Cell culture
PLAP positive cervical cancer (HeLa, SiHa and CaSki), PLAP negative hepatocellular carcinoma (HepG2) and non-PLAP, non-human Chinese hamster ovary (CHO) cell lines were used in this study. HeLa, HepG2 and CHO were obtained from American Type Culture Collection (ATCC) while SiHa and CaSki were obtained from National Centre for Cell Science (NCCS), Pune. Cells were cultured as per ATCC recommendations.
Construction of PLAP promoter/enhancer based reporter systems
Region of the PLAP promoter previously shown to drive tissue specific expression was cloned upstream to the pGl3 Basic luciferase plasmid (Promega, USA). In order to generate a hybrid clone of the above with NFκB enhancer, ten nucleotides of NFκB enhancer sequence (10 × 4 copies) were cloned upstream to the PLAP promoter. Details of the cloning are provided in the Additional file 1. NFκBEn–Pr+24-luc and PLAPPr+24-luc generated constructs were authenticated by restriction endonuclease digestion (Additional file 1: Figure S1A) and DNA sequencing. The sequence of PLAP promoter and enhancer elements are given in Additional file 2: Figure S2A.
Generation of TGS inducing system: PLAP promoter/enhancer + 2 HPV-16 E6/E7 shRNA
shRNA targeting NF-1 binding site on the enhancer region of HPV-16 LCR was designed using online siRNA wizard (http://www.sirnawizard.com/construct.php). 100 pico moles of sense and antisense oligonucleotides (with pre-added sticky ends; 5′BamHI and 3′HindIII) were annealed as described by Zakaria et al. [25]. Details of the directional cloning strategy so that the shRNA was located downstream to the (1) PLAP promoter alone or (2) NFκB–PLAP promoter are described in Additional file 1. All the clones were confirmed by restriction digestion and authenticated by DNA sequencing before being used for transfection (Additional file 1: Figure S1B, C). This generated the constructs—NFκBEn–Pr+2-HPV-16–E6/E7, PLAPPr+2-HPV-16–E6/E7, and their appropriate scrambled controls NFκBEn–Pr+2-HPV-16–E6/E7 Scr, PLAPPr+2-HPV-16–E6/E7 Scr. shRNA under CMV promoter, CMVPr–HPV-16–E6/E7, served as positive control.
Transfection
Cells were plated at 105 cells per well in a six-well plate, 3 × 105 cells per 25 cm2 flask or 106 cells per 75 cm2 flask (Corning, USA). Twenty-four hours later, they were transfected with different PLAP promoter based reporter or shRNA constructs using Lipofectamine™ 2000 (Invitrogen, USA). For preparing transfectants, required amount of plasmid DNA was mixed with opti MEM media in a microfuge tube and separately Lipofectamine™ 2000 was mixed with opti MEM keeping the final volume of each tube to 50 µl. Both the tubes were incubated for about 30 min followed by transferring the contents of DNA + opti MEM to the tube containing Lipofectamine + opti MEM. The tube was incubated again for 30 min. Meanwhile, cells were washed with opti MEM media and 900 µl of opti MEM was added to each well of a 6-well plate. The contents of the tube were then added into each well. Four hours later, DMEM containing 2× serum was added and the cells were incubated. The dose of Lipofectamine™ 2000 used per/µg of plasmid was 2.5 µl. The dose of the shRNA used was 1.8 µg/well of a 6-well plate.
Dual luciferase assay
All the three luciferase constructs: PLAPPr+24-luc, NFκBEn–Pr+24-luc and SV40-luc were transfected in a battery of cell lines. The passive lysis buffer, LAR II solution and Stop & Glo reagent were prepared as advised by the manufacturer (Promega, USA). Cells were plated onto 6-well plate and when 70% confluent, media was removed and cells were rinsed with PBS. 500 µl of passive lysis buffer was added into each well. Plate was kept on a rocker/shaker for 15 min to completely disrupt the cells. 20 µl of the resulting cell lysate was mixed with 100 µl of LAR II solution in a tube and luminescence was recorded. This was followed by addition of stop and glo solution (100 µl) and again the second readings were obtained. The firefly luciferase activity was normalized against Renilla luciferase activity and expressed relative to promoter-less pGl3-Basic control vector.
Real-time PCR
NFκBEn–Pr+2-HPV-16–E6/E7, PLAPPr+2-HPV-16–E6/E7, CMVPr–HPV-16–E6/E7 and their appropriate scrambled controls were transfected in cell lines by following transfection protocol as described above. Trizol (Sigma-Aldrich) reagent was used for isolation of RNA at requisite time points. In order to remove DNA contamination from the extracted RNA, it was treated with DNase (MBI Fermentas) and quantified by NanoDrop ND-1000 (Thermo Fisher Scientific). About 500–1,000 ng of RNA was used for preparing cDNA by using random decamer as primers. Moloney murine leukemia virus reverse transcriptase (MBI Fermentas) was used for preparing cDNA. Real time PCR was done on a RotorGene 6000 real-time PCR machine (Corbett Research, Australia). For quantitation of target genes, we used three reference genes as an internal control—18S, GAPDH and β-actin. Relative Expression Software Tool (REST) was used for relative quantitation. The list of primers used in all experiments is given in Additional file 2.
Cell proliferation assay
Overnight-cultured cells, 2 × 104 per well, in 24-well plates, were transfected with PLAP promoter/enhancer driven shRNA constructs or their respective scrambled controls. Cell proliferation was estimated on the 6th day. On the 6th day, 10 µl of MTT reagent (Sigma Aldrich) was added to each well and the plate was incubated for 2 h. 100 µl of solubilisation buffer was added and the plate was again incubated in the dark for 2 h. 100 µl of the solution from each well was transferred onto 96-well plate and absorbance was measured at 570 nm.
