Fig 1.
EpApt-siEp fabrication, in vitro processing by dicer enzyme, cell surface binding and internalization.
A. EpCAM aptamer secondary structure prediction from Mfold online. B. EpCAM aptamer siRNA chimeric construct carrying the siRNA targeting EpCAM (EpApt-siEp) is folded using Mfold online and the aptamer is indicated in blue box and the siRNA inside red box. C. EpCAM aptamer siRNA chimeric construct was incubated with the recombinant dicer enzyme at 37˚C for 18h. The reactions were performed without dicer as control reaction. Polyacrylamide gel electrophoresis of the reactions with and without dicer enzyme were run on 15% gel and stained with EtBr. The processed 21bp siRNA and unprocessed construct were observed. D. EpCAM aptamer siRNA chimeric construct was added to WERI-Rb1 and MCF7 cells in binding buffer and analyzed by flow cytometry. The overlay graph shows the uptake of the chimeric aptamer. E. Scatter plot showing the uptake of EpApt-siEp by the RB cell line, WERI-Rb1 and RB primary tumor cells. F. EpCAM aptamer siRNA chimeric construct was added to primary RB cells in media without serum for 2hr at 37˚C followed by washing with 1X PBS. Microscopic images were taken at 20X objective under phase and FITC channels of control cells alone and cells with EpApt-siEp. Data represents mean ± SD. Experiments were repeated 3 times independently with similar results. **P value of 0.01–0.001; *P value of 0.05–0.01.
Fig 2.
EpCAM knockdown using EpApt-siEp construct in WERI-Rb1 and MCF7 inhibits cell proliferation.
A. The EpCAM mRNA levels were detected from the total RNA of control, siEp and EpApt-siEp treated MCF7 cells by northern blotting. The total RNA was electrophoresed in formaldehyde agarose gel electrophoresis, blotted and developed by chemiluminescence based method. EpCAM targeting siRNA was used for synthesizing probe. On its right, the densitometry analysis of the bands were performed using imageJ software and plotted as graph with % mRNA expression against the 28s rRNA. B. The EpCAM mRNA levels were quantified by SYBR green based qPCR from the cDNA of control, siEp and EpApt-siEp treated WERI-Rb1 and MCF7 cells. C. Western blotting was performed on the siEp transfected and EpApt-siEp treated WERI-Rb1 and MCF7 cells for the EpCAM and b-tubulin. EpCAM targeting siRNA was used for synthesizing probe. D. The densitometry analysis of the western blotting bands was performed using imageJ software and plotted as graph with % protein (EpCAM) expression normalized to β-tubulin. E. The percentage cell proliferation was quantified by performing MTT assay on the control, siEp transfected, EpApt-siEp, EpApt and ScrApt treated WERI-Rb1 and MCF7 cells. The graph shows the % cell proliferation normalized to control cells. Data represents mean ± SD. Experiments were repeated 3 times independently with similar results. **P value of 0.01–0.001; *P value of 0.05–0.01.
Fig 3.
Expression of EpCAM Intracellular domain (EpICD) in RB and the effect of EpCAM knockdown on cancer stem cell markers.
A. Immunohistochemistry of the normal retina section showing no evident EpICD in the nucleus, RB tissue sections showing intense staining of nucleus. The expression of EpICD was majorly observed in nucleus of the tumor cells as shown by white arrows. B. The fold change in mRNA levels of SOX2, OCT4, Nanog, CD44S, CD133 and Survivin (BIRC5) were quantified by SYBR green based qPCR from the siEp and EpApt-siEp treated WERI-Rb1 cells and normalized to β-2-microglobulin as housekeeping gene. C. The fold change in mRNA levels of SOX2, OCT4 and Nanog were quantified by SYBR green based qPCR from the siEp and EpApt-siEp treated MCF7 cells and normalized to β-2-microglobulin as housekeeping gene.Data represents mean ± SD. Experiments were repeated 3 times independently with similar results.**P value of 0.01–0.001; *P value of 0.05–0.01.
Table 1.
Expression of EpICD in RB primary tumor by IHC.
Fig 4.
Tumor growth kinetics, changes in gene and protein expression in MCF7 Xenograft treated with EpApt-siEp.
