All cell culture reagents were purchased from Sigma Aldrich (St. Louis, MO, USA). PLGA was purchased from Lakeshore Biomaterials (Birmingham, AL, USA) as a 50:50 monomer ratio with a molecular weight of 58 kDa and inherent viscosity of 0.43 dl/g.
Human colon cancer HCT-116 cells were obtained from the American Type Culture Collection and propogated in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin in a humidified chamber at 37°C and 5% CO2.
Small interfering RNAs
DCAMKL-1 siRNA (si-DCAMKL-1) sequence targeting the coding region of DCAMKL-1 (accession No. NM_004734) (GGGAGUGAGAACAAUCUACtt) and scrambled siRNAs (si-SCR) not matching any of the human genes were obtained (Ambion Inc, Austin, TX) and transfected using siPORT™ NeoFX™ (Ambion).
Synthesis and characterization of DCAMKL-1 siRNA NPs
-glycolide) acid nanoparticles (PLGA NPs) were synthesized using a double emulsion solvent evaporation technique [44
]. First siRNA (DCAMKL-1 or scrambled) was condensed on the cationic polymer poly(ethyleneimine) (PEI, 5% w/v) to form an siRNA-PEI complex. siRNA-PEI (200 μl) was added to 30 mg PLGA in 1 ml chloroform (CHCl3
) and vortexed. This primary emulsion was then transferred into 5 ml of 2% (w/v) polyvinyl alcohol (PVA), which serves as a surfactant, and the entire solution was sonicated on ice for 1 min using a probe sonicator (Misonix XL-2000, Newtown, CT). The organic solvent in the final solution was allowed to evaporate overnight with continuous stirring. NPs were recovered by centrifugation at 20,000 ×g
for 20 min at 4°C. The supernatant was stored for later assay. The pellet consisting of aggregated NPs was washed three times in water to remove any residual PVA and free, i.e., non-encapsulated, siRNA. NPs were then resuspended in water, freeze-dried for 24 h and then stored at -20°C for later use. The amount of encapsulated siRNA was quantified using a spectrophotometer (DU-800, Beckman Coulter, Brea, CA). The size, polydispersity index, and zeta-potential measurements of synthesized siRNA NPs were determined using diffraction light scattering (DLS) utilizing Zeta PALS (Brookhaven Instruments, Holtsville, NY). Surface morphology of the NPs was examined using a JOEL-JSM-880 scanning electron microscope. Loading efficiency was calculated using the following formula:
where siRNANPs is the amount of siRNA encapsulated inside PLGA NPs, and siRNATot is the total amount of siRNA added.
Human multi-cancer tissue microarrays (Tissue Array Network, Rockville, MD) and tumor xenograft tissues were subjected to immunohistochemical analyses. Heat-Induced Epitope Retrieval was performed on 4 μm formalin-fixed paraffin-embedded sections utilizing a pressurized Decloaking Chamber (Biocare Medical) in citrate buffer (pH 6.0) at 99°C for 18 min. Brightfield: Slides were incubated in 3% hydrogen peroxide at room temperature for 20 min. After incubation with primary antibody [DCAMKL-1 C-terminal (Abcam Inc., Cambridge, MA) or c-Myc (Santa Cruz Biotechnologies Inc., Santa Cruz, CA) or Notch-1 (Santa Cruz Biotechnologies)], the slides were incubated in peroxidase-conjugated EnVision™+ polymer detection kit (DAKO). Slides were developed with diaminobenzidine (Sigma).
Slides were examined utilizing a Nikon 80i microscope and DXM1200C camera for brightfield analysis. Fluorescent images were taken with PlanFluoro objectives, utilizing CoolSnap ES2 camera (Photometrics). Images were captured utilizing NIS-Elements software (Nikon).
Xenograft tumor model
Male athymic nude mice (NCr-nu/nu) were purchased from the National Cancer Institute-Frederick Cancer Research and Development Center (Frederick, MD) and housed in pathogen-free conditions. They were cared for in accordance with guidelines set forth by the American Association for Accreditation of Laboratory Animal Care and the U.S. Public Health Service Commissioned Corps' "Policy on Human Care and Use of Laboratory Animals." All studies were approved and supervised by the Institutional Animal Care and Use Committee. HCT116 cells (6 × 106) were injected subcutaneously into the flanks of 4- to 6-wk-old male athymic nude mice (three mice per group). Tumors were measured using a caliper and the volume was calculated as (length × width2) × 0.5. The tumors reached 1000 mm3 15 days after injection of cells. NPs were reconstituted in sterile normal saline and injected directly into the tumors. DAPT was reconstituted in corn oil, which was injected intraperitoneally. In combination treatments, NPs were injected intratumorally and DAPT was injected i.p, at the same time points. Each animal bearing the tumor was injected on days 15, 18, 21, 24, and 27 with one of the following preparations - 50 μl (5 μM) of siRNA-NP preparation [either NP alone (control), NP-siScrambled (siSCR), or NP-siDCAMKL-1], or 10 mg/kg of DAPT alone, or a combination of NP-siDCAMKL-1 and DAPT. All mice were killed on day 30 .
