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Supplemental Materials and Methods:
Reagents: Oligo primers were purchased from Operon. Anti-MMP-14 and anti-TIMP-2
antibodies were purchased from EMD Millipore. The anti-tubulin antibody was purchased from
Cell Signaling Technology. EZ-Link Sulfo-NHS-SS-Biotin and Biotinylation Kits were
purchased from Pierce. Collagen type I was purchased from BD Biosciences. miScript Primer
Assay and miScript SYBR Green PCR Kit was purchased from Qiagen.
DNA construction: The DNA plasmids for MMP-14 with the 3'UTR (MMP-14/3'UTR),
MMP-14 without the 3'UTR (MMP-14), and MMP-14-green fluorescent protein (GFP)
(MMP14-GFP) chimeric cDNAs were generated and reported previously (1-3).
To study the effect of miR-181a-5p on MMP-14 expression via the 3’UTR, a reporter gene
system was generated. The pGL3 promoter-vector (Promega) containing firefly luciferase driven
by a pSV40 promoter was employed. A 1.5 kb DNA fragment of the MMP-14 3’UTR was
amplified by PCR using MMP-14/3'UTR as a template (forward primer #2962:
5’ATTCTAGATGCTCTACTGCCAGCGTTCC3’;
and
reverse
primer
#2963:
5’ATTCTAGAGTTCACCCCATTTCCCCTAC3’) and cloned into pGL3 at the XbaI site to
generate Luciferase/3’UTR (Luc/3'UTR). Another reporter utilizing a GFP was also generated by
cloning the MMP-14 3’ UTR (primers #2962 and #2963) into MMP14-GFP at the XbaI site to
generate MMP14-GFP/3'UTR. To determine the specificity of the miR-181a-5p response
element within the 3’UTR, a site direct mutagenesis approach was employed to convert the miR181a-5p binding sequence (TGAATG) to complementary nucleotides (ACTTAC) in the
Luc/3’UTR plasmid DNA (Luc/3'UTRmu).
To assess the ability of miR-181a-5p to regulate MMP-14 expression, we generated a
retroviral construct for expressing the miR-181a-5p precursor based on the MDH1-PGK-GFP 2.0
vector that harbors GFP as a reporter protein (Addgene). We amplified miR-181a-5p2 that
includes the approximately 22-nt mature miR-181a-5p and 140 nt of genomic sequence flanking
each side of the miR-181a-5p from human genomic DNA extracted from human umbilical vein
endothelial cells (HUVEC). The primers were used as follows: forward primer #2932:
5’ATCTCGAGTTTAAATACTCTCGACTTGA3’;
and
reverse
primer
#2933:
5’ATGAATTCTGGACCACATTTGGGGGATT3’. The resultant PCR fragment containing premiR-181a-5p was cloned into MDH1-PGK-GFP 2.0 vector at XhoI and EcoRI sites to generate
the miR-181a-5p/GFP construct. Similarly, we generated miR-128 (miRNA-128/GFP) (forward
primer
#2921:
5’ATCTCGAGCTAGCTGTTTTCTGTGTAGC3’;
and
reverse
primer
#2922 :5’ATGAATTCAGGTATTACAATTAATGAAA3’) that was not found to recognize
MMP-14 as a control. We also generated GFP-free miR-181a-5p and miRNA-control by
digesting the plasmid DNAs (miR-181a-5p/GFP and miR-control) with NcoI and NotI to remove
GFP cDNA.
To generate an inhibitor of miR-181a-5p
for downregulation of miR-181a-5p in less
invasive, high miR-181a-5p expressing cancer cells, we generated a miR-181a-5p sponge based
on a published report for miRNA sponge design (4). The miR-181a-5p sponge contains: 1) 22 nt
mismatched antisense of miR-181a-5p in each repeat (mature miR-181a-5p: 23 nt) ; 2) a bulge
area with a nt at position 9 missing and position 10-12 mismatched from the 5' end of miR-181a5p mature sequence; 3) four tandem repeats of the miR-181a-5p binding site with a 4 nt spacer
sequence (AATT) between the repeats; 4) 5' and 3' caps; and 5) a poly-A tail at the 3' end. The
first half of the miR-181a sponge was generated by annealing sense and anti-sense oligos (sense
oligo_3113: 5' gatcc GAC GGC GCT AGG ATC ATC AAC AAA CAT TCA GTA TGT CGG
TGA GTA ATT AAC ATT CAG TAT GTC GGT GAGT g 3’; and antisense_3114: 5’ aattc
ACT CAC CGA CAT ACT GAA TGT TAA TTA CTC ACC GAC ATA CTG AAT GTT TGT
TGA TGA TCC TAG CGC CGTC g 3’) containing BamHI and EcoRI sites. The double
stranded DNA insert was then cloned into pSIREN/RetroQ vector (Clontech). The resultant
construct then received the second half of the miR-181a-5p sponge with annealed doubled strand
oligos (sense_3115: 5’ [phos] aattc AAC ATT CAG TAT GTC GGT GAG TAA TTA ACA
TTC AGT ATG TCG GTG AGT AGA TGA TCC TAG CGC CGT CTT TTTT g 3’; and
antisense_3116: 5’ [phos] aattc AAA AAA GAC GGC GCT AGG ATC ATC TAC TCA CCG
ACA TAC TGA ATG TTA ATT ACT CAC CGA CAT ACT GAA TGTT g 3’) at the EcoRI
site to generate the full length sponge construct.