Tumor growth kinetics of MCF7 Xenograft treated with EpApt-siEp. Female nude mice (Hsd: Athymic Nude-Foxn1nu, bilaterally ovariectomized) housed in Individually Ventilated Cages (IVCs) were used for the present investigation. The tumorigenicity of the MCF7 cells in mice is estrogen-dependent. Twenty hours prior to MCF-7 cell injection, animals were implanted with 17β-estradiol pellets (0.36mg/pellet; 60-day release; Innovative Research of America, Sarasota, FL) into dorsal shoulder blade region of mice using trochar. MCF-7 tumor cells (5 x106 cells/animal) were injected subcutaneously in the flanks of the animals. After 7–10 days post injection of cells, animals were randomized based on tumor volume (TV≈80mm3) and dosing was initiated. Graph showing the (A) Tumor volume of the Vehicle control group injected with PBS subcutaneously near the tumor site, EpApt-siEp subcutaneously injected near the tumor site on alternate days. The orange arrows indicate the EpApt-siEp injections given and the blue asterisk indicates the day of sacrifice. On Day 21 and 33, due to experimental and ethical reasons animals from both the groups were sacrificed 50% at each time. Photographs of the representative mice (B) and excised tumors (C) of vehicle control and treated groups. D. Changes in protein expression by Western blotting of proteins extraction from the representative tissue of the control mice or mice treated with EpApt-siEp (0.6nmol) and terminated at 21 and 33days respectively (on its right, graph representing the relative expression calculated by imageJ software. E. Graph showing the changes in MCF7 xenograft tumor tissue EpCAM, CD44s, CD24 and MRP1 mRNA levels post treatment with EpApt-siEp construct. The vehicle control was used for normalizing the fold expression and the β-2 microglobulin was used as internal control. F. Graph showing the changes in STMN, BIRC5, Bcl2, Bax and ATM mRNA levels post treatment with EpApt-siEp aptamer construct normalization was done with both vehicle control / no treatment group. Data represents mean ± SE for in vivo experiment (n = 8) and mean ±SD for other experiments. Experiments were performed in triplicates and significance was calculated by t-test. # P<0.001; **P value of 0.01–0.001; *P value of 0.05–0.01.
Fig 5.
Immunohistochemical staining of xenograft tissues.
Immunohistochemistry of the tumor tissues excised from the vehicle control mice and the mice treated with EpApt-siEp by intraperitoneal mode of injection. The levels of EpCAM and PCNA were studied and images are taken under 20x and 40x objective for EpCAM, 40x for PCNA. The intensities of expression of the antigens were represented with “+” on the right hand corner of the respective section.
Fig 6.
Protein array for apoptotic markers, cytokine secretion and the IHC analysis on EpApt-siEp treated xenograft samples.
A. Mouse cytokine array performed on the vehicle control and EpApt-siEp treated mice serum collected on day 33 before sacrifice. The upper panel shows the MCF7 xenograft vehicle control serum and lower to it is the serum from mice treated with EpApt-siEp upto 24days and studied upto 33days. Graphs below shows the mean integrated pixel density of the each protein spot from the blot detected using chemiluminescence imaging and quantified using imageJ software using microarray profile plugin. B. The levels of apoptotic markers between the vehicle control mice and the EpApt-siEp treated mice upto 24days and studied upto 33days, was analyzed using 'human apoptotic array'. The labels next to the spot represent the protein and the integrated pixel density was quantified using imageJ software and plotted as graph (i, ii & iii). ‘i’ panel represents the targets that belongs to intrinsic apoptotic pathway, ‘ii’ panel represents extrinsic apoptotic pathway and ‘iii’ panel represents other key regulators of apoptosis. The error bar represents the standard deviation and the ** indicates P value of 0.01–0.001; * indicates P value of 0.05–0.01.
Fig 7.
Illustration summarizing the EpCAM aptamer siRNA chimera effect on the tumor growth inhibition.
The EpCAM aptamer siRNA chimeric construct (EpApt-siEp) binds to the EpCAM receptor and gets internalized (1) and released in the cytoplasm where gets into Ago complex with dicer enzyme (2) to generate siRNA. The siRNA loaded into RISC complex (3) binds to the EpCAM mRNA (4) and leads to mRNA degradation (5). The EpCAM proteolysis leads to release of EpICD (EpCAM intracellular domain), shedding of EpEx (EpCAM extracellular domain) from EpTM (EpCAM transmembrane domain) (6) and the EpICD complexes with Wnt signaling mediators, β-catenin, FHL2 and TCF (7) to translocate to nucleus and regulate the gene transcription of pluripotency markers, SOX2, OCT4, NANOG, EpCAM, CD133, CD44 and aids in proliferation (8) upon EpCAM silencing these markers are downregulated (9) and cell proliferation is hampered(10). The EpCAM silencing leads to apoptotic cell death by downregulation of pluripotency, CSC markers, survivin, IAPs, Bcl2, p27, HSP70, catalase and claspin, by affecting intrinsic apoptotic pathway (11) leading to cell death(12). Overall, the knockdown of EpCAM using EpApt-siEp chimera leads to the inhibition of the nuclear signaling mediated by the EpICD, thereby decreases the cancer stem cell marker expression and induces apoptosis that brings down the tumorigenicity.