Objective II:Evaluation Ofapotosis, Proliferation, Cellular Growthin Transfected Cells

2016 Phase II

Objective I: Transfection with specific siRNA/shRNA in human cervical carcinoma cell line Caski.

Objective II:Evaluation ofapotosis, proliferation, cellular growthin transfected cells.

Objective III:Transfection efficiency estimation (E6/E7 mRNA expression level in transfected cell culture).

Objective IV: Chromatin immunoprecipitation (ChIP) for MBD2, MBD3 in treated/untreated cells.

Objective I: Transfection with specific siRNA/shRNA in human cervical carcinoma cell line Caski.

In this phase one of the objectives was to silence E6 and E7HPV16 viral oncogenes in experimental model and for this purpose we started from the siRNA/shRNA sequences specifically build for targeted genes using bioinformatics methods in the previous phase and were designed and synthesized by LifeTechnology.

After analyzing the copy number of HPV16 DNA integrated in host genome/cell between two lines immortalized with HPV16 genotype, namely SiHa (1-2 copies/cell) and Caski (about 600 copies/cell) we chose as experimental model for further studies Caski cell line.

At this stage experiments were conducted in order to optimize silencing protocols and for this we tested two silencing strategies in order to obtain a higher percentage of knockdown.

The first strategy was to use shRNA molecules for E6 and E7 HPV16 knockdown in Caski cell line.

For this, shRNA specific sequenceswere cloned into a vector using BLOCK-iT U6 RNAi Entry Vectorkit (LifeTechnology). The kit allows a rapid and efficient cloning of shRNA sequences into a selected vector using as a method of cloning Gateway technology (Invitrogen) which is based on recombination site specific a property of bacteriophage λ.

The selected vector was pENTR/U6, that has 2854bp and contents a cassette that consists in RNA polymerase III (Pol III) special binding site a powerful promoter U6, this being the binding site for DNA insert sequence (fig 1).

Fig.1pENTRU6vector map (

The kit also contains a positive control that consists in a double-stranded oligo sequence, lacZthat can be clonated in pENTR/U6in order to generate a construct that express lacZ gene specific shRNA. By co-transferring the above generated vector and reporter plasmid pcDNA1.2/V5-GW/lacZinto mammalian cells, one can determine β-galactosidase geneknockdownpercent.

Shortly, the cloning work protocol in pENTR/U6 vector consists in following stages:

1. Generation and synthesis of two complementary single-stranded oligonucleotide sequences for each specific shRNA one of which encodes interest shRNA
2. Single-stranded oligonucleotide sequence annealing to generate double-stranded sequences (oligo ds).

Annealing reaction for each synthesised shRNA took place at room temperature by mixing together the following components (table 1):

Table 1. Reaction components list:

Component / Volum
oligoDNA“Top strand” (200 µM) / 5 µL
oligo DNA“Bottom strand” (200 µM) / 5 µL
10xoligo annealing buffer / 2 µL
DNA/RNA nuclease-free water / 8 µL
Final volume / 20 µL

The mixture was incubated at 94oC for 4 minutes, afterwards it was cooled at room temperature for 5-10 minutes, in this interval double-stranded oligonucleotides were synthesised. The samples were then centrifuged for 5 seconds and the mixture was diluted to 50µl concentration (at this concentration they can be stored at -20°C).The final concentration for cloning is 5nM.

For testing ds oligonucleotides integrity it was used electrophoretic migration in 4% agarose gel of5 ml aliquot of each of stock solution and5nM dilution for each sample.

Fig.24% agarose gel for testing interest shARN dc oligonucleotides (5 nM). Lane1- MW marker (10 pb); lane2 – shRNA-165 bottom strand oligo; lane3 – shRNA-165 top strand oligo; lane4 – shRNA-165 ds oligo; lane5 – shRNA-158 bottom strand oligo; lane6 – shRNA-158 top strand oligo; lane7 – shRNA-158 ds oligo; lane8 – shRNA-439 bottom strand oligo; lane9 – shRNA-439 top strand oligo; lane10 – shRNA-439 ds oligo; lane11 – shRNA-208 bottom strand oligo; lane12 – shRNA-208 top strand oligo; lane13 – shRNA-208 ds oligo; lane14 – shRNA-23 bottom strand oligo; lane15 – shRNA-23 top strand oligo; lane16 – shRNA-23 ds oligo; lane17 – shRNA-lacZ ds oligo; lane18 –MW marker (10 pb).