All constructs were confirmed by DNA sequencing.
Real-Time RT-PCR: Total RNA from cells was isolated using Qiagen miRNeasy Mini Kit
according to the manufacturer’s instructions. The RNA was reverse transcribed to generate
cDNA using the Bio-Rad iScript cDNA Synthesis Kit or Qiagen miScript II RT Kit. Quantitative
real-time PCR was performed using the Bio-Rad iQ SYBR Green Super Mix or Qiagen
QuantiTect SYBR Green PCR Master Mix on a BioRad iQ5 Real-Time PCR machine. Relative
expression was calculated using the Ct method. HPRT-1 or U6 were used as internal controls.
Gelatin Zymography: Gelatin zymography was carried out using 10% SDS-polyacrylamide
gels containing 0.1% gelatin (3). After electrophoresis, SDS was removed by incubation of the
gel with 2.5% Triton X-100 and gelatinase activity was recovered by incubation in a Tris-based
buffer. Gels were stained with Coomassie Brilliant Blue and cleared areas of gelatin signified
enzymatic activity.
Cell Surface Biotinylation Assay: Biotinylation of HT1080 cells using EZ-link Sulfo-NHSLC-biotin (Thermo) was performed according to the manufacturer’s instructions. Briefly,
transfected HT1080 cells were grown to 90% confluence and 2.5mM EZ-link sulfo-NHS-LCbiotin was added to the dishes after PBS washing. Samples were incubated at 4°C for 1hr,
rocking. The biotinylation reaction was terminated by addition of the quenching solution
followed by two washes with PBS. Cells were lysed in cold RIPA buffer containing 1% protease
inhibitors (Sigma). Clarified supernatants generated by centrifuging lysates at 10,000g at 4oC
were incubated with Pierce Streptavidin Agarose Resins (Thermo). After incubation, surface
proteins were collected by centrifugation at 10,000g at 4°C. The pellets were washed with cold
RIPA buffer and eluted by boiling with 2× SDS sample buffer with 10% 2-mercaptoethanol. For
the purpose of Western blotting analysis, input samples were loaded and probed for tubulin.
Transfected HT1080 cells without EZ-link sulfo-NHS-LC-biotin treatment were used as a
negative control.
Western Blotting: Immunoblotting was performed according to previous methods and
developed using a BioRad ChemiDoc (5).
Dual Luciferase Assay: To examine luciferase activity, cells were transiently transfected
with the appropriate constructs along with the renilla luciferase reporter gene using
polyethylenimine. Forty-eight hours after transfection, firefly and renilla luciferase activities
were measured using the Promega Dual-Glo Luciferase Assay System according to the
instructions.
Transwell Chamber Migration Assay: Polycarbonate membranes with 8-μm pore size were
assembled in blind well chemotactic chambers (Neuro Probe, MD). Cells were suspended in
complete media and placed in the upper portion of the chamber. Chambers were incubated for
18 h at 37 °C. The cells remaining on the top surface of the membrane were removed with
application of a cotton swab followed by three PBS washes. The cells on the bottom surface of
the membrane were fixed, stained, and quantified by counting 10 fields on the membrane under a
20X objective.
Three Dimensional (3D) Invasion Assay: The 3D invasion assay was carried out as
previously published (6). Briefly, cancer cells mixed with an equal volume of type I collagen (3
mg/ml) were dotted onto a 96-well plate followed by covering with an additional layer of type I
collagen gel (1.5 mg/ml). Complete media were added and incubated for 24 h, followed by
counting of invaded cells after Hoechst staining on a Nikon Eclipse TE2000-S equipped with a
Sutter Instruments SmartShutter System and a QiClick QImaging camera.
Data Mining: Expression of MMP-14 in human cancers was queried using the Oncomine
database (http://www.oncomine.org). This is a publicly available database summarizing gene
chip experiments across tissue types. Oncomine provides an infrastructure of data mining tools to
query genes and data sets of interest as well as to meta-analyze groups of studies. Studies were
included comparing normal tissues and corresponding cancers in order to determine the
expression level of MMP-14.
Laser capture microdissection (LCM): Cancer and normal epithelial cells in FFPE tissue
sections were isolated by LCM technique using a Leica Laser Microscope. UV-energy was set to
82 and UV-Focus was set to 76 for the collection of cells. Total RNA was extracted from the
isolated cells by using nano-RNA isolation kit (Epicentre) according to the manufacturer’s
instruction and amplified using Genisphere’s SenseAmp Plus for Low Molecular Weight RNA
followed by real-time RT-PCR.
Chicken Chorioallantoic Membrane Angiogenesis and Invasion Assay: The CAM assay was
performed as previously described (7). Fertilized white chicken eggs (SPF Premium, Charles
River Laboratory, CT) were incubated at 37oC in 70% humidity for three days. The embryos
were then incubated ex ovo in a sterile Petri dish for seven days. Gelatin sponges adsorbed with
cancer cells were implanted on the CAM surface (8) and neovasculature was counted on day four
post-implantation. For the invasion assay, 1x106 tumor cells were directly loaded onto the
surface of the CAM and invading cells were then examined by HE staining and IF using frozen
sections (9).
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