Reagents and antibodies
The compound ML264 was purchased from Sigma-Aldrich (SML1755), and its structure and synthesis pathway have been previously published. ML264 was dissolved in the Dimethyl sulfoxide (DMSO). DMSO was purchased from Sigma-Aldrich. Specific antibodies against E-cadherin (#3195), N-cadherin (#13116), phospho-FAKTyr397 (#3283), FAK (#3285), PARP (#9532), Caspase 3 (#9662), α-tubulin (#3873) and β-actin (#3700) were purchased from Cell Signaling Technology, antibodies against KLF5 (TA324928) were purchased from OriGene, antibodies against Cyclin D1 (sc-753) and Cyclin B1 (sc-245) were purchased from Santa Cruz, and antibodies against P21 (#556430) were purchased from BD Pharmingen.
Immunohistochemistry staining of KLF5
Nasopharyngeal carcinoma tissue array (NPC961) were purchased from Pantomics Inc. This tissue array had 50% missing rate, remaining 6 cases of primary tumors with paired normal tissues, 16 cases of primary tumors, 11 cases of lymph node metastatic tumors, 9 cases of normal/reactive nasopharyngeal mucosal tissues and one positive control. Precoated slides followed by deparaffinization, rehydration, and antigen retrieval as previously described . Endogenous peroxidase was blocked per the manufacturer’s protocol (Dako, Carpinteria, CA). The slides were incubated with an anti-KLF5 polyclonal antibody (TA324928, Origene) at a 1: 800 dilution at room temperature for 1 h. Primary antibodies were detected using the Dako ChemMate EnVision Kit (K5001, Dako, Carpinteria, CA). Finally, the slides were counterstained with hematoxylin and investigated by light microscopy.
Evaluation of immunohistochemical staining
Two qualified pathologists, who were blinded to the study, observed the immunohistochemical staining to classify the clinical status of the patients. The staining intensity (1, 2 or 3) was decided for each cell in a fixed field corresponding to the weak, intermediate and strong staining, respectively. The percentage of cells at each staining intensity expression was subsequently calculated, and H score (0–300) was assigned using the following formula: H score = 1 × (% of cells staining 1) + 2 × (% of cells staining 2) + 3 × (% of cells staining 3). H score for each case were calculated as the mean score of at least three individual section scores for each case, from which the mean score of all the individual field scores of each section was derived.
Nasopharyngeal carcinoma cells line HONE-1 and NPC-TW01 were kindly provided by Dr. Chang-Shen Lin at Kaohsiung Medical University. NPC-TW03 and NPC-TW04 were gifts from Prof. Chi-Ying Huang (Institute of Biopharmaceutical Sciences, National Yang Ming Chiao Tung University, Taiwan). Four cell lines were cultured in Dulbecco's Modified Eagle Medium (DMEM, Gibco™, Thermo Fisher Scientific, MA, USA) supplemented with antibiotic–antimycotic (Gibco™, Thermo Fisher Scientific, MA, USA) and 10% fetal bovine serum (FBS, Gibco™, Thermo Fisher Scientific, MA, USA) at 37 °C in a 5% CO2 air atmosphere. NP69 human nasopharyngeal epithelial cell line was purchased from Sigma-Aldrich (SCC197). NP69 was cultured in keratinocyte serum-free medium (Gibco Cat. No. 10744019) supplemented with Keratinocyte-SFM Growth Supplement and 2% FBS.
NPC cells were transfected with synthetic hsa-miR-145-5p mimics (mirVana® miRNA mimics, Thermo Scientific™, MA, USA) to overexpress miR-145-5p. Scramble form of miRNA was used as a control (mirVana® miRNA mimics, Thermo Scientific™, MA, USA). KLF5 overexpression plasmids CMV-KLF5 were purchased from Sino Biological, Wayne, PA, USA. Cell transfections were performed using TurboFect (Thermo Scientific™, MA, USA) according to the manufacturer’s protocol.