Figure 2 shows the foto image of electrophoretic migration of the samples; it is confirmed the ds oligonucleotide sequences presence – the larger MW (lane 4; lane 7; lane 10; lane 13; lane 16) for all the 5interest shRNA, as well as for the positive controllacZ (lane 17).

1. Ds oligo cloningin linearized by ligation reaction pENTR/U6 vector. Each shRNA generated ds oligonucleotidic sequences are introduced in cloningvector via ligation reaction.In table 2 are listed ligation reaction components.

Table 2. Reaction components list

Component / Volume
5x ligation buffer / 4 µL
pENTR/U6 vector(0,5 ng/ µL) / 2 µL
dsoligo insert (5 nM) / 1 µL
DNA/RNA nuclease-free water
T4 ligase( 1U/ µL) / 12 µL
1 µL
final volume / 20 µL

Reaction takes place by incubation at room temperaturefor 5 minutes up to 2 hours for a better ligation.

1. Insert transformation in E. coli competent cells (One Shot TOP10) followed by transformers kanamycin resistance selection.

The ligation reaction product is transformed afterwards in E. coli competent cells. As a positive control pUC19 plasmid is used - 10 pg (1µL) for one tube of competent cells.

For sample transformation, 2 µL ligation product is added in one tube of competent cells, mixed and incubated on ice 5-30 minutes.In the following step the cells are thermally shocked by incubation at 42oC for 30 seconds without mixing, then they are transferred on iceand 250µLS.O.Cmediumis added in each tube, followed by incubation at 37oC with shaking (200rpm) for an hour. 20-100 µLfrom each transformation product are seed in soft LB agar plates containing 50µg/ml kanamycinand incubated overnight at 37oC.

Transformers / positive clone analyses was performed selecting 5-10kanamycin resistant colonies incubated overnight (37oCand 300 rpm) in 2-5 mL LB medium continand containing 50µg/ml kanamycin, afterwards plasmid DNA was isolated and purified withHiSpeed plasmid purification maxi kit (Qiagen).

Working protocol was as follows:

1. Previous culture inoculum in 150 ml LB mediumis incubated for 12-16 hours at 37oC and 300 rpm , leading to a cellular density of approx. 3-4 x 109/ml.
2. Bacterial cells are harvested by centrifuging at 6000g/15 min./4oC.
3. For cellular lyses the bacterial pellet is resuspended in 10ml P1buffer by vortexing.
4. 10ml P2 buffer is added and mixed by 4-6 times inverting, followed by 5 minutes room temperature incubation.
5. 10ml cold P3 bufferis added over lysate and mixed by 4-6 times inverting, followed by 10 minutes room temperature incubation.
6. QIAfilter is prepared and the lysate is filtered.
7. HiSpeed Maxi filter is set and prepared by 10ml QBT buffer washing.
8. Lysate filtration.
9. Filter is washed with 60ml QC buffer.
10. The DNA is eluted with 15ml QF buffer in a 30ml capacity tube.
11. The DNA is precipitated by 10,5ml isopropanol followed by 5 minutes room temperature incubation.
12. DNA is syringed through QIAprecipitator.
13. 2ml 70% ethanol is added in QIAprecipitatorand the filter is air dried with the syringe.
14. The DNA is eluted with 1ml TE buffer.
15. Plasmid DNAconcentration is quantified by NanoDrop.
16. Plasmid DNAmay be stored at -20oC until use.

For construct specific shRNA sequences presence confirmation the samples were sequenced.

Fig.3shRNA-158 insert sequences presence confirmation by sequencing

Fig.4hRNA-208 insert sequences presence confirmation by sequencing

By sequencing it was confirmed the presence, sequence and the sense for all the 5 tested constructs and it was validated their further use in Caski cell line transfection experiment.

5.Transfection with specifficE6 and E7shRNAsin Caski cell line

“Pre-plate” technique was chosen for transfection, for this 1.5x105 Caski cells were cultured in 25 cm3flasks in RPMI medium(Gibco) with 10% foetal serumand 1% L-Glutamine, without antibiotic, until 70% confluence.

Caski cells were transfected with 3different concentrations(4, 6 si 8µg) from the 5 constructsLipofectamine 2000 (Invitrogen) and OptiMEM (Invitrogen) presence, following manufacturer's recommendations.In the same experiment untreated cells were used as cells controls and transfection medium treated cells were used as controls as well. In 4 hours following transfection, lipofectamine was removed for avoiding possible cytotoxic effects.