Real-time Reverse Transcription-Polymerase Chain Reaction analyses
Total RNA isolated from tumor xenografts and HCT116 cells was subjected to reverse transcription using Superscript™ II RNase H-Reverse Transcriptase and random hexanucleotide primers (Invitrogen, Carlsbad, CA). The complementary DNA (cDNA) was subsequently used to perform real-time polymerase chain reaction (PCR) by SYBR™ chemistry (SYBR Green I, Molecular Probes, Eugene, OR) for specific transcripts using gene-specific primers and JumpStart™ Taq DNA polymerase (Sigma-Aldrich). The crossing threshold value assessed by real-time PCR was noted for the transcripts and normalized with β-actin messenger RNA (mRNA). The quantitative changes in mRNA were expressed as fold-change relative to control with ± SEM value.
The following primers were used:
β-actin: forward: 5'-GGTGATCCACATCTGCTGGAA-3',
DCAMKL-1: forward: 5'- CAGCAACCAGGAATGTATTGGA -3',
reverse: 5'- ctcaactcggaatcggaagact-3';
ZEB1: forward: 5'-AAGAATTCACAGTGGAGAGAAGCCA-3',
ZEB2: forward: 5'-AGCCGATCATGGCGGATGGC-3',
E-cadherin: forward: 5'-CCTCCCATCAGCTGCCC-3',
Snail: forward: 5'-AAGGCCTTCTCTAGGCCCT-3',
Slug: forward: 5'-TGCTTCAAGGACACATTA-3',
Twist: forward: 5-GTCTGGAGGATGGAGGG-3,
c-Myc: forward: 5'-CACACATCAGCACAACTACGCA-3',
Notch-1: forward: 5'-CGGGTCCACCAGTTTGAATG-3',
Total RNA isolated from tumor xenografts and HCT116 cancer cells was subjected to reverse transcription with Superscript II RNase H-Reverse Transcriptase and random hexanucleotide primers (Invitrogen). The cDNA was subsequently used to perform real-time PCR by SYBR chemistry for pri-let-7a, pri-miR-144, and pri-miR-200a transcripts using specific primers and JumpStart Taq DNA polymerase. The crossing threshold value assessed by real-time PCR was noted for pri-let-7a, pri-miR-144, and pri-miR-200a miRNAs and normalized with U6 pri-miRNA. The changes in pri-miRNAs were expressed as fold-change relative to control with ± SEM values .
The following primers were used:
pri-U6: forward: 5'-CTCGCTTCGGCAGCACA-3',
pri-let-7a: forward: 5'-GAGGTAGTAGGTTGTATAGTTTAGAA-3',
pri-miR-144: forward: 5'-GCTGGGATATCATCATATACTG-3',
pri-miR-200a: forward: 5'-TTCCACAGCAGCCCCTG-3',
Western blot analysis
HCT116 cells or tumor xenograft samples treated with siRNA or siRNA-NPs were lysed and the concentration of protein was determined by the BCA protein assay kit (Pierce Biotechnology Inc., Rockford, IL). Forty μg of the protein was size separated in a 7.5-15% SDS polyacrylamide gel and transferred onto a nitrocellulose membrane with a semidry transfer apparatus (Amersham-Pharmacia, Piscataway, NJ). The membrane was blocked in 5% non-fat dry milk for 1 h and probed overnight with rabbit anti-DCAMKL-1 antibody (Abcam Inc) or with rabbit anti-c-Myc, rabbit anti-Notch1 or rabbit anti-HES1 antibody (Cell Signaling Danvers, MA). Actin, used as a loading control was identified using a goat polyclonal IgG (Santa Cruz Biotechnology Inc). Subsequently, the membrane was incubated with anti-rabbit or anti-goat IgG horseradish peroxidase-conjugated antibodies (Amersham-Pharmacia) for 1 h at room temperature. The proteins were detected using ECL™ Western Blotting detection reagents (Amersham-Pharmacia).
Luciferase reporter gene assay
HCT116 cells were transfected with a plasmid containing the firefly luciferase (Photinus pyralis) gene with a complementary let-7a (or miR-114) binding site at its' 3'untranslated region (UTR) obtained from Signosis Inc (Sunnyvale, CA). The cells were also co-transfected with the Renilla luciferase expressing plasmid pRL-TK (Promega) as an internal control. Following transfection, the cells were treated with NPs alone, NP-siSCR, or NP-siDCAMKL-1 and subjected to luciferase activity measurement. Luciferase activity was determined as per the manufacturer's instructions (Dual-Luciferase Reporter Assay System; Promega) using a Biotek Synergy III multi plate reader (BioTek, Winooski, VT) as described previously . The activity, normalized to Renilla luciferase activity, is presented as relative luciferase units relative to control with ± SEM values. Assays were performed in triplicate wells and experiments were repeated three times.
All experiments were performed in triplicate. Results are reported as average ± SEM unless otherwise indicated. Data were analyzed using the Student's t-test. Results were considered statistically significant when p < 0.01.