WST-1 cell proliferation assay
Cell proliferation was analyzed by Premix WST-1 Cell Proliferation Assay System (TaKaRa, Mountain View, CA, USA), at least, triplicate samples according to the manufacturer’s protocol. Briefly, HONE-1 (1 × 103 cells/well), NPC-TW01 (1 × 103 cells/well), NPC-TW03 (1.2 × 103 cells/well) and NPC-TW04 (1.2 × 103 cells/well) cells, after transfection, were seeded in 96-well plates in 100 μL medium and were incubated at 37 °C. After incubation for 24, 48, and 72 h, 10 µL of WST-1 solution was added to each well and incubated for 1 h. Finally, cell proliferation was analyzed by measuring absorbance at 450 nm in a spectrophotometer.
BrdU ELISA cell proliferation assay
Cell proliferation was analyzed by Cell Proliferation ELISA, BrdU (colorimetric) (Roche, Basel, Switzerland), at least, triplicate samples according to the manufacturer’s protocol. Briefly, HONE-1 (1 × 103 cells/well), NPC-TW01 (1 × 103 cells/well), NPC-TW03 (1.2 × 103 cells/well), NPC-TW04 (1.2 × 103 cells/well) cells and NP69 (1.2 × 103 cells/well) cells after transfection, were seeded in 96-well plates in 100 μL medium and were incubated at 37 °C. After incubation for 72 h, 10 µL of BrdU labeling solution was added to each well and incubated for 2 h at 37 °C. Removed labeling medium from adherent cells by tapping off and then added 200 μl/well FixDenat to each well and incubated for 30 min at room temperature. Next, removed FixDenat solution and added 100 μl/well Anti-BrdU-POD working solution and incubated for 90 min at room temperature. Removed antibody conjugate and rinsing wells three times with 200 to 300 μl/well Washing solution. Finally, added 100 μl Substrate Solution to each well, and cell proliferation was analyzed by measuring absorbance at 370 nm and 492 nm (reference wavelength) in a spectrophotometer.
Cell migration was estimated by wound-healing assay. Briefly, NPC and NP69 cells were counted and seeded in 2-well culture-inserts (ibidi, Gräfelfing, Germany) after transfection. After 24 h of incubation, the insert was removed to create the gap. The gap of NPC cells was maintained in DMEM medium with 1% FBS and then photographed at different time points using a phase contrast microscope. The wound healing area was analyzed by Image J software, and wound closure percentage was calculated as reported by Ayman Grada el. .
Cell invasion assays
Cell invasion ability was measured by QCM ECMatrix cell invasion assay (Merck, Darmstadt, Germany) according to the manufacturer’s protocol. Briefly, NPC and NP69 cells were transfected and seeded (1.5 × 105) into the upper chamber with serum-free medium and incubated onto the lower chamber having serum-containing medium for 24 h at 37 °C. The cells migrated through the ECM layer and clung to the bottom of the polycarbonate membrane. Invaded cells were incubated with cell detachment buffer, and then lysed and stained with CyQuant GR® dye (Merck, Darmstadt, Germany). Finally, the fluorescence was measured using a fluorescence plate reader through 480/520 nm filter set.
Protein lysate preparation and Western blot analysis
The cell lines were plated in 6 cm dish using a density of 2 × 105 cells and were allowed to grow to 60% confluence. Forty-eight hour after transfection of microRNA mimics, the cell lines were washed twice with cold PBS, lysed in cell lysis buffer (Cell signaling) for 10 min and scraped. The extracts were centrifuged at 13,500 g for 10 min at 4 °C. Protein concentrations were measured and equalized using Bio-Rad protein assay (Bio-Rad Laboratories) according to the manufacturer's instructions. Equivalent amounts of protein (30 μg) were then separated by SDS-PAGE and then transferred to polyvinylidene difluoride membranes (PVDF). In order to detect the protein expression of different molecules on the same membrane, the blots were cut prior to hybridization with antibodies. The PVDF membranes were blocked for 1 h in blocking buffer (1X Tris-buffered saline, 5% nonfat dry milk, and 0.1% Tween 20), which was subsequently replaced by the primary antibody in blocking buffer, overnight at 4 °C. After incubation, the membranes were washed three times in washing buffer (1X Tris-buffered saline and 0.1% Tween 20). Primary antibody was detected using AffiniPure Mouse or Rabbit Anti-Human IgG (Jackson ImmunoResearch, USA) and visualized with Immobilon™ Western Chemiluminescent HRP Substrate (Merck Millipore, USA). The images were acquired by ChemiDoc Touch Imager with Image Lab™ Software (BioRad). The Fig. 4a, 5b, c and 6d have been done in triplicate. The band quantification was performed with the ImageJ software and the data shown in the histograms represent the average of at least three independent experiments.