Apoptosis study
105 cells were seeded in 25 cm2 cell culture flask (Corning, USA) followed by transfection with various shRNA constructs. On the 6th day, 70% ice-cold ethanol was utilized for fixing the cells. Propidium Iodide (PI; Sigma-Aldrich, Germany) was used for staining and Flow cytometer (BD Biosciences, USA) helped to capture the fluorescence. Cell cycle analysis was done using WinMDI software (http://winmdi.software.informer.com/2.8/)
Western blotting
On the 6th day post transfection/immuno-virosomal delivery of shRNA constructs, cells were washed with PBS followed by lysis using triple lysis buffer [50 mmol/L Tris–Cl (pH 7.4), 150 mmol/L NaCl, 0.02% sodium azide, 0.1% SDS, 1% NP40, and 0.5% sodium deoxycholate]. Supernatants were extracted by centrifuging the lysates for 10 min. 5–12% SDS–PAGE gels were used for resolving equal quantities of protein followed by electro transfer on to nitrocellulose membranes. Blocking was done at room temperature using 4% BSA. Immunoblotting antibodies used were anti-actin (sc-8432) and anti-p53 (sc-126; Santa Cruz Biotechnology). Detection was done by ECL detection system (Applied Biosystems, USA) by using horseradish peroxidase labelled secondary antibodies.
Preparation of chimeric scFv targeted fusion (F) Sendai virosomes and loading of shRNA constructs
This is fully described by Kumar et al. [24]. In brief, the following process went into the generation of the scFv targeted Sendai virosome. (1) The scFv antibody that had been demonstrated to bind specifically to PAP isozyme was fused in frame with a portion of the Sendai virosome’s F protein containing a segment of its membrane spanning region. (2) The virosome was then reconstituted to include both the scFv linked F protein and the wild type F protein in a ratio of 1:5. (3) The DNA constructs were incorporated as required within the virosome during the process of reconstitution of the virosome components. (4) The appropriate cells were exposed to the scFv targeted virosomes loaded with DNA constructs as described by Kumar et al. [24] and Zakaria et al. [25]. A schematic diagram is given in Additional file 3. In brief, targeting by scFv results in the juxtaposition of the virosome to the PLAP expressing cell. The membrane of the virosome and the cell then fuse as a result of the wild type F protein, which then results in direct cytoplasmic delivery of the packaged DNA constructs.
Live cell fusion: kinetics of chimeric scFv-F-virosome fusion
1 mg/ml of Triton X-100 containing dialyzed and reduced Sendai virus envelope was mixed with 10 µl of ethanolic solution of octadecyl Rhodamine (R18) (1 mg/ml). This was vortexed and incubated in dark at room temperature for 30 min. Ultra-centrifugation, at 1,00,000g, was done to remove unbound R18 for 1 h at 4°C. Cells (1 × 106) were incubated with 2 μg of R18 labelled scFv virosomes for 1 h at 4°C and then centrifuged at 2,000 rpm for 5 min to remove unbound virosomes. The pellet was then suspended in 100 µl of cold 10 mM PBS. 50 µl of the labeled scFv-cell complex suspension was placed in a cuvette containing 3 ml of PBS with 1.5 mM Ca2+ (pre-warmed to 37°C). Kinetics of fusion was recorded online by a spectrofluorimeter (Horiba, USA). This is based on dequenching of a fluorescent dye R18 after fusion, with extent of dequenching being directly proportional to the virosome cell fusion (Additional file 3).
CpG methylation study
DNA was isolated post virosomal delivery of NFκBEn–Pr+2-HPV-16–E6/E7 or its scrambled control on the 6th day using Gen Elute Mammalian genomic DNA Miniprep Kit (Sigma-Aldrich, Germany). 500 ng of genomic DNA was bisulphite treated using EpiTect Bisulphite Kit (Qiagen, Germany). Bisulphite primers were designed from http://bisearch.enzim.hu/. Primers were M13-tagged for sequencing of PCR products.
Chromatin immunoprecipitation (ChIP) assay
ChIP assay for H3K9Me2 and H3K27Me3 was done using EZ ChIP kit (Millipore, USA) as per manufacturer’s protocol. Immunoprecipitated DNA was amplified using primers specific for target region of HPV-16 LCR. Immunoprecipitation percentage was calculated as described earlier [26]. Cells were pre-treated with Trichostatin A (TSA; Sigma-Aldrich, Germany; 300 nM) for 48 h followed by virosomal delivery of the NFκBEn–Pr+2-HPV-16–E6/E7 or its scrambled control.
Caspase 3/7 assay
Caspase-3/7 activity was determined post virosomal delivery of NFκBEn–Pr+2-HPV-16–E6/E7 or NFκBEn–Pr+2-HPV-16–E6/E7 Scr using caspase-3/7 assay kit (Promega, USA).
Statistical analysis
All experiments like dual luciferase assay, cell proliferation assay and RT-PCR were performed in triplicates and repeated thrice. Western blotting, fluorescence dequenching assay, Flow cytometric analysis, Bisulfite PCR, ChIP assay and capase 3/7 assay were repeated at least twice. Student’s t test was utilized to calculate the significance in all experiments and p < 0.05 was considered significant whereas p < 0.001 as highly significant. The data are shown as mean ± SD.