Transfection with specifficE6 and E7siRNAsin Caski cell line

The second method used for E6 and E7 viral oncogenes silencing was specific siRNA transfection of Caski cell line using Silencer siRNA kit (Ambion; Lifetechnologies) and lipofectamine as transfection agent (siPORT NeoFX, Ambion; Lifetechnologies)in “pre-plate” technique (this technique consists in adherent cells culturing 24 hours before transfection). The experiment started with 2x105 Caski cellsand two different specific siRNA concentrations (75nM and 100nM). As controls were used transfection medium (Opti-MEM I) treated cells, siRNA control treated cells and untreated cells.

Transfection cells protocol using siRNA was as follows:

1. Growing cells (2x105 celule Caski) inRPMI (Gibco) 6-well plates with 10% foetal serum and 1% L-Glutamine, without antibiotic, 24 hours before transfectionuntil 50-60% confluence.
2. Preparing siRNA transfection agent complexes as follows:

• siPORT NeoFX transfection agent is vortex before use and diluted Opti-MEM Iby adding 3μL siPORT NeoFXdrop-by-drop in Opti-MEM Iuntil 25μL final volume. The mixture is vortexed and incubated at room temperature for 10-15 minutes.
• 0,25-7,5μL of 2μM siRNA stoc solution is diluted (until 1-100 nM) in Opti-MEM I to 25μL final volume.
• Diluted siRNAis mixed withdilutedsiPORT NeoFX by inverting the tube or by pippeting and the mixture is incubated at room temperature for 10-15 minutes.

1. Caski cells transfection is realised by adding the previous obtained mixture drop-by-drop to the cells (until 500µL final transfection volume). The plate is afterwards gently shacked to assure an evan distribution and then the cells are incubated in normal culture conditions for 24/48 hours; during this time (after 8-48 hours) 0,5-1ml normal culture medium can be added in each well to facilitate cellular growth and avoid a possible cytotoxicity of the complexes.

Activity II: Evaluation of cellular growt, proliferation, apoptosis and senescence in transfected cells.

The apoptosis rate in transfected Caski cells was estimated by the method of incorporation of propium iodide (PI); stained cells were then analyzed in flow cytometry.The results showed that siRNA/shRNA treatement may induce an early apoptosis vs. cells controls. La Rezultatele au aratat ca tratamentul cu poate induce o crestere a apoptozei timpurii prin comparatie cu controlul de celule. Cells undergoing late apoptosis/necrosis had unsignificant changes (figura 5).

Fig5. Flow cytometry results

Activitate III: Transfection efficiency detection (E6/E7 mRNA expression level in transfected cells cultures).

Transfection efficiency estimation was done comparingthe two tested silencing methods by assessing E6 and E7 mRNA expression levels in transfected cell cultures vs controls at 24 and 48 hours.

Total RNA was isolated from cells using Trizol (Invitrogen) and qualitatively and quantitatively assessed for each sample.

For cDNA synthesis Transcriptor First Strand cDNA Synthesis kit (Roche) was used. E6/E7 mRNAswere quantified in Real-Time PCR using specific primers and FastStart SYBR Green (Roche).

Table 3.Sequences of the used primers

Gene / primer sense / primer antisense
E6 HPV16 / 5’-GCATAAATCCCGAAAAGCAA-3’ / 5’-AGCGACCCAGAAAGTTACCA-3’.
E7 HPV16 / 5’-GCTCAGAGGAGGAGGATGAAATAG-3’ / 5’-TCCGGTTCTGCTTGTCCAG-3’

All samples were tested in triplicate. In order to determine E6 mRNA level and the remained E7HPV16 following treatment with specific shRNA/siRNA, relative quantification method was used (Ct) using GAPDH as reference gene. The obtained values were further normalized also to controls (untreated cells Caski) (Ct).

The shRNA/siRNA trasnsfection efficiency was used with the formula:

% knockdown = 100 – 100 X 2–Ct E7/E6 HPV16

Ct = Ct E7/E6 HPV16– Ct GAPDH;

Ct = Ct E7/E6 HPV16–Ct cells controle

Diferent knockdown percentages (46,5 - 61,2%) were obtained for siRNA-E7 HPV16 and siRNA-E6 HPV16 transfection in Caski cells in two diferent concentrations and two different time intervals (figure6). The best results were obtained for E7 gene at 75 nM and 24 hours post-transfection. (figure6B).

Fig.6Knockdown percentage in Caski cell line treated with siRNA-E6HPV16 (A) and siRNA-E7HPV16 (B) at 24/48 hours post-transfectionat 75 and100 nM concentrations.

