2.1 Materials. The transfection reagents Prime-Fect, Leu-Fect A, Leu-Fect C and Trans-Booster (will be referred as ‘additive’ from hereon) were purchased from RJH Bioscience Inc. (Edmonton, AB, Canada). Lipofectamine™ 2000 transfection reagent was purchased from ThermoFisher. Hank’s Balanced Salt Solution (HBSS), Dulbecco’s Modified Eagle Medium (DMEM) and Roswell Park Memorial Institute (RPMI) medium were purchased from Gibco (USA). 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyltetrazoliumbromide (MTT), and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO). Matrigel was obtained from Corning (Oneonta, NY). SensiFAST™ cDNA synthesis kit and SensiFAST™ SYBR Hi-ROX kit were purchased from Bioline (ON, CA). Mcl-1 siRNA (5’-CGCCGAAUUCAUUAAUUUA[dT][dT]-3’) was purchased from Sigma-Aldrich (St. Louis, MO). Survivin siRNA (Cat. No.HSC.RNAI.N001012271.12.1), CDC20 siRNA (5’-GGAUCAAAGAGGGCAACUACUUGGC-3’), HSP90 siRNA (5’-AUCUGAUUACCUUGAAUUGGAUACA-3’), the negative control scrambled siRNA (CsiRNA; Cat. no. 308480258), 6-carboxyfluorescein (FAM)-labeled scrambled siRNA, primers for Mcl-1 (forward: 5’-CCTTTGTGGCTAAACACTTGAAG-3’; reverse: 5’-CGAGAACGTCTGTGATACTTTCTG-3’), Survivin (BIRC5) (forward: 5’-TGAGAACGAGCCAGACTTGG-3’; reverse: 5’-ATGTTCCTCTATGGGGTCGT-3’) and Beta-Actin (forward: 5’-CCACCCCACTTCTCTCTAAGGA-3’; reverse: 5’-AATTTACACGAAGCAATGCTATCA-3’) were purchased from IDT Technologies (Coralville, IA).
2.2 Cell Culture. MDA-MB-436 breast cancer cell line was cultured in DMEM medium containing 10% fetal bovine serum (FBS), 100 U/mL of penicillin, and 100 µg/mL of streptomycin under a humidified atmosphere with 5% CO2 at 37°C. When cells were ~ 80% confluent, the spent medium was removed followed by rinsing the monolayer once with sterile 1X HBSS (pH 7.4). Cell monolayers were incubated at 37°C for 3 mins with 1 mL of 0.25% trypsin/EDTA to promote cell dissociation. The suspended cells were centrifuged at 600 rpm for 5 min and resuspended in 10 mL of fresh medium after removing the supernatant. The cells were sub-cultured at a split ratio of 1:9 for subsequent passage and remaining cells were seeded into 96-well plates for transfection experiments.
2.3 siRNA Complex Preparation. The Trans-Booster additive was added to siRNA that was dissolved in RPMI at a 1:1 siRNA:additive ratio (w/w) with a final siRNA concentration of 40 or 80 nM in the tissue culture medium. The desired transfection reagents were then added at a 1:5 or 1:10 (w/w) transfection reagent:siRNA ratio and the mixture was then incubated for 30 min at room temperature to allow for optimal complexation[22]. Table 1 summarizes preparation of typical complexes.
Table 1
The examples of siRNA polyplex preparation. The amounts are indicated for one well of 96-well plate and multiplied based on the total numbers of wells to be treated.
siRNA Conc, siRNA/Transfection reagent/ PAA
|
Transfection volume
(µL)
|
RPMI
(µL)
|
siRNA
(0.14 µg/µL)
|
PAA
(0.14 µg/µL)
|
Transfection reagent
(1 µg/µL)
|
80 nM, 1:10:0
|
15
|
11.83
|
1.32
|
0
|
1.85
|
80 nM, 1:10:1
|
15
|
8.66
|
1.32
|
1.32
|
3.7
|
40 nM, 1:10:0
|
15
|
13.42
|
0.66
|
0
|
0.92
|
40 nM, 1:10:1
|
15
|
11.83
|
0.66
|
0.66
|
1.85
|
2.4 Complex Size and Zeta (ζ)-Potential. The ζ-potential of siRNA complexes was measured by LITESIZER 500 (Anton Paar, Austria). Mcl-1 and scrambled siRNA were formulated into various complexes using siRNA:Prime-Fect:additive ratios of 1:10:0, 1:10:1, 1:5:0, and 1:5:1 (w/w/w) and yielding a final concentration of 80 nM siRNA in 20 µL RPMI. The complexes were then incubated for 30 mins at room temperature. Prior to the size and ζ-potential measurements, the prepared complexes were further diluted with RPMI to a final volume of 1 mL before each measurement. The instrument was set at a 175° back scatter and 25°C.
