Silencing of Phosphoinositide-specific Phospholipase C ε Remodulates the Expression of the Phosphoinositide Signal Transduction Pathway in Human Osteosarcoma Cell Lines

Materials and Methods

Cell culture. 143B and Hs888 human osteosarcoma cell lines, obtained from the American Type Culture Collection (Rockville, MD, USA), were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10%-15% foetal bovine serum (FBS), 1 mM sodium pyruvate, 100 U/ml of penicillin, and 100 mg/ml of streptomycin. Confluent monolayer cells were detached with 0.25% trypsin and counted using a Neubauer haemocytometer (Weber Scientific International Ltd., Middlesex, UK).

Cell survival trypan blue test. The number of viable cells was determined by adding 0.4% trypan blue staining (Sigma Aldrich, Dorset, UK) to an equal volume of cell suspension; a growth curve was designed counting the cells per square centimeter at different times. The following equation was used to calculate the number of viable cells in 1 ml suspension: number of viable cells in 1 ml , when is the average of the cell counts from the squares of the haemocytometer grid, and 2 is the dilution factor (1:1). Student's t-test was used to evaluate the statistical significance between the survival rate of transfected cells versus metafectamine-transfected cells or versus non-transfected cells.

Cell transfection for PLCE silencing. Cells were transiently transfected using METAFECTENE SI+ (Biontex, Munich, Germany). silencing RNA (siRNA) sequences targeting PLCE and negative control siRNA, obtained from Invitrogen (Life Technologies, Foster City, CA, USA), according to PLCE complementary DNA (cDNA) sequence (PLCE Gene ID: 51196). A suspension containing 1.5×10⁵ cells/ml of 143B cells or 3×10⁵ cells/ml of Hs888 cells was mixed with150 μl of 1× SI+ buffer, 72 μl of METAFECTENE® SI+ and 540 pMol of RNA stock solution within one hour from seeding. PLCE transcription was measured by reverse transcription–polymerase chain reaction (RT-PCR) and PI-PLC protein by western blot analysis 24, 48 and 72 h after transfection.

RNA extraction. RNA was extracted using SV Isolation System (Promega, Madison, WI, USA) according to the manufacturer's instructions. Cells suspension was incubated with 175 μl of Lysis Buffer, then Dilution Buffer (350 μl) was added and centrifuged for 10 min at 14,000 × g. After the addition of 200 μl 95% ethanol, the mixture was transferred to a spin-column assembly, and centrifuged for one minute. The liquid was discarded, 600 μl of Wash Solution was added, centrifuged for one minute, and the collection tube was emptied. DNase incubation mixture (40 μl of Yellow Core Buffer, 5 μ1 of 0.09 M MnCl₂ and 5 μl of DNase I enzyme) was added. After 15 min incubation, 200 μl of Stop Solution was added, the mixture centrifuged for 1 min, and 600 μl of Wash Solution was added and centrifuged for one minute. The collection tube was emptied, 250 μl of Wash Solution was added, and the mixture centrifuged for 2 minutes. Nuclease-Free Water was added to an elution tube containing the spin basket, centrifuged for 1 min and RNA was eluted into a sterile tube with RNase-free water. The concentration and quality of the RNA was monitored using a NanoDrop ND-1000 Spectrophotometer (Thermo Fisher Scientific, Inc., Waltham, MA, USA).

RT-PCR. RNA was reverse-transcribed into cDNA using High-Capacity cDNA Reverse Transcription Kit (Life Technologies). Briefly, 2μg RNA were incubated with the master mix (2 μl of 10× Reverse Transcription Buffer, 0.8 μl of 25 x dNTPs (100 mM), 2 μl of 10× random primers, 1 μl of MultiScribe™ Reverse Transcriptase (50 U/μl) and 3.2 μl of DNase-free water). Ten microlitres of diluted RNA was added to 20 μl final volume and reverse transcribed for 10 min at 25°C, 120 min at 37°C and 5 minutes at 85°C in a Gene Amp® PCR System 9700 (Applied Biosystems) thermocycler. Standard analytical PCR reaction was performed with GoTaq Master Mix (Promega) with 5× GoTaq buffer, 0.2 μM primers pairs (Bio Basic Inc, Amherst, NY, USA (Table I), 0.2 mMdNTPs, 0.5 mM MgCl₂, 1.25 U GoTaq and 3.5 μl (about 35 μg) of template cDNA to 50 μl final volume. Cycling was performed with a 95°C initial denaturation step, followed by 40 cycles of denaturation at 95°C (30 sec), annealing (30 seconds) at the appropriate temperature for each primer pair and 72°C extension (1 min). PCR products were visualized by 1.5% ethidium bromide-stained agarose gel electrophoresis at 100 V using UV light transilluminator.

Real-time PCR. TaqMan® primers and probes for each gene, as well as the glyceraldehyde 3 phosphate dehydrogenase gene (GAPDH) reference gene, were obtained from Applied Biosystems. Reaction mixtures contained: 5 μl TaqMan® mastermix (2×; Applied Biosystems), 0. 5 μl primer/probe mix specific for each analyzed gene, 1 μl PCR-grade water and cDNA (3.5 μl, 35 ng). After incubation for 2 min at 50°C and 10 min at 95°C, the reaction was carried out for 40 cycles at 95°C for 15 seconds and 60°C for 1 minute. The reaction was carried out in triplicate on 96-well plate using a 7500 Real-Time PCR ABI PRISM (Applied Biosystems™) and products were evaluated using ABI PRISM 7500 software. The cycle threshold (Ct) values for each set of three reactions were averaged for calculations. The 2^−ΔΔIS method was used to calculate relative changes in gene expression. PCR product concentrations of transfected and non-transfected cells were compared with student's one tailed t-test using Prism 5.0a software (GraphPad Software, San Diego, CA, USA). A p-value of less than 0.05 was considered significant.

Western blot. Western blot analyses were conducted 24 and 48 hours from transfection and in non-transfected controls. Cells were processed in lysis buffer [50 mMTris-HCl, 150 mMNaCl, 2 mM EDTA, 1% NP-40, 2 mM sodium fluoride, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate (SDS) containing protease inhibitors]. Fifty micrograms of protein was separated by 10% SDS–polyacrylamide gel electrophoresis (PAGE) and transferred onto nitrocellulose membranes (Invitrogen), blocked with 5% skimmed milk for 1 hour and incubated overnight with primary antibodies directed against PI-PLC enzymes. Membranes were stained adding appropriate secondary antibodies (Jackson Immunoresearch Laboratories, Newmarket, Suffolk, UK) withchemiluminescence. Expression of β-actin was used as an internal control to normalize results. The densities of the bands on the membrane were scanned and analysed with ImageJ software (NIH Image J version 1.38 software;http://rsb.info.nih.gov/ij/).

Immunofluorescence analysis of subcellular distribution of target molecules. Immunofluorescence detection of PI-PLCε, PI-PLCβ1, PI-PLCγ2, PI-PLCδ4 and ezrin was performed on coverslip-cultured transfected and non-trasfected cells, fixed with 4% paraformaldehyde, incubated with primary antibodies directed against each PI-PLC isoform (Santa Cruz Biotechnologies Inc, Santa Cruz, CA, USA) for 1 h and then with the specific secondary antibody Texas Red or fluorescein-conjugated for 1 hour. Slides were counterstained with 4’,6-diamidino-2-phenylindole (DAPI) and visualized using an inverted microscope.