The results for Caski cells transfection with the 5 specific shRNA constructs varied between 59,7- 82,5% efficiency, the best conditions were 6 µg concentration, 24 hours post-transfection.(figures 7-11).

Fig.7Knockdown percentage in Caski cell line treated with 4, 6 and 8 µg shRNA-165 at 24/48 hours post-transfection.

Fig.8Knockdown percentage in Caski cell line treated with 4, 6 and 8 µg shRNA-158 at 24/48 hours post-transfection.

Fig.9 Knockdown percentage in Caski cell line treated with 4, 6 and 8 µg shRNA-439 at 24/48 hours post-transfection.

Fig.10Knockdown percentage in Caski cell line treated with 4, 6 and 8 µg shRNA-208 at 24/48 hours post-transfection.

Fig.11Knockdown percentage in Caski cell line treated with 4, 6 and 8 µg shRNA-23 at 24/48 hours post-transfection.

Comparing all these results of the two E6 / E7 HPV16 silencing methods in experimental modelCaski cell linethe following conclusion emerges: using shRNA molecules in gene silencing leads to a higher knockdown percentage vs siRNA molecules, this recommanding this approach for gene silencing experiments.

Activitate IV: ChiP experiments for MBD2, MBD3 in treated and untreated cells.

Cell Culture Cross-linking and Sample Preparation: For optimal ChIP results, use approximately 4 X 106 cells for each immunoprecipitation to be performed.

Before starting:

• Remove and warm 200X Protease Inhibitor Cocktail (PIC) #7012 and 10X Glycine

Solution #7005. Make sure PIC is completely thawed.

• Prepare 2 ml of Phosphate Buffered Saline (PBS) + 10 μl 200X PIC per 15 cm dish

to be processed and place on ice.

• Prepare 40 ml of PBS per 15 cm dish to be processed and place on ice.

• Prepare 540 μl of 37% formaldehyde per 15 cm dish of cells to be processed and

keep at room temperature.

To crosslink proteins to DNA, add 540 μl of 37% formaldehyde to each 15 cm culture

dish containing 20 ml medium. Swirl briefly to mix and incubate 10 min at room

temperature. Final formaldehyde concentration is 1%. Addition of formaldehyde may

result in a color change of the medium.

Add 2 ml of 10X glycine to each 15 cm dish containing 20 ml medium, swirl briefly

to mix, and incubate 5 min at room temperature.

For suspension cells, transfer cells to a 50 ml conical tube, centrifuge at 1,500 rpm

in a benchtop centrifuge 5 min at 4°C and wash pellet two times with 20 ml ice-cold

PBS. Remove supernatant and immediately continue with Nuclei Preparation and

Chromatin Digestion (Section II).

For adherent cells, remove media and wash cells two times with 20 ml ice-cold 1X

PBS, completely removing wash from culture dish each time.

Add 2 ml ice-cold PBS + PIC to each 15 cm dish. Scrape cells into cold buffer.

Combine cells from all culture dishes into one 15 ml conical tube.

6. Centrifuge cells at 1,500 rpm in a benchtop centrifuge for 5 min at 4°C.

II. Nuclei Preparation and Chromatin Digestion

Before starting:

• Remove and warm 200X Protease Inhibitor Cocktail (PIC) #7012. Make sure it is

completely thawed prior to use.

• Prepare 1 M DTT (192.8 mg DTT #7016 + 1.12ml dH2O). Make sure DTT crystals

are completely in solution.

• Remove and warm 10X ChIP Buffer #7008 and ensure SDS is completely in solution.

• Prepare 1 ml 1X Buffer A (250 μl 4X Buffer A #7006 + 750 μl water) + 0.5 μl 1M

DTT + 5 μl 200X PIC per IP prep and place on ice.

• Prepare 1.1 ml 1X Buffer B (275 μl 4X Buffer B #7007 + 825 μl water) + 0.55 μl 1M

DTT per IP prep and place on ice.

• Prepare 100 μl 1X ChIP Buffer (10 μl 10X ChIP Buffer #7008 + 90 μl water) + 0.5 μl

200X PIC per IP prep and place on ice.

Resuspend cells in 1 ml ice-cold 1X Buffer A + DTT + PIC per IP prep. Incubate on ice for 10 min. Mix by inverting tube every 3 min.

Pellet nuclei by centrifugation at 3,000 rpm in a benchtop centrifuge for 5 min at 4°C.

Remove supernatant and resuspend pellet in 1 ml ice-cold 1X Buffer B + DTT per IP

prep. Repeat centrifugation, remove supernatant, and resuspend pellet in 100 μl 1X

Buffer B +DTT per IP prep. Transfer sample to a 1.5 ml microcentrifuge tube, up to

1 ml total per tube.