2.5 siRNA Binding by SYBR Green Dye Exclusion Assay. The SYBR Green II stain was used to test the ability of the transfection reagents to bind siRNA. In black 96-well plates, 200 µL of SYBR Green II (1X) was added to each well, followed by 4 µL of 0.025 µg/µL scrambled siRNA. Different volumes of 0.005 µg/µL of the transfection reagent (diluted from 1 mg/mL stock solution) were added to wells to achieve transfection reagent:siRNA ratios ranging from 1:1 to 70:1 (w/w). The plate was incubated for 30 mins in the dark at room temperature and the fluorescence of the samples was then measured with a multiwell plate reader (Thermo Ascent; λex 485 nm, λem 527 nm) to determine the amount of free siRNA. The percentage of bound siRNA vs. transfection reagent:siRNA ratio (w/w) was plotted to compare the relative siRNA binding efficiency of different transfection reagents.
2.6 Cell Viability Assay. MTT Assay was used to assess the effect of Mcl-1 siRNA treatment in MDA-MB-436 cells. The treatment was carried out in complete medium (DMEM/10% FBS/Pen-Strep) in triplicates in 96-well plates. 6×103 cells in a volume of 150 µL were seeded in wells and incubated for 24 h at 37°C in 5% CO2 atmosphere. The siRNA complexes were prepared as described above and 15 µL of siRNA polyplexes were added to the wells to give a final siRNA concentration of 40 to 80 nM. The cells were typically incubated for 3 or 6 days after polyplex treatment. At the desired timepoint, MTT solution (5 mg/mL) was added to the cells so as to attain a final concentration of 1 mg/mL in each well. The plates were incubated for an additional 2 h at 37°C. The residual MTT medium was removed, and the formazan crystals formed were dissolved with DMSO. The absorbance (450 nm) was measured using a microplate reader (Molecular Devices, Spectramax 250) with pure DMSO serving as a blank solution. Percent cell viability was calculated with respect to the absorbance of non-treated cells. Analysis of Variance (one-way ANOVA) was applied to determine statistically significant differences among the study groups, with a confidence interval of 95% (p-value < 0.05) (GraphPad, version 8.0, San Diego, CA). Tukey’s post hoc test was also performed for comparing all possible pairs of means in all groups.
2.7 Analysis of Apoptosis. MDA-MB-436 cells were seeded (8×104 cells/well) in 24-well plates for 24 hours before being treated with 40 nM and 80 nM control, Mcl-1, survivin and Mcl-1/survivin siRNA complexes for 72 h. The cells were washed once with HBSS, then collected and transferred to 1.5 mL centrifuge tubes after Accutase® digestion, washed (x2) with 1X binding buffer, and centrifuged for 5 mins at 900 rpm. Cells were then resuspended in 300 µL of binding buffer and transferred to 5 mL polystyrene round-bottom tubes. Each sample was incubated with 2.5 µL of FITC-Annexin V and 2.5 µL of Propidium Iodine (Apoptosis kit from BD Biosciences) for 15 mins before fluorescence measurement by flow cytometry.