RNA extraction and real-time PCR analyses
Total RNA was extracted from NPC cells using Trizol reagent (Invitrogen, Carlsbad, CA, USA) and then reverse transcribed to cDNA with reverse transcriptase and random primers. Real-time PCR was performed by StepOnePlus™ Real-Time PCR System (Applied Biosystems™, Thermo Fisher Scientific, MA, USA) using SYBR Green master mixture (Thermo Fisher Scientific, MA, USA). The following primers were used in the Real-Time-PCR experiment: KLF5 sense, 5'-CCCTTGCACATACACAATGC-3'; KLF5 antisense 5'- GGATGGAGGTGGGGTTAAAT-3'; GAPDH sense, 5'-GCACCACCAACTGCTTAGCA-3'; and GAPDH antisense, 5'-TCTTCTGGGTGGCAGTGATG-3'. The relative levels of KLF5 mRNA are expressed as the inverse log of the ΔΔCt value and normalized with the internal control gene, GAPDH .
Quantitative real-time PCR (qRT-PCR)
Total RNA was extracted from NPC cells using Trizol reagent (Invitrogen, Carlsbad, CA, USA) and then reverse transcribed to microRNA with TaqMan™ MicroRNA Reverse Transcription Kit (Applied Biosystems™, Thermo Fisher Scientific, MA, USA) and random primers. Quantitative real-time PCR was performed by StepOnePlus™ Real-Time PCR System (Applied Biosystems™, Thermo Fisher Scientific, MA, USA) using TaqMan MicroRNA Assays (Catalog number: 4427975, Assay ID: 002,278, Applied Biosystems™, Thermo Fisher Scientific, MA, USA). The reactions were incubated at 95 °C for 20 s, followed by 45 cycles at 95 °C for 5 s and 60 °C for 35 s. An RT reaction containing a miRNA specific stem-loop reverse-transcription primer and a separate specific TaqMan miRNA Assay. The RT stem-loop primer provides the specificity for the mature miRNA target; it does not detect its precursor. The relative levels of hsa-miR-145-5p are expressed as the inverse log of the ΔΔCt value and normalized with the internal control, U6.
Dual-luciferase reporter assay
For dual-luciferase reporter assay, NPC cells were seeded in a 96-well plate and co-transfected with pmirGLO dual-luciferase miRNA target expression vector (catalog number: E1330, Promega, Madison, USA) using TurboFect reagent (Thermo Scientific™, MA, USA). The samples were wild-type and mutant KLF5, scramble control and miR-145-5p mimics. Renilla and Firefly luciferase activities were measured 48 h post-transfection using the dual-luciferase reporter assay kit (catalog number: E2920, Promega, Madison, USA) and Microplate Luminometer (BioTeck, Winooski, VT, USA).
MiRNA target-predicting and bioinformatics
For predicting miRNA and mRNA direct interaction, we used miRNA target-predicting tools. The prediction tools contained Targetscan , miRDB , Miranda  and mirDIP . To find potential transcription factor binding to FAK, we used PROMO tool to investigate putative transcription factor binding sites (TFBS) in FAK sequence . To search potential molecular networks between KLF5 and FAK by Ingenuity Pathway Analysis software (QIAGEN Digital Insights).
Experiments were performed independently at least three times. The results are shown as mean ± SD. All statistical analyses were performed using the SPSS 19.0 (IBM SPSS, Armonk, NY, USA) and Microsoft Office Excel for PC. Statistical significance of dissimilarity between groups was confirmed by two-tailed student's t-test. P-values less than 0.05 were regarded as statistically significant.