKCNH2-PANC-1 cells were injected into the tail of the pancreas of nude mice as previously described[13, 14]. Eight weeks after inoculation, mice were sacrificed, the pancreatic tumors were removed and weighed, and metastatic liver nodules were counted. All animal experiments were performed in compliance with the Guidelines of the Institutional Animal Care and Use Committee at the Affiliated Hospital of Qingdao University. This study was reported in accordance with ARRIVE guidelines.
Patients’ samples and immunohistochemistry
The use of clinical samples was approved by the Ethics Committee of the Affiliated Hospital of Qingdao University. All patients provided written informed consent. Tissue samples were obtained from 83 patients who were diagnosed with PC between November 2015 and December 2017 in our hospital. Samples were fixed with 4% formalin, embedded with paraffin, sectioned, and stained with hematoxylin and eosin. The 5-µm-thick tissue sections were incubated with 3% H2O2 in methanol for 20 min. After incubation with blocking buffer, the sections were then stained with primary anti-KCNH2 antibody (Abcam) for four nights, followed by incubation with a secondary antibody (Santa Cruz Biotechnology) for 30 min at room temperature and then with the DAB (DAB) kit (Gene Tech). Tissue sections were washed three times with TBS for more than 10 min after each incubation. The staining results were evaluated according to the following criteria: (1) Percentage of positively-stained tumor cells: 0 (0–10%), 1 (11–25%), 2 (26–50%), 3 (51–75%), 4 (76–100%); (2) Signal intensity: 0 (no signal), 1 (weak), 2 (medium), 3 (strong). The immunoreactivity score (range 0–12) was calculated by multiplying the score of positive cells by the intensity score. The final score was as follows: – (0 point), + (1–4 points), ++ (5–8 points), +++ (9–12 points). In this study, – and + were considered as low expression, while ++ and +++ were high expression.
Written informed consent was obtained from all patients whose tissues were used in this study. The study was approved by the Institutional Review Board of the Affiliated Hospital of Qingdao University, Qingdao, China. All methods were performed in accordance with the relevant guidelines and regulations.
Pearson’s Chi-squared test was used to evaluate the relationship between KCNH2 expression and clinicopathological characteristics. Overall survival (OS) was defined as the time from surgery to death. The Kaplan-Meier method was used to evaluate OS and the log-rank test was used for comparison. A Cox regression model was used to perform multivariate analysis on all significant parameters in univariate analysis. A two-sided p-value of less than 0.05 was considered statistically significant.