2.8 Real-time RT-qPCR for Gene Silencing. MDA-MB-436 cells (3.2 × 105 cells/well) were seeded in triplicates in 12 well plates and incubated for 24 h at 37°C in 5% CO2. The cells were treated with siRNA complexes in complete media for 3 days. After washing (x2) cells with HBSS, 0.5 mL TRIzol reagent was added to isolate total RNA. The lysates were incubated at room temperature for 5 mins, followed by addition of 100 µL chloroform, and centrifuged for 15 mins at 8900 rpm at 4°C. The upper phase was transferred to a new tube and mixed with 250 µL of isopropanol. The mixture was centrifuged for 15 mins at 8900 rpm at 4°C[27]. The total RNA pellet was stored in 1 mL of 75% ethanol overnight at -20°C and then centrifuged at 7500 rcf for 5 mins. The pellet was then air-dried for 30 mins, resuspended in nuclease-free water, and then placed in a water bath at 60°C for 10 mins. One µg of extracted RNA was converted into cDNA by using the SensiFAST™ cDNA Synthesis kit as per the instructions of the manufacturer (Meridian Bioscience, OH, USA). For real time RT-qPCR analysis, human Beta-actin was used as an endogenous housekeeping gene. All RT-qPCR reactions were performed by the reaction mixtures incubated at 95°C for 10 mins followed by 40 amplification cycles at 95°C for 15 s, 65°C for 1 min using StepOnePlus (Applied Biosystems, CA, USA) real time thermal cycler. The fold change was calculated by the ΔΔCt method. Significant differences among study groups were analyzed using one-way ANOVA (GraphPad, v8.0; San Diego, USA), followed by Tukey’s multiple comparison tests with significance level set at p < 0.05.
A microarray analysis was used to further inspect human apoptotic gene expression (RT2 Profiler™ PCR Array Human Apoptosis; PAHS-012ZC) in treated cells. RNA extraction from treated cells and conversion of extracted RNA to cDNA was carried out as mentioned above. The RT2 Profiler™ PCR ArrayHuman Apoptosis plate was loaded with the SensiFAST™ SYBR Hi-ROX mastermix and cDNA template (5 ng/µL). Using a StepOnePlus (Applied Biosystems, CA, USA) thermal cycler, real time RT-qPCR was performed as outlined above. The ΔΔCt method was used to calculate the fold change.
2.9. Cellular Uptake of siRNA Polyplexes. To quantify siRNA uptake, MDA-MB-436 cells were transfected with 30 nM FAM-labeled siRNA at siRNA:reagent:additive ratios of 1:10:0 and 1:10:1 (w/w/w). After 24 h of transfection, cells were trypsinized and fixed with 3.7% formaldehyde. The mean fluorescence in cells and FAM-positive cell population were quantified using BD Accuri C6 Plus flow cytometer (BD Biosciences, Franklin Lakes, NJ). In addition, the mean fluorescence of the recovered cell population and the percentage of cells showing FAM-fluorescence were determined after gating a representative portion of nontreated cells for autofluorescence (typically set at 1% of total population)[21].
A fluorescence microscopy analysis of MDA-MB-436 cells transfected with FAM- labeled siRNA complexes was performed after 24 h and 72 h of cell uptake. Cells were washed (x2) with HBSS, fixed with 3.7% formaldehyde, and stained with DAPI (Brunschwig Chemie, Amsterdam, the Netherlands) to visualize the nuclear border using the fluorescent microscope model FSX100 Bio-imaging navigator (Olympus America, Center valley, PA).
2.10 Mcl-1 and Survivin Silencingin vivo. 15 NCG male mice, aged 6–8 weeks, were purchased from Charles River Labs (Quebec City, Canada) and used in this study. The animal study was carried out in compliance with the Canadian Council on Animal Care guidelines, with approval from the University of Alberta's Animal Care and Use Committee (AB, Canada). The MDA-MB-436 cell line was injected subcutaneously (2 × 106 cells in 50 µL of PBS and 50 µL of Matrigel) to generate tumor xenografts, and the mice were treated after the tumors reached a size of ≥ 50 mm3. Mice were treated via intravenous (tail-vein) injection of CsiRNA and 1:1 combination of Mcl-1 and survivin siRNA polyplexes. The siRNA dose was 20 µg, formulated with 20 µg of Trans-Booster additive and 200 µg of Prime-Fect (giving a siRNA:Trans-Booster:Prime-Fect ratio (w/w) of 1:1:10) in 100 µL of RPMI media. The siRNA polyplexes were administered every third day and mice were observed daily. Tumor size was measured using a caliper on days 0, 3, 6 and 9 and the mean/standard error of relative tumor volume was determined in each group using the formula: 0.5LW2 (the tumor's long diameter is L, and its short diameter is W.)
The level of gene knockdown in the tumor was measured using real-time RT-qPCR. RNA from the tumors was extracted using the TRizol method and converted to cDNA using the SensiFAST™ cDNA Synthesis kit. The real time RT-qPCR reactions were performed as outlined above. The ΔΔCt technique was used to calculate the fold change.