Approval was obtained from the Princess Margaret Cancer Centre (PMCC) Institutional Research Ethics Board. Diagnostic formalin-fixed paraffin-embedded (FFPE) blocks were obtained from NPC patients treated at the PMCC between 1993 to 2009, as previously described . RNA was isolated using the Recover All Total Nucleic Acid Isolation Kit for FFPE (Ambion, Austin, TX, USA). Total RNA (200 ng) was assayed using the nCounter Human miRNA Assay v1.0 (Nanostring; 734 unique human and viral miRNAs). FFPE blocks from patients who did not have NPC (n=17) were used for normal nasopharyngeal epithelial tissues.
Cell Culture, Transfection, and Infection
The EBV-positive NPC cell line C666-1, the human normal nasopharyngeal cell lines NP69 (SV40-immortalized) and NP460 (hTert-immortalized), and HEK293T cells (used for virus purification and functional analysis), were cultured as previously described . Mycoplasma and STR analyses were also performed .
Polyplus-transfection JetPRIME (Graffenstaden, France) was used for C666-1, NP69, NP460, and HEK293T transfections, according to manufacturer’s specifications. C666-1, NP69, and NP460 cells were transfected with pre-miR-34c or pre-miR negative control (20 nM; Ambion, Austin, TX, USA). Lentiviral infection was used to generated stable cell lines as previously described. pLV-miRNA-34c (Biosettia, San Diego, CA, USA), pLV-miR-34c-lockers (Biosettia, San Diego, CA, USA), and their respective control vectors were used.
SB431542 (#S1067, SelleckChem, Houston, TX, USA), a TGFβ receptor I (TGFβR1, also known as ALK5) inhibitor, was used as indicated. Human TGFβ1 (#8915; Cell Signaling, Danvers, MA, USA) was used where indicated, after overnight starvation in Minimum Essential Media (MEM) supplemented with 0.5% FBS.
Quantitative Real-Time PCR (qRT-PCR)
The Total RNA Purification Kit (Norgen Biotek, Thorold, ON, Canada) was used for RNA isolation. Reverse-transcription of total RNA (1 μg) was performed using the iScript cDNA Synthesis Kit (BioRad, Hercules, CA, USA). qRT-PCR utilized SYBR Green (Roche, Basel, Switzerland) and the primers listed in Table 1. mRNA expression was normalized to the average expression of two housekeeping genes (β-actin and GAPDH, as in ). MicroRNA levels were assessed using the TaqMan MicroRNA Assay (Applied Biosystem, Foster City, CA, USA). RNU44 and RNU48 were used to normalize miR-34c expression. The 2-ΔΔ Ct method was used to calculate relative expression .
Immunoprecipitation buffer (150 mM NaCl, 5 mM EDTA, 50 mM Hepes pH 7.6, 1-2% Nonidet P-40; with protease inhibitor cocktail, Roche), was used for protein extraction. Electrophoresis was performed with a Bolt 4-20% Gel (Life Technologies, Carlsbad, CA, USA).
The Epithelial-Mesenchymal Transition Antibody Sampler Kit (Cell Signaling; #9782; 1/1 000 each), anti-TGFβ1 (Cell Signaling; #3711; 1/1 000), and anti-β-actin (Sigma: 1/5 000) antibodies were used. The SuperSignal West Femto ECL (Pierce, #34095, Thermo Scientific, Waltham, MA, USA) was used for ZEB1, CDH1 and ZO-1 detection. Otherwise, the Pierce ECL (#32209) was used.
RNA-Sequencing (RNA-Seq) and Data Analysis
RNA was isolated (200 ng/sample), processed (Ribo-Zero Gold rRNA Removal Kit (Illumina, San Diego, CA, USA)), and sequenced as previously described . Library preparation was performed using the TruSeq Stranded Total RNA Sample Prep Kit (Illumina, San Diego, CA, USA). Sequencing used the Illumina HiSeq 2000 to >100 million paired-end 100 bp reads. STAR (v2.4.2a) was used to align the reads , and RSEM (v1.2.21) was used to summarize expression values .
Luciferase Reporter Assay for MiR-34c/SOX4 Target Activity
miR-34c was predicted to target the wild-type (WT) 3’-untranslated region (3’UTR) of SOX4 in silico. This region was inserted into the pMIR-REPORT vector (Ambion). JetPRIME was used to reverse transfect HEK293T cells with pre-miR-control or pre-miR-34c. After one day, JetPRIME was used to co-transfect pRL-SV Renilla vector (Promega, Madison, WI, USA) with either pMIR-SOX4 3’UTR WT (CTAGTGCTCAGCTCAAGTTCACTGCCTGTCAGAT) or pMIR-SOX4 3’UTR Mutant (CTAGTGCTCAGCTCAAGTTTCTGTAAAGTCAGAT). One day later, the Dual-Luciferase Reporter Assay (Promega) was used to measure luciferase activity.
Cell Viability Assays
Cells were seeded in 96-well plates (2,000 cells/well). After one day, they were exposed to increasing concentrations of cisplatin (CDDP) for 72 h as indicated. Then, the ATPlite 1 Step Luminescence Assay System (PerkinElmer, Waltham, MA, USA) was used.
Sections from FFPE blocks were subject to IHC using microwave antigen retrieval. Citric acid (0.01 M, pH 6.0) and the LSAB+ System-HRP (Dako, Les Ulis, France) were used. Rabbit polyclonal anti-SOX4 (PA5-41442, lot#SB2344261A, Invitrogen: 1/40) antibody was used, but omitted for negative control staining. Positive nuclear SOX4 localization was detected by light microscopy. The percentage of positive tumor cells was quantified by evaluating a total of at least 300 tumor cells from the three most densely staining fields (magnification ×400). A final score was calculated as the product of percentage of positive tumor cell and staining intensity (0=negative; 1=weak; 2=moderate; 3=strong) as previously described . All scoring was performed blinded to any knowledge of clinical or pathological parameters. Every section was scored at least twice.
All experiments were performed at least three times. In order to maintain independence between replicates, new frozen batches of cells were used each time. Data are presented as the mean + SEM. GraphPad Prism (GraphPad Software, San Diego, CA, USA) was used for statistical analyses. Inter-group statistical significance was determined using the ANOVA test, with the Bonferroni post-test (if applicable), or the Mann-Whitney U test (socscistatistics.com).