Add 0.5 μl of Micrococcal Nuclease #10011 per IP prep, mix by inverting tube several times and incubate for 20 min at 37°C with frequent mixing to digest DNA to length

of approximately 150-900 bp. Mix by inversion every 3 to 5 min.

4. Stop digest by adding 10 μl of 0.5 M EDTA #7011 per IP prep and placing tube on ice.

5. Pellet nuclei by centrifugation at 13,000 rpm in a microcentrifuge for 1 min at 4°C and remove supernatant. Resuspend nuclear pellet in 100 μl of 1X ChIP Buffer + PIC per IP prep and incubate on ice for 10 min. Sonicate up to 500 μl of lysate per 1.5 ml microcentrifuge tube with several pulses to break nuclear membrane. Incubate samples for 30 sec on wet ice between pulses. Optimal conditions required for complete lysis of nuclei can be determined by observing nuclei under light microscope before and after sonication.

8.Clarify lysates by centrifugation at 10,000 rpm in a microcentrifuge for 10 min at 4°C.

9.Transfer supernatant to a new tube. This is the cross-linked chromatin preparation, which should be stored at -80°C until further use. Remove 50 μl of the chromatin preparation for Analysis of Chromatin Digestion and Concentration (Section III).

III. Analysis of Chromatin Digestion and Concentration

1. To the 50 μl chromatin sample (from Step 9 in Section II), add 100 μl nuclease-free

water, 6 μl 5 M NaCl #7010, and 2 μl RNAse A #7013. Vortex to mix and incubate

samples at 37°C for 30 min.

1. To each RNAse A-digested sample, add 2 μl Proteinase K. Vortex to mix and incubate samples at 65°C for 2 h.
2. Purify DNA from samples using DNA purification spin columns as described in

Section VI.

1. After purification of DNA, remove a 10 μl sample and determine DNA fragment size

by electrophoresis on a 1% agarose gel with a 100 bp DNA marker. DNA should be

digested to a length of approximately 150-900 bp.

1. To determine DNA concentration, transfer 2 μl of purified DNA to 98 μl nuclease-free water to give a 50-fold dilution and read the OD260. The concentration of DNA in μg/ml is OD260 x 2,500. DNA concentration should ideally be between 50 and 200 μg/ml

IV Chromatin Immunoprecipitation

Before starting:

• Remove and warm 200X Protease Inhibitor Cocktail (PIC) #7012. Make sure PIC is

completely thawed.

• Remove and warm 10X ChIP Buffer #7008 and ensure SDS is completely in solution.

• Thaw digested chromatin preparation (from Step 9 in Section II) and place on ice.

• Prepare low salt wash: 3 ml 1X ChIP Buffer (300 μl 10X ChIP Buffer #7008 + 2.7 ml

water) per immunoprecipitation. Store at room temperature until use.

• Prepare high salt wash: 1 ml 1X ChIP Buffer (100 μl 10X ChIP Buffer #7008 +

900 μl water) + 70 μl 5M NaCl #7010 per immunoprecipitation. Store at room

temperature until use.

1.In one tube, prepare enough 1X ChIP Buffer for the dilution of digested chromatin into

the desired number of immunoprecipitations: 400 μl of 1X ChIP Buffer (40 μl of 10X

ChIP Buffer #7008 + 360 μl water) + 2 μl 200X PIC per immunoprecipitation. When

determining the number of immunoprecipitations, remember to include the positive

control Histone H3.

2.To the prepared 1X ChIP Buffer, add the equivalent of 100 μl (5 to 10 μg of chromatin)

of the digested, cross-linked chromatin preparation (from Step 9 in Section II) per

immunoprecipitation. For example, for 10 immunoprecipitations, prepare a tube

containing 4 ml 1X ChIP Buffer (400 μl 10X ChIP Buffer + 3.6 ml water) + 20 μl 200X

PIC + 1 ml digested chromatin preparation.

3.Remove a 10 μl sample of the diluted chromatin and transfer to a microfuge tube. This

is your 2% Input Sample, which can be stored at -20°C until further use (Step 1 in

Section V).

4.For each immunoprecipitation, transfer 500 μl of the diluted chromatin to a 1.5 ml

microcentrifuge tube and add the immunoprecipitating antibody. The amount of

antibody required per IP varies and should be determined by the user. For the positive

control Histone H3.Incubate IP samples 4 h to overnight at 4°C with rotation.