Tissue samples
Fifteen paired LSCC and adjacent normal tissues were collected from the Department of Pathology in Jingling Hospital between 2011 and 2013, after informed consent had been obtained. The ethics committee of Jiangsu Province Medical Association approved the study protocol. LSCC diagnosis was determined according to the latest WHO criteria and TNM stage classification (UICC 2002). None of the patients had received chemotherapy or radiotherapy before surgery. All tissue samples were snap-frozen in liquid nitrogen, and transferred to 500 ml TRIzol solution (Invitrogen, Carlsbad, CA, USA) immediately after harvesting to avoid mRNA degradation. Samples were stored in a biobank at −80 °C until processed.
Cell culture
Two LSCC cell lines (Hep-2 and AMC-HN-8) and a normal human keratinocyte cell line (HaCaT) were purchased from Shanghai Institute Chinese Academy of Science (Shanghai, China), cultured in RPMI 1640 media (Invitrogen) supplemented with 10 % fetal bovine serum (FBS), and 100 μM each of penicillin and streptomycin in a humidified atmosphere of 5 % CO2 at 37 °C.
Transfection of plasmids
For ectopic expression of miR-24 or knockdown of XIAP, pGCMV/miR-24 (or pGCMV/miR-NC vector) or pSil/shXIAP (pSil/shcontrol) were purchased from GenePharm (Shanghai, China). Transfections were performed using Lipofectamine™ 2000 (Invitrogen) according to the manufacturer’s instructions. Cells were transfected with recombinant DNA vectors containing a G418 selection marker and selected on G418 (Sigma, St. Louis, MO, USA) at 600 mg/ml for 4 weeks. Single clones were maintained in G418 at 100 mg/ml.
qRT-PCR assay
Total RNA was isolated using Trizol (Invitrogen), and 10 μg RNA used to synthesize cDNA with Super-Script II First-Strand Synthesis System (Invitrogen), or TaqMan® MicroRNA Reverse Transcription Kit (Applied Biosystems). Aliquots of the reaction mixture were used for real-time PCR with Power SYBR Green PCR Master Mix or with the TaqMan® 2 × Universal PCR Master Mix. PCR reaction conditions: 50 °C for 20 s, 95 °C for 10 min followed by 40 cycles of 95 °C for 15 s, 60 °C for 1 min. We calculated a △Ct (target-reference), which is equal to the difference between threshold cycles for miR-24 (target) and the threshold cycle for U6 RNA (reference) (△Ct (target-reference) = Ct target-Ct reference). The fold-change between patient or cell sample and a normal control for miR-24 or XIAP was calculated by the 2ˉΔΔCt method.
Western blotting assay
Cells were lysed using the mammalian protein extraction reagent RIPA (Beyotime, Beijing, China) supplemented with a protease inhibitor cocktail (Roche, Pleasanton, CA, USA), and phenylmethylsulfonyl fluoride (PMSF) (Roche). Approximately 50 μg protein extract was separated by 10 % SDS-PAGE, transferred to 0.22 μm nitrocellulose (NC) (Sigma), and incubated with specific antibodies. Autoradiograms were quantified by densitometry using Quantity One software (Bio-Rad, Brea, CA, USA). Rabbit anti-XIAP, cleaved caspase-3, total caspase-3, cleaved PARP, and total PARP were obtained from Cell Signaling Technology (Danvers, MA, USA). GAPDH antibody was used as a control.
Cell growth assay
Cell growth was measured by MTT assay (Sigma). In brief, cells were seeded into five 96-well culture plates with each plate having all three kinds of cells (6-parallel wells/group). Each day, 200 μL MTT (5 mg/mL) was added to each well, and the cells incubated at 37 °C for 4 h. Reactions were stopped by lysing cells with 150 μL DMSO for 5 min. Optical density was determined on a Versamax microplate reader (Molecular Devices, Sunnyvale, CA, USA) at 490 nm.
Colony formation assay
Cells were trypsinized to single cell suspensions and seeded to 6-well plates at 600/well. After 14 days culture in RPMI 1640 medium, colonies were stained with Giemsa solution and the number of colonies counted. Each experiment was performed in triplicate.
Flow cytometric detection of apoptosis
The cells were harvested, washed twice with cold PBS, fixed in ice-cold 70 % ethanol, and incubated overnight at −20 °C. Cells were then stained with 40 μg/mL of propidium iodide (PI) for 30 min. A minimum of 1.0 × 106 cells were collected and analyzed using Cell Quest software (Becton Dickinson Co., Franklin Lakes, NJ, USA), and the percentage of cells with apoptotic nuclei (% apoptosis) calculated.
Clonogenic survival assay
Cells were seeded into 6-well plates and cultured overnight, radiated (0.0, 2.0, 4.0, 6.0 and 8.0 Gy), and cultured for another 14 days. Colonies (>50 cells) were fixed with chilled methanol and stained with crystal violet, and counted on an inverted microscope. The surviving fraction was calculated as follows: number of colonies/number of plated cells. All the procedures were repeated in triplicate.
Dual-luciferase reporter assay
To validate XIAP as a direct target of miR-24, we performed dual-luciferase reporter assays using a pLUC target reporter plasmid containing XIAP/3′-UTR (pLUC/XIAP/3′-UTR-wt). Additionally, we generated a mutant XIAP/3′-UTR reporter construct by site-directed mutagenesis of the putative miR-24 target site in wild-type XIAP/3′-UTR (pLUC/XIAP/3′-UTR-mut) using Stratagene QuikChange® Site-Directed Mutagenesis Kit (Stratagene, Heidelberg, Germany). Cells were transiently cotransfected for 24 h with reporter plasmids (200 ng) and pGCMV/miR-24 (or pGCMV/miR-NC) and harvested in reporter lysis buffer. Both firefly and Renilla luciferase activities were measured using the Dual-Luciferase assay kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Luciferase activity was normalized against protein concentration and expressed as a ratio of firefly to Renilla luciferase unit.
Statistical analysis
Statistical analysis was performed with SPSS version 13.0 (SPSS Inc., Chicago, IL, USA). Values are expressed as the mean ± SD. χ
2 test and t-test were applied as appropriate. Correlations were evaluated by Spearman’s rank correlation coefficients. A P < 0.05 was considered statistically significant.